EP1395285A2 - Reagents and methods for modulating dkk-mediated interactions - Google Patents
Reagents and methods for modulating dkk-mediated interactionsInfo
- Publication number
- EP1395285A2 EP1395285A2 EP02744162A EP02744162A EP1395285A2 EP 1395285 A2 EP1395285 A2 EP 1395285A2 EP 02744162 A EP02744162 A EP 02744162A EP 02744162 A EP02744162 A EP 02744162A EP 1395285 A2 EP1395285 A2 EP 1395285A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- dkk
- hbm
- protein
- lrp6
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
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- A01K2217/00—Genetically modified animals
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- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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- A—HUMAN NECESSITIES
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- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
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- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to signal transduction, bone development, bone loss disorders, modulation of lipid-related conditions, research reagents, methods of screening drug leads, drug development, treatments for bone and/or lipid disorders, screening and development of therapies, molecular, cellular, and animal models of bone and/or lipid development and maintenance, which are mediated by Dkk, including Dkk-1 , and/or LRP5, LRP6, HBM or other members of the Wnt pathway.
- Osteoporosis affects both men and women, and, taken with other abnormalities of bone, presents an ever-increasing health risk for an aging population.
- the most common type of osteoporosis is that associated with menopause.
- Most women lose between 20-60% of the bone mass in the trabecular compartment of the bone within 3-6 years after the cessation of menses. This rapid bone loss is generally associated with an increase of bone resorption and formation. However, the resorptive cycle is more dominant and the result is a net loss of bone mass.
- Osteoporosis is a common and serious disease among postmenopausal women. There are an estimated 25 million women in the United States alone who are afflicted with this disease. The results of osteoporosis are personally harmful, and also account for a large economic loss due to its chronicity and the need for extensive and long-term support (e.g., hospitalization and nursing home care) from disease sequelae. This is especially true in elderly patients. Additionally, while osteoporosis is generally not thought of as a life-threatening condition, a 20-30% mortality rate is related to hip fractures in elderly women. A large percentage of this mortality rate can be directly associated with postmenopausal osteoporosis.
- the most vulnerable tissue in the bone to the effects of postmenopausal osteoporosis is the trabecular bone.
- This tissue is often referred to as spongy bone and is particularly concentrated near the ends of the bone, near the joints, and in the vertebrae of the spine.
- the trabecular tissue is characterized by small structures which inter-connect with each other as well as the more solid and dense cortical tissue which makes up the outer surface and central shaft of the bone. This cris- cross network of trabeculae gives lateral support to the outer cortical structure and is critical to the biomechanical strength of the overall structure.
- osteoporosis In postmenopausal osteoporosis, it is primarily the net resorption and loss of the trabeculae which lead to the failure and fracture of the bone. In light of the loss of the trabeculae in postmenopausal women, it is not surprising that the most common fractures are those associated with bones which are highly dependent on trabecular support, e.g., the vertebrae, the neck of the femur, and the forearm. Indeed, hip fracture, Colle's fractures, and vertebral crush fractures are indicative of postmenopausal osteoporosis. Osteoporosis affects cortical as well as trabecular bone. Alterations in endosteal bone resorption and Haversian remodeling with age affect cortical thickness and structural integrity contributing the increased risk for fracture.
- estrogen replacement therapy One of the earliest generally accepted methods for treatment of postmenopausal osteoporosis was estrogen replacement therapy. Although this therapy frequently is successful, patient compliance is low, primarily due to the undesirable side-effects of chronic estrogen treatment. Frequently cited side-effects of estrogen replacement therapy include reinitiation of menses, bloating, depression, and, potentially, increased risk of breast or uterine cancer. In order to limit the known threat of uterine cancer in women who have not had a hysterectomy, a protocol of estrogen and progestin cyclic therapy is often employed. This protocol is similar to that used in birth control regimens, and often is not tolerated by women because of the side-effects characteristic of progestin.
- raloxifene See, U.S. Patent No. 5,393,763; Black et al, J. Clin. Invest, 93:63-69 (1994); and Ettinger et al., JAMA 282:637-45 (1999)).
- tamoxifen a widely used clinical agent for treating breast cancer, has been shown to increase bone mineral density in post menopausal women suffering from breast cancer (Love et al., N. Engl. J. Med., 326:852-856 (1992)).
- Calcitonin is a naturally occurring peptide which inhibits bone resorption and has been approved for this use in many countries (Overgaard et al., Br. Med. J., 305:556-561 (1992)). The use of calcitonin has been somewhat limited, however. Its effects are very modest in increasing bone mineral density, and the treatment is very expensive.
- Another therapy for the treatment of postmenopausal osteoporosis is the use of bisphosphonates. These compounds were originally developed for treating Paget's disease and malignant hypercalcemia. They have been shown to inhibit bone resorption.
- Alendronate a bisphosphonate
- These agents may be helpful in the treatment of osteoporosis, but these agents also have potential liabilities which include osteomalacia, extremely long half-life in bone (greater than 2 years), and possible "frozen bone syndrome,” e.g., the cessation of normal bone remodeling.
- Senile osteoporosis is similar to postmenopausal osteoporosis in that it is marked by the loss of bone mineral density and resulting increase in fracture rate, morbidity, and associated mortality. Generally, it occurs in later life, i.e., after 70 years of age. Historically, senile osteoporosis has been more common in females, but with the advent of a more elderly male population, this disease is becoming a major factor in the health of both sexes. It is not clear what, if any, role hormones such as testosterone or estrogen have in this disease, and its etiology remains obscure. Treatment of this disease has not been very satisfactory. Hormone therapy, estrogen in women and testosterone in men, has shown equivocal results; calcitonin and bisphosphonates may be of some utility.
- Peak skeletal mass is the most powerful determinant of bone mass in elderly years (Hui et al, Ann. Int. Med., 111 :355-361 (1989)), even though the rate of age-related bone loss in adult and later life is also a strong determinant (Hui et al., Osteoporosis Int., 1 :30- 34 (1995)). Since bone mass is the principal measurable determinant of fracture risk, the inherited peak skeletal mass achieved at maturity is an important determinant of an individual's risk of fracture later in life. Thus, study of the genetic basis of bone mass is of considerable interest in the etiology of fractures due to osteoporosis.
- the present invention relates to modulation of lipid levels.
- Cardiovascular disease is the most common cause of mortality in the United States, and atherosclerosis is the major cause of heart disease and stroke. It is widely appreciated that cholesterol plays an important role in atherogenesis. Normally, most cholesterol serves as a structural element in the walls of cells, whereas much of the rest is in transit through the blood or functions as the starting material for the synthesis of bile acids in the liver, steroid hormones in endocrine cells and vitamin D in skin. The transport of cholesterol and other lipids through the circulatory system is facilitated by their packaging into lipoprotein carriers.
- spherical particles comprise protein and phospholipid shells surrounding a core of neutral lipid, including unesterified (“free”) or esterified cholesterol and triglycehdes.
- Reasterified low density lipoprotein
- HDL high- density lipoprotein
- LDL metabolism began in 1974 by Michael Brown and Joseph Goldstein.
- the liver synthesizes a precursor lipoprotein (very low density lipoprotein, VLDL) that is converted during circulation to intermediate density lipoprotein (IDL) and then to LDL.
- VLDL very low density lipoprotein
- IDL intermediate density lipoprotein
- the majority of the LDL receptors expressed in the body are on the surfaces of liver cells, although virtually all other tissues ("peripheral tissues”) express some LDL receptors.
- peripheral tissues express some LDL receptors.
- the receptor- lipoprotein complex is internalized by the cells via coated pits and vesicles, and the entire LDL particle is delivered to lysosomes, wherein it is dissembled by enzymatic hydrolysis, releasing cholesterol for subsequent cellular metabolism.
- This whole- particle uptake pathway is called "receptor-mediated endocytosis.”
- Cholesterol-mediated feedback regulation of both the levels of LDL receptors and cellular cholesterol biosynthesis help ensure cellular cholesterol homeostasis.
- Genetic defects in the LDL receptor in humans results in familial hypercholesterolemia, a disease characterized by elevated plasma LDL cholesterol and premature atherosclerosis and heart attacks.
- One hypothesis for the deleterious effects of excess plasma LDL cholesterol is that LDL enters the artery wall, is chemically modified, and then is recognized by a special class of receptors called macrophage scavenger receptors, that mediate the cellular accumulation of the LDL cholesterol in the artery, eventually leading to the formation of an atherosclerotic lesion.
- the major lipoprotein classes include intestinally derived chylomicrons that transport dietary fats and cholesterol, hepatic-derived VLDL, IDL, and LDL that can be atherogenic, and hepatic- and intestinally-derived HDL that are antiatherogenic.
- Apoprotein B is necessary for the secretion of chylomicrons (ApoB48) and VLDL, IDL, and LDL (ApoBIOO).
- Plasma levels of VLDL triglycehdes are determined mainly by the rates of secretion in LDL lipolytic activity.
- Plasma levels of LDL cholesterol are determined mainly by the secretion of ApoBIOO into plasma, the efficacy with which VLDL are converted to LDL and by LDL receptor-mediated clearance. Regulation of HDL cholesterol levels is complex and is affected by rates of synthesis of its Apo proteins, rates of esterification of free cholesterol to cholesterol ester by LCAT, levels of triglyceride-rich lipoproteins and CETP-mediated transfer of cholesterol esters from HDL, and clearance from plasma of HDL lipids and Apo proteins.
- lipoprotein transport is associated with low levels of triglycerides and LDL cholesterol and high levels of HDL cholesterol.
- lipoprotein levels can change in ways that predispose individuals to atherosclerosis and arteriosclerosis (see Ginsburg, Endocrinol. Metab. Clin. North Am., 27:503-19 (1998)).
- lipoprotein receptors may be involved in cellular lipid uptake. These receptors include: scavenger receptors; LDL receptor-related protein/ ⁇ 2- macroglobulin receptor (LRP); LDL receptor; and VLDL receptor. With the exception of the LDL receptor, all of these receptors are expressed in atherosclerotic lesions while scavenger receptors are mostly expressed in macrophages, the LRP and VLDL receptors may play an important role in mediating lipid uptake in smooth muscle cells (Hiltunen et al, Atherosclerosis, 137 suppl.:S81-8 (1998)).
- LRP LDL receptor-related protein/ ⁇ 2- macroglobulin receptor
- VLDL receptor VLDL receptor
- 3-hydroxy-3-methylglutaryl-CoA reductase HMG CoA reductase inhibitory drugs.
- 3-hydroxy-3-methylglutaryl-CoA reductase is the rate controlling enzyme in cholesterol biosynthesis, and its inhibition in the liver stimulates LDL receptor expression.
- both plasma LDL cholesterol levels and the risk for atherosclerosis decrease.
- the discovery and analysis of the LDL receptor system has had a profound impact on cell biology, physiology, and medicine. HDL is thought to remove unesterified, or "free" cholesterol (FC) from peripheral tissues, after which most of the cholesterol is converted to cholesterol ester (CE) by enzymes in the plasma.
- HDL cholesterol is efficiently delivered directly to the liver and steroidogenic tissues via a selective uptake pathway and the HDL receptor, SR-BI (class B type I scavenger receptor) or, in some species, transferred to other lipoproteins for additional transport in metabolism (see Krieger, Proc. Natl. Acad. Sci. USA, 95:4077-4080 (1998)).
- SR-BI class B type I scavenger receptor
- the present invention provides reagents, compounds, compositions and methods relating to novel interactions of the extracellular domain of LRP5, HBM (a variant of LRP5), and/or LRP6 with Dkk proteins.
- LRP5 is also referred to as Zmaxl or Zmax.
- the invention is also to be understood to encompass embodiments relating to interactions between Dkk and LRP6 and Dkk and HBM.
- the methods, reagents, compounds, and compositions of the present invention include the Dkk family members, including but not limited to Dkk-1 , Dkk-2, Dkk-3, Dkk-4 and Soggy.
- the invention encompasses novel fragments of Dkk-1 which demonstrate a binding interaction between the ligand binding domain (LBD) of LRP5 and additional proteins and/or which can modulate an interaction between LRP5, or a variant or fragment thereof, and a Dkk protein.
- LBD ligand binding domain
- the invention provides assays, methods, compositions, and compounds relating to Dkk-Wnt signaling.
- Wnt proteins are compatible with the present invention, including Wnt1-Wnt19, and particularly, Wntl , Wnt3, Wnt3a, and WntlOb.
- the present invention further provides reagents, compounds, compositions and methods modulating interactions between one or more other proteins and Dkk-1.
- the present invention also provides a series of peptide aptamers which bind to Dkk-1 or to LRP5 (or HBM and/or LRP6).
- polypeptides of the invention for example in the form of peptide oligomers, aptamers, proteins, and protein fragments as well as the nucleic acids of the invention, for example in the form of nucleic acids which encode the polypeptides of the invention as well as antisense, or complimentary nucleic acids, are useful as reagents for the study of bone mass and lipid level modulation.
- the polypeptides and nucleic acids of the invention are also useful as therapeutic and diagnostic agents.
- the present invention provides useful reagents for the modulation of Dkk proteins with LRP5, LRP6, and/or HBM, the modulation Dkk-1 and/or Dkk-1 interacting protein activity, and modulation of LRP5/Dkk-1 , LRP6/Dkk1 and HBM/Dkk-1 interactions and Dkk-1 /Dkk-1 interacting protein interactions.
- the present invention provides a series of peptide aptamers which bind Dkk-1 or LRP5, LRP6, and/or HBM.
- An object of the invention is to provide for a method of regulating LRP5/LRP6/HBM/HBM-like activity in a subject comprising administering a therapeutically effective amount of a composition which modulates Dkk activity.
- the subject can be a vertebrate or an invertebrate organism, but more preferably the organism is a canine, a feline, an ovine, a primate, an equine, a porcine, a caprine, a camelid, an avian, a bovine, or a rodent organism.
- a more preferred organism is a human.
- the Dkk protein is Dkk-1.
- Dkk-1 activity is decreased.
- Dkk activity modulates bone mass and/or lipid levels.
- bone mass is increased and/or lipid levels are decreased.
- the modulation in bone mass is an increase in bone strength determined via one or more of a decrease in fracture rate, an increase in areal bone density, an increase in volumetric mineral bone density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density or thickness, an increase in bone diameter, and an increase in inorganic bone content.
- the invention further provides such a method wherein the composition comprises a Dkk, Dkk-1 or a LRP5/LRP6/HBM binding fragment thereof, such as those depicted in Figure 6 or a mimetic of those fragments depicted in Figure 6.
- the invention further provides such a method wherein the composition comprises one or more of the proteins which interact with Dkk, including Dkk-1 , such as those depicted in Figure 5, or a Dkk-binding fragment thereof, or an antisense, siRNA, or shRNA molecule which recognizes and binds to a nucleic acid encoding one or more Dkk interacting or Dkk-1 interacting proteins.
- the invention further provides such a method wherein the composition comprises an LRP5/LRP6/Zmax1 antibody, Dkk antibody, a Dkk-1 antibody or an antibody to a Dkk-1 interacting protein.
- the invention further provides such a method wherein the compositions comprise an aptamer of Dkk or Dkk-1 , such as those depicted in Figure 3 (SEQ ID NOs:171-188), or a mimetic of such an aptamer.
- the method further provides that invention further provides such a method wherein the compositions comprise an aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- a composition of the present invention may modulate activity either by enhancing or inhibiting the binding of Dkk to LRP5/LRP6/Zmax1 , particularly Dkk-1 , or the binding of Dkk-1 to a Dkk-1 interacting protein, such as those shown in Figure 5.
- a composition of the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figures 4 (SEQ ID NOs:189-192) (particularly, peptide (SEQ ID NO:191 ) and 13 (including SEQ IDNOs:204-214), or a mimetic of such an aptamer.
- Preferred compositions of the present invention also comprise LRP5 antibodies.
- Another aspect of the invention is to provide for a method of regulating Dkk- Wnt pathway activity in a subject comprising administering a therapeutically effective amount of a composition which modulates Dkk-Wnt pathway activity.
- the Dkk protein is Dkk-1.
- Dkk- 1 activity is decreased.
- Dkk activity modulates bone mass and/or lipid levels.
- bone mass is increased and/or lipid levels are decreased.
- the modulation in bone mass is an increase in bone strength determined via one or more of a decrease in fracture rate, an increase in areal bone density, an increase in volumetric mineral bone density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density or thickness, an increase in bone diameter, and an increase in inorganic bone content.
- the Wnt is Wnt1-Wnt19.
- the Wnt is Wntl , Wn3, Wnt3a, or Wnt10b.
- Preferred compositions comprise Dkk-modulating or Dkk-1 - modulating compounds or one or more Dkk interacting or Dkk-1 interacting proteins, or a Dkk-binding fragment thereof.
- Dkk modulating compositions comprise a Dkk or Dkk-1 antibody or an antibody to a Dkk interacting or Dkk-1 interacting protein.
- antisense, siRNA, and shRNA molecules which recognize and bind to a nucleic acid encoding one or more Dkk-1 interacting proteins.
- the invention further provides such a method wherein the composition comprises a biologically active or LRP5/LRP6/HBM binding fragment of Dkk, including Dkk-1 , such as those depicted in Figure 6 or a mimetic of those fragments depicted in Figure 6.
- the Dkk modulating composition may also comprise a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- a composition of the present invention may modulate activity either by enhancing or inhibiting the binding of Dkk, including Dkk-1 , to LRP5, LRP6, or HBM or the binding of Dkk, including Dkk-1 , to a Dkk interacting protein, such as those shown in Figure 5.
- the invention further provides such a method wherein the composition comprises an aptamer of Dkk or Dkk-1 , such as those depicted.
- a composition of the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208).
- Preferred compositions of the present invention also comprise LRP5 antibodies.
- a further aspect of the invention is to provide for a method of modulating Wnt signaling in a subject comprising administering a therapeutically effective amount of a composition which modulates Dkk activity or modulates Dkk interaction with LRP5 (or LRP6 or HBM).
- the Dkk protein is Dkk-1.
- Dkk-1 activity is decreased.
- Dkk activity modulates bone mass and/or lipid levels.
- bone mass is increased and/or lipid levels are decreased.
- the modulation in bone mass is an increase in bone strength determined via one or more of a decrease in fracture rate, an increase in areal bone density, an increase in volumetric mineral bone density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density or thickness, an increase in bone diameter, and an increase in inorganic bone content.
- the Wnt is Wnt1-Wnt19.
- the Wnt is Wntl , Wnt3, Wnt3a, or Wnt10b.
- Preferred Wnt modulating compositions comprise one or more Dkk interacting or Dkk-1 interacting proteins, or a biologically active or LRP5/LRP6/HBM binding fragment thereof.
- compositions which recognize and bind to a nucleic acid encoding one or more Dkk interacting or Dkk-1 interacting proteins.
- the invention further provides such a method wherein the composition comprises a biologically active or LRP5/LRP6/HBM binding fragment of Dkk or Dkk- 1 , such as those depicted in Figure 6 or a mimetic of those fragments depicted in Figure 6.
- the Dkk modulating composition may also comprise a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- a composition of the present invention may modulate activity either by enhancing or blocking the binding of Dkk, including Dkk-1 , to LRP5, LRP6, or HBM or the binding of Dkk or Dkk-1 to a Dkk interacting or Dkk-1 interacting protein, such as those shown in Figure 5.
- the invention further provides such a method wherein compositions comprising an aptamer of Dkk or Dkk-1 , such as those depicted in Figure 3 (SEQ ID NOs: 171 -188), or a mimetic of such an aptamer.
- the composition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkk interacting or Dkk-1 interacting protein.
- the invention further provides such a method wherein compositions of an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figures 4 (SEQ ID NO:189-192 (particularly peptide (SEQ ID NO:191 ) and Figure 13 (including SEQ ID NOs:204- 214), or a mimetic of such an aptamer.
- Additional preferred compositions of the present invention also comprise LRP5 antibodies.
- the invention provides for a method of modulating bone mass and/or lipid levels in a subject comprising administering to the subject a composition which modulates Dkk activity or Dkk interaction with LRP5 in an amount effective to modulate bone mass and/or lipid levels, wherein bone mass and/or lipid levels are in need of modulation.
- the Dkk protein is Dkk-1.
- Dkk-1 activity is decreased.
- Dkk activity modulates bone mass and/or lipid levels.
- bone mass is increased and/or lipid levels are decreased.
- the modulation in bone mass is an increase in bone strength determined via one or more of a decrease in fracture rate, an increase in areal bone density, an increase in volumetric mineral bone density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density or thickness, an increase in bone diameter, and an increase in inorganic bone content.
- Preferred bone mass and/or lipid modulating compositions comprise one or more Dkk interacting or Dkk-1 interacting proteins, or a biologically active or LRP5/LRP6/HBM binding fragment thereof.
- antisense, siRNA, and shRNA molecules which recognize and bind to a nucleic acid encoding one or more Dkk interacting or Dkk-1 interacting proteins.
- the invention further provides such a method wherein the composition comprises a biologically active or LRP5/LRP6/HBM binding fragment of Dkk, including Dkk-1 , such as those depicted in Figure 6 or a mimetic of those fragments depicted in Figure 6.
- the Dkk modulating composition may also comprise a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- the invention further provides such a method wherein the composition comprises an aptamer of Dkk or Dkk-1 , such as those depicted in Figure 3 (SEQ ID NOs:171-188), or a mimetic of such an aptamer.
- a composition of the present invention may modulate activity either by enhancing or inhibiting the binding of Dkk, including Dkk-1 , to LRP5, LRP6, or HBM or the binding of Dkk, including Dkk-1 , to a Dkk interacting protein, such as those shown in Figure 5.
- the invention further provides such a method wherein the composition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkk interacting or Dkk-1 interacting protein.
- a composition of the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figures 4 (SEQ ID NOs:189-192 (particularly peptide 13 (SEQ ID NO:191)) and 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- LRP5 peptide aptamer such as OST262 (SEQ ID NO:208), Figures 4 (SEQ ID NOs:189-192 (particularly peptide 13 (SEQ ID NO:191)) and 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- Preferred compositions of the present invention also comprise LRP5 antibodies.
- lipid-modulated diseases include a cardiac condition, atherosclerosis, familial lipoprotein lipase deficiency, familial apoprotein CM deficiency, familial type 3 hyperlipoproteinemia, familial hypercholesterolemia, familial hypertriglyceridemia, multiple lipoprotein-type hyperlipidemia, elevated lipid levels due to dialysis and/or diabetes, and an elevated lipid level of unknown etiology.
- Bone disorders contemplated for treatment and/or diagnosis by the methods and compositions disclosed herein include a bone development disorder, a bone fracture, age related loss of bone, a chondrodystrophy, a drug-induced bone disorder, high bone turnover, hypercalcemia, hyperostosis, osteogenesis imperfecta, osteomalacia, osteomyelitis, osteoporosis, Paget's disease, osteoarthritis, and rickets.
- the Dkk is Dkk-1.
- the binding of Dkk-1 to LRP5/LRP6/HBM is decreased. It is a further object of the invention to provide a method of screening compounds or compositions which modulate the interaction of DKK with LRP5, LRP6, HBM, or a DKK-finding fragment thereof comprising:
- the Dkk is Dkk-1.
- the interaction of Dkk-1 with LRP5/LRP6/HBM is decreased.
- Dkk or a biologically active fragment thereof is attached to a solid substrate.
- LRP5/LRP6/HBM, or a biologically active fragment thereof (such as the ligand binding domain), is exposed to the compound.
- Another aspect of the invention provides for compounds and compositions identified by the disclosed methods.
- a preferred embodiment of the invention provides that the compound screened in an afore-mentioned method is one or more proteins which interact with Dkk, particularly Dkk-1 , as depicted in Figure 5, or a LRP5/LRP6/HBM-binding fragment thereof.
- the compound comprises a Dkk or Dkk-1 peptide aptamer, such as those depicted in Figure 3 (SEQ ID NOs:171-188), or a mimetic of such aptamers.
- the compound may also comprise a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- the method further provides that the compound comprises a Dkk or Dkk-1 antibody or an antibody to a Dkk-1 interacting protein.
- the invention further provides that the compound may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 )) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- Preferred compounds of the present invention also comprise LRP5 antibodies. It is a further object of the invention to provide a method of screening for compounds or compositions which modulate the interaction of Dkk and a Dkk interacting protein comprising:
- Dkk interacting proteins or the Dkk-binding fragment thereof In a preferred embodiment, the Dkk is Dkk-1.
- Dkk is Dkk-1.
- the Dkk interacting proteins particularly Dkk-1 interacting proteins, or a Dkk-binding fragment thereof are attached to a solid substrate.
- Another aspect of the invention provides for compounds and compositions identified by the disclosed methods.
- the compound comprises a Dkk or Dkk-1 peptide aptamer, such as those depicted in Figure 3 (SEQ ID NOs: 171 -188), or a mimetic of such aptamers.
- the compound may also comprise a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- the compound may also comprise an antibody to a Dkk interacting or Dkk-1 interacting protein.
- LRP5/LRP6/HBM modulating compounds include Dkk or Dkk-1 or a LRP5/LRP6/HBM binding fragment thereof.
- compounds which comprise monoclonal or polyclonal antibodies or immunologically active fragments thereof which bind to Dkk, including Dkk-1 , and a pharmaceutically acceptable excipient and/or carrier are also contemplated.
- the modulating compound comprises one or more Dkk interacting or Dkk-1 interacting proteins, or a biologically active fragment thereof.
- the modulating compound comprises an antisense, siRNA, and shRNA molecule which recognizes and binds to a nucleic acid encoding a Dkk interacting or Dkk-1 interacting protein.
- the modulating compound comprises a Dkk or Dkk-1 peptide aptamer, a mimetic of a Dkk or Dkk-1 peptide aptamer, a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- the compound comprises an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs: 189-192) (particularly peptide) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- Preferred compounds of the present invention also comprise LRP5 antibodies.
- compositions for treating a Dkk-mediated disease or condition comprising a compound which modulates Dkk activity and a carrier therefor, including pharmaceutically acceptable excipients.
- Such compositions include those wherein the compound comprises an antisense, siRNA, and shRNA molecule or an antibody which binds to Dkk, including Dkk-1 , and thereby prevents it from interacting with LRP5, LRP6, or HBM.
- compositions include one or more of Dkk interacting or Dkk-1 interacting proteins, such as those depicted in Figure 5, or a Dkk-binding fragment thereof, or a monoclonal or polyclonal antibody, or immunologically active fragment thereof, which binds to a Dkk interacting or Dkk-1 interacting protein or Dkk-binding fragment thereof.
- Dkk interacting or Dkk-1 interacting proteins such as those depicted in Figure 5, or a Dkk-binding fragment thereof, or a monoclonal or polyclonal antibody, or immunologically active fragment thereof, which binds to a Dkk interacting or Dkk-1 interacting protein or Dkk-binding fragment thereof.
- contemplated compositions include antisense, siRNA, and shRNA molecules which recognize and bind to a nucleic acid encoding a Dkk interacting or Dkk-1 interacting protein.
- compositions include Dkk and Dkk-1 peptide aptamers, such as those depicted in Figure 3 (SEQ ID NOs;171-188), mimetics of such aptamers, a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- Other contemplated compositions comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 )) and Figure 13 (including SEQ ID NO:204-214), or a mimetic of such an aptamer.
- compositions of the present invention comprise LRP5 antibodies.
- said composition comprises an antisense, siRNA, or shRNA molecule which recognizes and binds to a nucleic acid encoding a Dkk interacting or Dkk-1 interacting protein.
- One aspect of the invention provides for a method of modulating at least one activity of Dkk or a Dkk-1 interacting protein comprising administering an effective amount of a composition which modulates at least one activity of Dkk or a Dkk-1 interacting protein.
- the invention provides for a composition comprising a Dkk interacting or Dkk-1 interacting protein, such as those shown in Figure 5, or a biologically active fragment thereof.
- Other agents contemplated for this method are antisense, siRNA, or shRNA molecules which recognize and bind to a nucleic acid encoding a Dkk interacting or Dkk-1 interacting protein.
- the method further provides that the composition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkk interacing or Dkk-1 interacting protein.
- the composition comprises a Dkk or Dkk-1 peptide aptamer, a mimetic of a Dkk or Dkk-1 peptide aptamer, a peptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimetic of such an aptamer.
- a composition of the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NO:189-192) (particularly peptide including (SEQ ID NO:191 )) and Figure including (SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- compositions of the present invention also comprise LRP5 antibodies.
- the modulated Dkk activity is lipid modulation or bone mass modulation.
- HBM can be used as a control in the absence of a test sample or compound.
- the effect of a test sample of compound on Wnt signaling through the interaction of Dkk with LRP5/LRP6/HBM does not necessarily require a direct measurement of an association or interaction of Dkk and LRP5/LRP6/HBM.
- One aspect of the invention provides for a method of identifying binding partners for a Dkk protein comprising the steps of:
- the Dkk is Dkk-1.
- step (b) providing a second group of control animals respectively for the group of transgenic animals in step (a);
- the Dkk is Dkk-1.
- the Dkk is Dkk-1.
- the binding domain is attached to a solid substrate.
- the invention further provides for compounds identified by this method.
- the invention provides that the Dkk interacting or Dkk-1 interacting protein is detected by antibodies.
- the solid substrate is a microarray.
- Another preferred embodiment provides that the ligand binding domain of Dkk and/or Dkk interacting protein is fused or conjugated to a peptide or protein.
- the compounds include Dkk and Dkk-1 peptide aptamers, mimetics of Dkk and Dkk-1 peptide aptamers, Dkk and Dkk-1 interacting proteins peptide aptamers, or mimetics of such aptamers.
- An aspect of the invention provides a composition comprising one or more polypeptide sequences of one or more Dkk-1 interacting proteins, or a biologically active fragment thereof, one or more Dkk proteins, or a biologically active fragment thereof, or LRP5/LRP6/HBM polypeptide sequences or a biologically active fragment thereof (for example, the ligand binding domain) and a pharmaceutically acceptable excipient and/or carrier.
- the composition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkk interacting or Dkk-1 interacting protein and a pharmaceutically acceptable excipient.
- a composition of the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 )) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- LRP5 peptide aptamer such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 )) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- a composition of the present invention may comprise a Dkk peptide aptamer, for example as shown in Figure 3 (SEQ ID NOs: 171 -188).
- compositions of the present invention also comprise LRP5 antibodies.
- Another aspect of the invention is to provide an antibody or immunologically active antibody fragment which recognizes and binds to a Dkk-1 amino acid sequence selected from the group consisting of: Asn34-His266 (SEQ ID NO:110), Asn34-Cys245 (SEQ ID NO:111 ), Asn34-Lys182 (SEQ ID NO: 112), Cys97-His266 (SEQ ID NO:113), Val139-His266 (SEQ ID NO:114), Gly183-His266 (SEQ ID NO:115), Cys97-Cys245 (SEQ ID NO:116), or Val139-Cys245 (SEQ ID NO:117) of human Dkk-1.
- Asn34-His266 SEQ ID NO:110
- Asn34-Cys245 SEQ ID NO:111
- Asn34-Lys182 SEQ ID NO: 112
- Cys97-His266 SEQ ID NO:113
- Additional antibodies may bind to any of the sequences depicted in Figures 3 (SEQ ID NOs:171-188) and Figure 4 (SEQ ID NOs:189-192).
- Another aspect of the invention is to provide for polyclonal antibodies to one or more amino acid sequences: Peptide 1 -GNKYQTIDNYQPYPC (SEQ ID NO: 118), Peptide 2 - LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO.119), Peptide 3 - RIQKDHHQASNSSRLHTCQRH (SEQ ID NO:120), Peptide 4 - RGEIEETITESFGND (SEQ ID NO:121 ), and Peptide 5 - EIFQRCYCGEGLSCRIQKD (SEQ ID NO: 122).
- the scaffold protein is thioredoxin (trxA), S1 nuclease from Staphylococcus or M13.
- Other preferable embodiments include Dkk-1 amino acid sequences selected from Figure 6.
- Another aspect of the invention includes a method of detecting the modulatory activity of a compound on the binding interaction of a first peptide and a second peptide of a peptide binding pair that bind through extracellular interaction in their natural environment, comprising:
- culturing at least one eukaryotic cell wherein the eukaryotic cell comprises; a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein; b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element produces a selected phenotype; (ii) incubating a compound with the eukaryotic cell under conditions suitable to detect the selected phenotype; and (iii) detecting the ability of the compound to affect the binding
- the eukaryotic cell is a yeast cell.
- the yeast cell is Saccharomyces.
- Saccharomyces cell is Saccharomyces cerevisiae.
- the compound may comprise a Dkk interacting or Dkk-1 interacting protein, or a biologically active fragment thereof.
- the Dkk interacting or Dkk-1 interacting protein, or a Dkk-binding fragment thereof is added directly to the assay.
- the Dkk interacting or Dkk-1 interacting protein, or a Dkk-binding fragment thereof is recombinantly expressed by the eukaryotic cell in addition to the first and second peptides.
- the compound comprises a Dkk or Dkk-1 aptamer, a mimetic of a Dkk or Dkk-1 peptide aptamer, a Dkk interacting or Dkk-1 interacting protein aptamer, or a mimetic of a Dkk-1 interacting protein aptamer.
- the compound comprises an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 ) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- the present invention also provides that the compound may comprise LRP5 antibodies or Dkk antibodies.
- the yeast cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the DNA binding domain of a transcriptional activation protein, a nucleotide sequence encoding the transcriptional activation domain of a transcriptional activation protein, and a nucleotide sequence encoding the reporter element, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.
- the peptide binding pair comprises a ligand and a receptor to which the ligand binds.
- the transcriptional activation protein is Gal4, Gcn4, Hap1 , Adr1 , Swi5, Ste12, Mcm1 , Yap1 , Ace1 , Ppr1 , Arg81 , Lac9, Qa1 F, VP16, or a mammalian nuclear receptor.
- at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.
- the DNA binding domain comprises a heterologous DNA-binding domain of a transcriptional activation protein.
- the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.
- the reporter element is selected from the group consisting of lacZ, a polynucleotide encoding luciferase, a polynucleotide encoding green fluorescent protein (GFP), and a polynucleotide encoding chloramphenicol acetyltransferase.
- the reporter element is lacZ
- the invention further provides for a rescue screen for detecting the activity of a compound for modulating the binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:
- yeast cell comprises; a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein; b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype;
- the invention provides that the yeast cell is
- the Saccharomyces cell is Saccharomyces cerevisiae.
- the compound comprises one or more Dkk interacting or Dkk-1 interacting proteins, or a Dkk-binding fragment thereof.
- Compounds used in the present invention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191 )) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer.
- the compound may comprise LRP5 antibodies or Dkk antibodies.
- the yeast cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the DNA binding domain of a transcriptional activation protein, a nucleotide sequence encoding the transcriptional activation domain of a transcriptional activation protein, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.
- the transcriptional activation protein is Gal4, Gcn4, Hap1 , Adr1 , Swi5, Ste12, Mcm1 , Yap1 , Ace1 , Ppr1 , Arg81 , Lac9, Qa1 F, VP16, or a mammalian nuclear receptor.
- at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.
- the DNA binding domain is a heterologous DNA-binding domain of a transcriptional activation protein. The invention also provides for a rescue screen for detecting the modulatory activity of a compound on the binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:
- yeast cell comprises; a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein; b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element prevents exhibition of a selected phenotype; (ii) incubating a compound with the yeast cell under conditions suitable to detect the selected phenotype; and (iii) detecting the ability of the compound to affect the binding interaction of the peptide binding pair by
- the yeast cell is Saccharomyces.
- Saccharomyces cell is Saccharomyces cerevisiae.
- the yeast cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the DNA binding domain of a transcriptional activation protein, a nucleotide sequence encoding the transcriptional activation domain of a transcriptional activation protein, and a nucleotide sequence encoding the reporter gene, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.
- the transcriptional activation protein is Gal4, Gcn4, Hap1 , Adr1 , Swi5, Ste12, Mcm1 , Yap1 , Ace1 , Ppr1 , Arg81 , Lac9, Qa1 F, VP16, or a mammalian nuclear receptor.
- at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.
- the DNA binding domain is a heterologous DNA-binding domain of a transcriptional activation protein.
- the invention also provides for a method for identifying potential compounds which modulate Dkk activity comprising: a) measuring the effect on binding of one or more Dkk interacting protein, or a Dkk-binding fragment thereof, with Dkk or a LRP5/LRP6/HBM binding fragment thereof in the presence and absence of a compound; and b) identifying as a potential Dkk modulatory compound a compound which modulates the binding between one or more Dkk interacting proteins or Dkk-binding fragment thereof and Dkk or LRP5/LRP6/HBM fragment thereof.
- the Dkk is Dkk-1.
- the invention further provides for any of the Dkk peptide aptamers of Figure 3 (SEQ ID NOs:171-188).
- the invention also provides for any of the LRP peptide aptamers of Figure 4 (SEQ ID NOs: 189-192).
- Another aspect of the invention provides for a method of identifying agents which modulate the interaction of Dkk with the Wnt signaling pathway comprising:
- the agent may be chosen from among mRNA encoding Dkk interacting proteins, fragments thereof, siRNA, shRNA, antisense nucleotides, and antibodies.
- Dkk is Dkk-1.
- mRNA of HBM, LRP5/6, any Wnt (including Wnt1-Wnt19, particularly Wntl , Wnt3, Wnt3a, and Wnt10b), Wnt antagonist, or combination of these is co-injected into the Xenopus blastomere.
- the marker gene analyzed could include Siamois, Xnr3, slug, Xbra, HNK-1 , endodermin, Xlhbox ⁇ , BMP2, BMP4, XLRP6, EF-1 , or ODC.
- the present invention provides for a method for identifying agents which modulate the interaction of Dkk with the Wnt signaling pathway comprising:
- the cells may be HOB-03- CE6, HEK293, or U20S cells.
- the Wnt-responsive promoter is TCF or LEF.
- the cells are co-transfected with CMV beta- galactosidase or tk-Renilla.
- the present invention further provides for a LRP5/HBM monoclonal or polyclonal antibody to one or more peptides of amino acid sequences
- MYWTDWVETPRIE SEQ ID NO:123
- MYWTDWGETPRIE SEQ ID N0.124
- KRTGGKRKEILSA SEQ ID NO:125
- ERVEKTTGDKRTRIQGR SEQ ID NO:126
- KQQCDSFPDCIDGSDE SEQ ID N0.127
- the present invention provides a method for identifying compounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising:
- the Dkk is Dkk-1.
- the epitope tag is alkaline phosphatase, histidine, myc, or a V5 tag.
- Another embodiment of the present invention provides for a method for identifying compounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising:
- the Dkk is Dkk-1.
- the present invention also provides for a method of diagnosing low or high bone mass and/or low or high lipid levels in a subject comprising examining expression of Dkk, LRP5, LRP6, HBM or HBM-like variant in the subject and determining whether Dkk, LRP5, LRP6, or HBM or a HBM-like variant is over- or under-expressed to determine whether subject has (a) high or low bone mass and/or (b) high or low lipid levels.
- the invention further provides for a transgenic animal wherein Dkk is knocked out in a tissue-specific fashion.
- the Dkk is Dkk-1.
- the tissue specificity is bone tissue.
- the tissue specificity is liver or other tissues or cells involved in regulating lipid metabolism or cancer tissue.
- the present invention further provides a method of screening for compounds which modulate the interaction of Dkk with LRP5, LRP6, or HBM comprising:
- the Dkk is Dkk-1.
- the compound comprises an LRP5 peptide aptamer.
- Other preferred compositions include the peptide aptamer, OST262 (SEQ ID NO:208), Figure 4 (SEQ ID NOs:189- 192) (particularly peptide 13 (SEQ ID NO:191 ) and Figure 13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer, and an LRP5 antibody.
- the present invention also provides a method for identifying compounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising:
- the solid surface (b) treating the solid surface with a secreted Dkk protein or a secreted epitope-tagged Dkk and a test compound; and (c) determining whether the compound regulates binding between Dkk and LRP5/LRP6/HBM using antibodies to Dkk or the epitope tag or by directly measuring activity of an epitope tag.
- the epitope tag is alkaline phosphatase, histidine, myc or a V5 tag.
- the Dkk is Dkk-1.
- the invention also provides for a method for identifying compounds which modulate the interaction of Dkk with the Wnt signaling pathway comprising:
- the Dkk is Dkk-1.
- the Wnt is any of Wnt1-Wnt19.
- the Wnt is Wntl , Wnt3, Wnt3a, or Wnt10b.
- the Wnt construct contains Wnt3a.
- the Wnt construct contains Wntl .
- the Wnt construct encodes for a Wnt that signals through the canonical Wnt pathway.
- both Wnt3a and Wntl constructs are co-transfected into the cells.
- the cells may be U2-OS, HOB-03-CE6, or HEK293 cells.
- the reporter element used is TCF-luciferase, tk-Renilla, or a combination thereof. The invention also provides for a method of testing compounds that modulate
- Dkk-mediated activity in a mammal comprising:
- step (a) providing a group of transgenic animals having (1 ) a regulatable one or more Dkk genes, (2) a knock-out of Dkk genes, or (3) a knock-in of one or more Dkk genes; (b) providing a second group of control animals respectively for the group of transgenic animals in step (a); and
- the Dkk is Dkk-1.
- the invention further provides variants of LRP5 which demonstrate HBM biological activity, i.e., that are "HBM-like.”
- variants G171 F, M282V, G171 K, G171Q, A65V, G171V, G171 I, and A214V of LRP5 are provided.
- the invention further provides for the use any of these variants in the forgoing methods.
- FIGURES show a schematic of the components of the Wnt signal transduction pathway. Schematic obtained from: http://www.stanford.edu/ ⁇ rnusse/pathways/cell2.html
- Figure 2 show bait sequences (SEQ ID NOs:168-170) utilized in yeast two hybrid (Y2H) screens for protein-protein interactions.
- Figure 3 shows a table of peptide aptamer insert sequences (SEQ ID NOs: 171-192) identified in Y2H screen with a Dkk-1 bait sequence.
- Figure 4 shows a table of peptide aptamer insert sequences identified in a Y2H screen using a LRP5 ligand binding domain bait sequence.
- Figure 5 shows a table of proteins identified in a Y2H screen using a Dkk-1 bait sequence. These proteins are identified by both their nucleic acid and amino acid accession numbers.
- Figure 6 shows the results of a minimum interaction domain mapping screen of Dkk-1 with LRP5.
- a map of Dkk-1 showing the location of the signal sequence, and cysteine rich domains 1 and 2.
- scoring of the binding results observed in the experiment To the right, scoring of the binding results observed in the experiment.
- Figure 7 shows a diagram of the Xenopus Embryo Assay for Wnt activity.
- Figure 8 shows the effects of Zmax LRP5 and HBM on Wnt signaling in the
- Figure 9 shows the effects of Zmax/LRP5 and HBM on induction of secondary axis formation in the Xenopus embryo assay.
- Figure 10 shows the effects of human Dkk-1 on the repression of the canonical Wnt pathway.
- Figure 11 shows the effects of human Dkk-1 on Zmax LRP5 and HBM- mediated Wnt signaling.
- Figure 12 shows pcDNA3.1 construct names with nucleotide sequences (including SEQ ID NOs:193-203) for LRP5-binding peptide aptamers, Dkk-1 peptides and control constructs.
- Figure 13 shows the amino acid sequences (including SEQ ID NOs:204-214) for the corresponding LRP5-binding peptides, Dkk-1 peptide aptamers and control constructs in Figure 12.
- Figure 14 shows the effects of Dkk-1 and Dkk-2 on Wntl signaling with coreceptors LRP5, HBM, and LRP6 in HOB03CE6 cells.
- Figure 15 shows the effects of Dkk-1 and Dkk-2 on Wnt3a signaling with coreceptors LRP5, HBM, and LRP6 in HOB03CE6 cells.
- Figure 16 demonstrates that the LRP5-LBD peptide aptamer 262 activates Wnt signaling in the presence of Wnt3a in U20S cells.
- Figure 17 shows the differential binding of an antibody generated to a sequence (a. a. 165-177) containing the HBM mutation in LRP5 in LRP5 and HBM virus-infected cells.
- Figure 18 shows data generated from a Y2H interaction trap where a mutant Dkk-1 (C220A) is unable to bind to LRP5 and demonstrating the window of capability of detecting small molecule effects on LRP and Dkk interactions.
- Figure 19 shows that Dkk-1 represses Wnt3a-mediated Wnt signaling in U20S bone cells using the cell-based reporter gene assay for high throughput screening.
- Figure 20 demonstrates that Wnt1-HBM generated signaling is not efficiently inhibited by Dkk-1 in U20S bone cells while LRP5 and LRP6-mediated signaling are using the cell-based reporter gene assay for high throughput screening.
- Figure 21 shows that the TCF signal in the cell-based reporter gene assay for high throughput screening can be modulated by Dkk-1 and Dkk-1 -AP without Wnt DNA transfection.
- Figure 22 shows the morphological results in the Xenopus assay using aptamers 261 and 262 from the LRP5-LBD to activate Wnt signaling.
- Figure 23 demonstrates that LRP5-LBD aptamers 261 and 262 induce Wnt signaling over other LRP5 aptamers.
- Figure 24 shows that the mutation G171 F in LRP5 produces a greater activation of the Wnt pathway than LRP5 which is consistent with HBM activity.
- Figure 25 shows that the mutation M282V in LRP5 produces an activation of the Wnt pathway which is consistent with HBM activity in U20S cells.
- Figure 26 shows the amino acid sequence of the various peptides of dkk-1 selected to generate polyclonal antibodies, their relationship to the Dkk-1 amino acid sequence and identities of polyclonal antibodies generated.
- Figure 27 shows a Western blot demonstrating that polyclonal antibody #5521 to amino acids 165-186 of Dkk-1 was able to detect Dkk1-V5 and Dkk1-AP from conditioned medium.
- Figure 28 shows a Western blot demonstrating that polyclonal antibody #74397 to amino acids 147-161 was able to detect Dkk1-V5 in both conditioned medium and immunoprecipitated conditioned medium.
- Gene refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide.
- the term “gene” includes intervening, non-coding regions, as well as regulatory regions, and can include 5' and 3' ends.
- nucleic acid is meant to include single stranded and double stranded nucleic acids including, but not limited to DNAs, RNAs (e.g., mRNA, tRNAs, siRNAs), cDNAs, recombinant DNA (rDNA), rRNAs, antisense nucleic acids, oligonucleotides, and oligomers, and polynucleotides.
- the term may also include hybrids such as triple stranded regions of RNA and/or DNA or double stranded RNA:DNA hybrids.
- the term also is contemplated to include modified nucleic acids such as, but not limited to biotinylated nucleic acids, tritylated nucleic acids, fluorophor labeled nucleic acids, inosine, and the like.
- Gene sequence refers to a nucleic acid molecule, including DNA which contains a non-transcribed or non-translated sequence, which comprises a gene.
- the term is also intended to include any combination of gene(s), gene fragment(s), non-transcribed sequence(s) or non-translated sequence(s) which are present on the same DNA molecule.
- the nucleic acid sequences of the present invention may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA or combinations thereof. Such sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions and/or poly (A) sequences. The sequences, genomic DNA or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
- cDNA refers to complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
- a "cDNA clone” means a duplex DNA sequence for which one strand is complementary to an RNA molecule of interest, carried in a cloning vector or PCR amplified.
- cDNA can also be single stranded after first strand synthesis by reverse transcriptase. In this form, it is a useful PCR template and does not need to be carried in a cloning vector. This term includes genes from which the intervening sequences have been removed.
- the term “gene”, as sometimes used generically, can also include nucleic acid molecules comprising cDNA and cDNA clones.
- Recombinant DNA means a molecule that has been engineered by splicing in vitro a cDNA or genomic DNA sequence or altering a sequence by methods such as PCR mutagenesis.
- Codoning refers to the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to use methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
- cDNA library refers to a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire or a partial repertoire of genes expressed in a particular tissue or cell source.
- a cDNA library can be prepared by methods known to one skilled in the art and described by, for example, Cowell and Austin, "cDNA Library Protocols," Methods in Molecular Biology (1997).
- Codoning vehicle refers to a plasmid or phage DNA or other DNA sequence which is able to replicate in a host cell. This term can also include artificial chromosomes such as BACs and YACs.
- the cloning vehicle is characterized by one or more endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the DNA, which may contain a marker suitable for use in the identification of transformed cells.
- “Expression” refers to the process comprising transcription of a gene sequence and subsequent processing steps, such as translation of a resultant mRNA to produce the final end product of a gene.
- the end product may be a protein (such as an enzyme or receptor) or a nucleic acid (such as a tRNA, antisense RNA, or other regulatory factor).
- expression control sequence refers to a sequence of nucleotides that control or regulate expression of structural genes when operably linked to those genes. These include, for example, the lac systems, the trp system, major operator and promoter regions of the phage lambda, the control region of fd coat protein and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells.
- Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host, and may contain transcriptional elements such as enhancer elements, termination sequences, tissue- specificity elements and/or translational initiation and termination sites.
- “Expression vehicle” refers to a vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
- the cloned gene is usually placed under the control of (i.e., operably linked to) an expression control sequence.
- “Operator” refers to a DNA sequence capable of interacting with the specific repressor, thereby controlling the transcription of adjacent gene(s).
- Promoter refers to a DNA sequence that can be recognized by an RNA polymerase. The presence of such a sequence permits the RNA polymerase to bind and initiate transcription of operably linked gene sequences.
- Promoter region is intended to include the promoter as well as other gene sequences which may be necessary for the initiation of transcription. The presence of a promoter region is sufficient to cause the expression of an operably linked gene sequence.
- promoter is sometimes used in the art to generically indicate a promoter region. Many different promoters are known in the art which direct expression of a gene in a certain cell types. Tissue-specific promoters can comprise nucleic acid sequences which cause a greater (or decreased) level of expression in cells of a certain tissue type.
- “Operably linked” means that the promoter controls the initiation of expression of the gene.
- a promoter is operably linked to a sequence of proximal DNA if upon introduction into a host cell the promoter determines the transcription of the proximal DNA sequence(s) into one or more species of RNA.
- a promoter is operably linked to a DNA sequence if the promoter is capable of initiating transcription of that DNA sequence.
- "Prokaryote” refers to all organisms without a true nucleus, including bacteria.
- Eukaryote refers to organisms and cells that have a true nucleus, including mammalian cells.
- “Host” includes prokaryotes and eukaryotes, such as yeast and filamentous fungi, as well as plant and animal cells.
- the term includes an organism or cell that is the recipient of a replicable expression vehicle.
- animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
- Preferred animals include higher eukaryotes such as avians, rodents (e.g., mice, rabbits, rats, chinchillas, guinea pigs, hamsters and the like), and mammals.
- Preferred mammals include bovine, equine, feline, canine, ovine, caprine, porcine, buffalo, humans, and primates.
- a "transgenic animal” is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation or by inheritance from a manipulated progenitor at a subcellular level, such as by microinjection or infection with a recombinant viral vector (e.g., adenovirus, retrovirus, herpes virus, adeno-associated virus, lentivirus).
- a recombinant viral vector e.g., adenovirus, retrovirus, herpes virus, adeno-associated virus, lentivirus.
- This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA.
- Embryonic stem cells or “ES cells” as used herein are cells or cell lines usually derived from embryos which are pluripotent meaning that they are un- differentiated cells. These cells are also capable of incorporating exogenous DNA by homologous recombination and subsequently developing into any tissue in the body when incorporated into a host embryo. It is possible to isolate pluripotent cells from sources other than embryonic tissue by methods which are well understood in the art.
- mice Embryonic stem cells in mice have enabled researchers to select for transgenic cells and perform gene targeting. This allows more genetic engineering than is possible with other transgenic techniques.
- mouse ES cells are relatively easy to grow as colonies in vitro.
- the cells can be transfected by standard procedures and transgenic cells clonally selected by antibiotic resistance. See, for example, Doetschman et al.., 1994, Gene transfer in embryonic stem cells. In Pinkert (Ed.) Transgenic Animal Technology: A Laboratory Handbook. Academic Press Inc., New York, pp.115-146.
- the efficiency of this process is such that sufficient transgenic colonies (hundreds to thousands) can be produced to allow a second selection for homologous recombinants.
- Mouse ES cells can then be combined with a normal host embryo and, because they retain their potency, can develop into all the tissues in the resulting chimeric animal, including the germ cells. The transgenic modification can then be transmitted to subsequent generations.
- Methods for deriving embryonic stem (ES) cell lines in vitro from early preimplantation mouse embryos are well known. See for example, Evans et al,
- ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells or a differentiation inhibiting source is present.
- the term "somatic cell” indicates any animal or human cell which is not a sperm or egg cell or is capable of becoming a sperm or egg cell.
- the term “germ cell” or “germ-line cell” refers to any cell which is either a sperm or egg cell or is capable of developing into a sperm or egg cell and can therefore pass its genetic information to offspring.
- the term “germ cell-line transgenic animal” refers to a transgenic animal in which the genetic information was incorporated in a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.
- the genetic alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
- fragment of a gene refers to any portion of a gene sequence.
- a “biologically active fragment” refers to any portion of the gene that retains at least one biological activity of that gene. For example, the fragment can perhaps hybridize to its cognate sequence or is capable of being translated into a polypeptide fragment encoded by the gene from which it is derived.
- Variant refers to a gene that is substantially similar in structure and biological activity or immunological characteristics to either the entire gene or to a fragment of the gene. Provided that the two genes possess a similar activity, they are considered variant as that term is used herein even if the sequence of encoded amino acid residues is not identical.
- the variant is one of LRP5, HBM or LRP6.
- the variant preferably is one that yields an HBM-like phenotype (i.e., enhances bones mass and/or modulates lipid levels).
- variants include missense mutations, single nucleotide polymorphisms (SNPs), mutations which result in changes in the amino acid sequence of the protein encoded by the gene or nucleic acid, and combinations thereof, as well as com in the exon domains of the HBM gene and mutations in LRP5 or LRP6 which result in an HBM like phenotype.
- SNPs single nucleotide polymorphisms
- Amplification of nucleic acids refers to methods such as polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known in the art and described, for example, in U.S. Patent Nos. 4,683,195 and 4,683,202. Reagents and hardware for conducting PCR are commercially available. Primers useful for amplifying sequences from the HBM region are preferably complementary to, and hybridize specifically to sequences in the HBM region or in regions that flank a target region therein. HBM sequences generated by amplification may be sequenced directly. Alternatively, the amplified sequence(s) may be cloned prior to sequence analysis.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- Antibodies may refer to polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, that can bind to the HBM proteins and fragments thereof or to nucleic acid sequences from the HBM region, particularly from the HBM locus or a portion thereof.
- Preferred antibodies also include those capable of binding to LRP5, LRP6 and HBM variants.
- the term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
- Proteins may be prepared synthetically in a protein synthesizer and coupled to a carrier molecule and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the HBM protein or fragment.
- Monoclonal antibodies may be made by injecting mice with the proteins, or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with HBM protein or fragments thereof. Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988) and Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999). These antibodies will be useful in assays as well as pharmaceuticals.
- antibody is meant to include but not limited to polyclonal, monoclonal, chimeric, human, humanized, bispecific, multispecific, primatizedTM antibodies.
- HBM protein refers to a protein that is identical to a Zmaxl (LRP5) protein except that it contains an alteration of glycine 171 to a valine.
- An HBM protein is defined for any organism that encodes a Zmaxl (LRP5) true homolog.
- a mouse HBM protein refers to the mouse Zmaxl (LRP5) protein having the glycine 170 to valine substitution.
- HBM-like is meant a variant of LRP5, LRP6 or HBM which when expressed in a cell is capable of modulating bone mass, lipid levels, Dkk activity, and/or Wnt activity.
- HBM gene refers to the genomic DNA sequence found in individuals showing the HBM characteristic or phenotype, where the sequence encodes the protein indicated by SEQ ID NO: 4.
- the HBM gene and the Zmaxl (LRP5) gene are allelic.
- the protein encoded by the HBM gene has the property of causing elevated bone mass, while the protein encoded by the Zmaxl (LRP5) gene does not.
- the HBM gene and the Zmaxl (LRP5) gene differ in that the HBM gene has a thymine at position 582, while the Zmaxl gene has a guanine at position 582.
- the HBM gene comprises the nucleic acid sequence shown as SEQ ID NO: 2.
- the HBM gene may also be referred to as an "HBM polymorphism.”
- Other HBM genes may further have silent mutations, such as those discussed in Section 3 below.
- HBM gene may also refer to any allelic variant of Zmaxl (LRP5) or LRP6 which results in the HBM phenotype.
- variants may include alteration from the wild-type protein coding sequence as described herein and/or alteration in expression control sequences of Zmaxl (LRP5) or contains an amino acid mutation in LRP5 or LRP6, such that the resulting protein produces a phenotype which enhances bone mass and/or modulates lipid levels.
- a preferred example of such a variant is an alteration of the endogenous Zmaxl (LRP5) promoter region resulting in increased expression of the Zmaxl (LRP5) protein.
- LRP5 has also been referred to LRP7 in mouse.
- Zmaxl , LRP5 and Zmax may be used interchangeably throughout the specification and are meant to be the same gene, perhaps only relating to the gene in a different organism.
- the Zmaxl gene has a guanine at position 582 in the human sequence.
- the Zmaxl gene of human comprises the nucleic acid sequence shown as SEQ ID NO: 1.
- Normal wild-type
- HBM human serum
- Bone development generally refers to any process involved in the change of bone over time, including, for example, normal development, changes that occur during disease states, and changes that occur during aging. This may refer to structural changes and dynamic rate changes such as growth rates, resorption rates, bone repair rates, and etc.
- Bone development disorder particularly refers to any disorders in bone development including, for example, changes that occur during disease states and changes that occur during aging. Bone development may be progressive or cyclical in nature. Aspects of bone that may change during development include, for example, mineralization, formation of specific anatomical features, and relative or absolute numbers of various cell types.
- Bone modulation or “modulation of bone formation” refers to the ability to affect any of the physiological processes involved in bone remodeling, as will be appreciated by one skilled in the art, including, for example, bone resorption and appositional bone growth, by, inter alia, osteoclastic and osteoblastic activity, and may comprise some or all of bone formation and development as used herein.
- Bone is a dynamic tissue that is continually adapting and renewing itself through the renewal of old or unnecessary bone by osteoclasts and the rebuilding of new bone by osteoblasts. The nature of the coupling between these processes is responsible for both the modeling of bone during growth as well as the maintenance of adult skeletal integrity through remodeling and repair to meet the everyday needs of mechanical usage.
- Bone is measured by several different forms of X-ray absorptiometry. All of the instruments measure the inorganic or bone mineral content of the bone.
- Standard DXA measurements give a value that is an areal density, not a true density measurement by the classical definition of density (mass/unit volume). Nevertheless, this is the type of measurement used clinically to diagnose osteoporosis.
- BMD is a major contributing factor to bone strength, as much as 40% of bone strength stems from other factors including: 1) bone size (i.e., larger diameters increase organ-level stiffness, even in the face of lower density); 2) the connectivity of trabecular structures; 3) the level of remodeling (remodeling loci are local concentrators of strain); and 4) the intrinsic strength of the bony material itself, which in turn is a function of loading history (i.e., through accumulated fatigue damage) and the extent of collagen cross-linking and level of mineralization.
- osteoporotic fractures there is good evidence that all of these strength/fragility factors play some role in osteoporotic fractures, as do a host of extraskeletal influences as well (such as fall patterns, soft tissue padding, and central nervous system reflex responsiveness).
- Additional analytical instruments can be used to address these features of bone.
- the pQCT allows measurement of separate trabecular and cortical compartments for size and density and the ⁇ CT provides quantitative information on architectural features such as trabecular connectivity.
- the ⁇ CT also gives a true bone density measurement.
- the important non-BMD parameters can be measured for diagnosing the extent of disease and the efficacy of treatments. Current treatments for osteoporosis are based on the ability of drugs to prevent or retard bone resorption.
- bone modulation may be assessed by measuring parameters such as bone mineral density (BMD) and bone mineral content (BMC) by pDXA X-ray methods, bone size, thickness or volume as measured by X-ray, bone formation rates as measured for example by calcien labeling, total, trabecular, and mid-shaft density as measured by pQCT and/or ⁇ CT methods, connectivity and other histological parameters as measured by ⁇ CT methods, mechanical bending and compressive strengths as preferably measured in femur and vertebrae respectively.
- BMD bone mineral density
- BMC bone mineral content
- bone quality may be assessed by the compressive strength of vertebra when such a measurement is appropriate.
- Bone modulation may also be assessed by rates of change in the various parameters. Most preferably, bone modulation is assessed at more than one age.
- Normal bone density refers to a bone density within two standard deviations of a Z score of 0 in the context of the HBM linkage study. In a general context, the range of normal bone density parameters is determined by routine statistical methods. A normal parameter is within about 1 or 2 standard deviations of the age and sex normalized parameter, preferably about 2 standard deviations. A statistical measure of meaningfulness is the P value which can represent the likelihood that the associated measurement is significantly different from the mean. Significant P values are P ⁇ 0.05, 0.01 , 0.005, and 0.001 , preferably at least P ⁇ 0.01.
- HBM refers to "high bone mass” although this term may also be expressed in terms of bone density, mineral content, and size.
- the "HBM phenotype” and “HBM-like phenotype” may be characterized by an increase of about 2 or more standard deviations, preferably 2, 2.5, 3, or more standard deviations in 1 , 2, 3, 4, 5, or more quantitative parameters of bone modulation, preferably bone density and mineral content and bone strength parameters, above the age and sex norm for that parameter.
- the HBM phenotype and HBM-like phenotype are characterized by statistically significant increases in at least one parameter, preferably at least 2 parameters, and more preferably at least 3 or more parameters.
- the HBM phenotype and the HBM-like phenotype may also be characterized by an increase in one or more bone quality parameters and most preferably increasing parameters are not accompanied by a decrease in any bone quality parameters. Most preferably, an increase in bone modulation parameters and/or bone quality measurements is observed at more than one age.
- the HBM phenotype and HBM-like phenotype also includes changes of lipid levels, Wnt activity and/or Dkk activity.
- isolated and purified refer to a substance altered by hand of man from the natural environment.
- An isolated peptide may be for example in a substantially pure form or otherwise displaced from its native environment such as by expression in an isolated cell line or transgenic animal.
- An isolated sequence may for example be a molecule in substantially pure form or displaced from its native environment such that at least one end of said isolated sequence is not contiguous with the sequence it would be contiguous with in nature.
- Bioly active refers to those forms of proteins and polypeptides, including conservatively substituted variants, alleles of genes encoding a protein or polypeptide fragments of proteins which retain a biological and/or immunological activity of the wild-type protein or polypeptide.
- the activity is one which induces a change in Dkk activity, such as inhibiting the interaction of Dkk with a ligand binding partner ⁇ e.g., LRP5 or LRP6 or Dkk-1 with a Dkk-1 interacting protein such as those shown in Figure 5).
- biologically active is also meant to include any form which modulates Wnt signaling.
- module and “regulate” is meant methods, conditions, or agents which increase or decrease the wild-type activity of an enzyme, inhibitor, signal transducer, receptor, transcription activator, co-factor, and the like.
- This change in activity can be an increase or decrease of mRNA translation, mRNA or DNA transcription, and/or mRNA or protein degradation, which may in turn correspond to an increase or decrease in biological activity.
- modulated activity is meant any activity, condition, disease or phenotype which is modulated by a biologically active form of a protein. Modulation may be effected by affecting the concentration or subcellular localization of biologically active protein, i.e., by regulating expression or degradation, or by direct agonistic or antagonistic effect as, for example, through inhibition, activation, binding, or release of substrate, modification either chemically or structurally, or by direct or indirect interaction which may involve additional factors.
- effective amount or dose effective amount” or “therapeutically effective amount” is meant an amount of an agent which modulates a biological activity of the polypeptide of the invention.
- immunoglobulin protein or fragment thereof which recognizes and binds to an antigen.
- Dkk is meant to refer to the nucleic acids and proteins of members of the
- Dkk Dickkopf family. This includes, but is not limited to, Dkk-1 , Dkk-2, Dkk-3, Dkk- 4, Soggy, and related Dkk proteins.
- Dkk-1 is a preferred embodiment of the present invention.
- the Dkk proteins have substantial homology and one skilled in the art will appreciate that all of the embodiments of the present invention utilizing Dkk-1 may also be utilized with the other Dkk proteins.
- Dkk-1 is meant to refer to the Dkk-1 protein and nucleic acids which encode the Dkk-1 protein.
- Dkk-1 refers to Dickkopf-1 , and in Xenopus it is related to at least Dkk-2, Dkk-3, and Dkk-4 (see Krupnik et al, Gene 238:301-313 (1999)).
- Dkk-1 was first identified in Xenopus (Glinka et al, Nature 391 :357-62 (1998)). It was recognized as a factor capable of inducing ectopic head formation in the presence of inhibition of the BMP pathway. It was then also found to inhibit the axis- inducing activity of several Xenopus Wnt molecules by acting as an extracellular antagonist of Wnt signaling.
- Dkk-1 Mammalian homologs have been found including Dkk- 1 , Dkk-2, Dkk-3, Dkk-4 and soggy (Fedi et al, 1999 and Krupnick et al. 1999). Human Dkk-1 was also referred to as sk (Fedi et al. 1999). As used herein, Dkk-1 is meant to include proteins from any species having a Wnt pathway in which Dkk-1 interacts. Particularly preferred are mammalian species (e.g., murine, caprine, canine, bovine, feline, equine, primate, ovine, porcine and the like), with particularly preferred mammals being humans. Nucleic acid sequences encoding Dkk-1 include, but are not limited to human Dkk-1 (GenBank Accession Nos. AH009834,
- the genomic sequences with exon annotation are GenBank Accession Nos. AF261157 and AF261158.
- Dkk-1 amino acid sequences include, but are not limited to human dickkopf homolog 1 (GenBank Accession Nos.
- Dkk mediated disorder condition or disease is any abnormal state that involves Dkk activity.
- the abnormal state can be induced by environmental exposure or drug administration.
- the disease or disorder can be due to a genetic defect.
- Dkk mediated diseases, disorders and conditions include but are not limited to bone mass disorders or conditions and lipid disorders and conditions.
- bone mass disorders/conditions/diseases which may be mediated by Dkk, include but are not limited to age related loss of bone, bone fractures (e.g., hip fracture, Colle's fracture, vertebral crush fractures), chondrodystrophies, drug- induced disorders (e.g., osteoporosis due to administration of glucocorticoids or heparin and osteomalacia due to administration of aluminum hydroxide, anticonvulsants, or glutethimide), high bone turnover, hypercalcemia, hyperostosis, osteogenesis imperfecta, osteomalacia, osteomyelitis, osteoporosis, Paget's disease, osteoarthritis, and rickets.
- age related loss of bone e.g., bone fractures (e.g., hip fracture, Colle's fracture, vertebral crush fractures), chondrodystrophies, drug- induced disorders (e.g., osteoporosis due to administration of glucocorticoids or hepar
- Lipid disorders/diseases/conditions which may be mediated by Dkk, include but are not limited to familial lipoprotein lipase deficiency, familial apoprotein Cll deficiency, familial type 3 hyperlipoproteinemia, familial hypercholesterolemia, familial hypert glyceridemia, multiple lipoprotein-type hyperlipidemia, elevated lipid levels due to dialysis and/or diabetes, and elevated lipid levels of unknown etiologies
- the term "recognizes and binds,” when used to define interactions of antisense nucleotides, siRNAs (small inhibitory RNA), or shRNA (short hairpin RNA) with a target sequence means that a particular antisense, siRNA, or shRNA sequence is substantially complementary to the target sequence, and thus will specifically bind to a portion of an mRNA encoding polypeptide.
- sequences will be highly complementary to the mRNA target sequence, and will have no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence.
- highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
- Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or " % exact-match ' ) to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds.
- the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to the target mRNA to which the designed oligonucleotide specifically binds.
- Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
- the preferred default parameters for the GAP program include: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non- identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
- mimetic is meant a compound or molecule that performs the same function or behaves similarly to the compound mimicked.
- reporter element is meant a polynucleotide that encodes a poplypeptide capable of being detected in a screening assays.
- reporter elements include, but are not limited to, lacZ, GFP, luciferase, and chloramphenicol acetyltransferase.
- a polymorphism in LRP5 (Zmax), G171V, designated as HBM, has been identified as conferring a high bone mass phenotype in a population of related subjects as described in co-pending applications International Patent Application PCT/US 00/16951 , and U.S. Patent Application Nos. 09/543,771 and 09/544,398, which are hereby incorporated by reference in their entirety (Little et al, Am J Hum Genet. 70:11-19 (2002)).
- LRP5 is also described in International Patent Application WO 98/46743, which is incorporated by reference in its entirety. Loss of LRP5 function has been shown to have a deleterious effect on bone (Gong et al, Cell 107:513-523 (2001 )).
- the HBM polymorphism and LRP5 may also be important in cardiac health and lipid-mediated disorders. Thus, methods of regulating their activity can serve as methods of treating and/or preventing cardiac and lipid-mediated disorders.
- Wnt proteins are secreted proteins which interact with the transmembrane protein Frizzled (Fz).
- LRP proteins such as LRP5 and LRP6, are believed to modulate the Wnt signal in a complex with Fz (Tamai et al, Nature 407:530-5 (2000)).
- the Wnt pathway acts intracellularly through the Disheveled protein (Dsh) which in turn inhibits glycogen synthetase kinase-3 (GSK3) from phosphorylating ⁇ -catenin.
- Dsh Disheveled protein
- GSK3 glycogen synthetase kinase-3
- TCF T-cell factor
- the protein dickkopf-1 (Dkk-1 ) is reported to be an antagonist of Wnt pathway. Dkk-1 is required for head formation in early development. Dkk-1 and its function in the Wnt pathway are described in e.g., Krupnik, et al, Gene 238:301-13 (1999); Fedi et al, J. Biol. Chem. 274:19465-72 (1999); see also for Dkk-1 and the Wnt pathway, Wu et al, Curr. Biol. 10:1611-4 (2000), Shinya et al, Mech. Dev. 98:3- 17 (2000), Mukhopadhyay et al, Dev Cell 1 :423-434 (2001 ) and in PCT Patent Application No.
- Dkk-1 acts upstream of Dsh, however the nature of the mechanism of inhibition by Dkk-1 is just beginning to be elucidated. Dkk-1 is expressed in the mouse embryonic limb bud and its disruption results in abnormal limb morphogensis, among other developmental defects (Gotewold et al, Mech. Dev. 89:151-3 (1999); and, Mukhopadhyay et al, Dev Cell 1 :423-434 (2001 )).
- LRP6 can titrate Dkk-1's effects of inhibiting Wnt signaling using the commercial TCF-luciferase reporter gene assay (TOPFLASH).
- TOPFLASH commercial TCF-luciferase reporter gene assay
- the present invention provides an explanation for the mechanism of Dkk-1 inhibition of the Wnt pathway and provides a mechanism whereby the Wnt pathway may be modulated.
- the present application and related provisional application 60/291 ,311 describe Dkk- 1/LRP5 interactions and demonstrate that the interaction between LRP5/LRP6/HBM and Dkk can be used in a method as an intervention point in the Wnt pathway for an anabolic bone therapeutic or a modulator of lipid metabolism.
- Dkk-1 is able to repress LRP5-mediated Wnt signaling but not HBM-mediated Wnt signaling.
- HBM mutation in LRP5 is a gain of function or activation mutation. That is, Wnt signaling, via the canonical pathway, is enhanced with HBM versus LRP5.
- the present data suggest the mechanism of this functional activation: the inability of Dkk- 1 to repress HBM-mediated Wnt signaling.
- the present data reveal the importance and functional consequence for the potential structural perturbation of the first beta-propeller domain of LRP5.
- Our data identified the ligand binding domain of LRP5 as the interacting region with Dkk-1 while the Mao et al. publication demonstrated the functional role of propellers 3 and 4 in their LRP6/Dkk-1 studies.
- the involvement of position 171 of propeller 1 may be direct or indirect with Dkk-1. Direct involvement could arise from perturbations of the 3-dimensional structure of the HBM extracellular domain that render Dkk-1 unable to bind.
- residue 171 of propeller 1 may directly interact with Dkk-1 ; however, by itself, it is insufficient to bind and requires other LRP5 domains.
- Potential indirect candidate molecules may be among the proteins identified the Dkk-1 yeast-two- hybrid experiments.
- Dkk-1 binds LRP5, LRP6, and HBM. It is able to effectively inhibit LRP6, and to a slightly lesser extent, LRP5 activity. Further, has been observed that different members of the Dkk family differentially affect LRP5/LRP6/HBM activity. For example, Dkk-1 inhibits LRP5/LRP6/HBM activity while another Dkk may enhance LRP5/LRP6/HBM activity.
- An endpoint to consider is the modulation of the LRP5/LRP6/HBM activity, not simply binding.
- a therapeutic agent of the invention may be a small molecule, peptide or nucleic acid aptamer, antibody, or other peptide/protein, etc.
- Methods of reducing Dkk-1 expression may also be therapeutic using methodologies such as: RNA interference, antisense oligonucleotides, morpholino oligonucleotides, PNAs, antibodies to Dkk-1 or Dkk-1 interacting proteins, decoy or scavenger LRP5 or LRP6 receptors, and knockdown of Dkk-1 or Dkk-1 interactor transcription.
- the activity of Dkk-1 or the activity of a Dkk-1 interacting protein may be modulated for example by binding with a peptide aptamer of the present invention.
- LRP5 activity may be modulated by a reagent provided by the present invention (e.g., a peptide aptamer).
- the Dkk-1 /LRP5 interaction may be modulated by a reagent of the present invention (e.g., a Dkk-1 interacting protein such as those identified in Figure 5).
- the Wnt signal transduction pathway may be modulated by use of one or more of the above methods.
- the Dkk-1 mediated activity of the Wnt pathway may be specifically modulated by one or more of the above methods.
- the Wnt signal transduction pathway may be stimulated by down-regulating Dkk-1 interacting protein activity; such down-regulation could, for example, yield greater LRP5 activity.
- LRP5 activity bone mass regulation may be stimulated to restore or maintain a more optimal level.
- lipid metabolism may be stimulated to restore or maintain a more optimal level.
- Alternative embodiments provide methods for screening candidate drugs and therapies directed to correction of bone mass disorders or lipid metabolism disorders.
- preferred embodiments of the present invention provide drugs and therapies developed by the use of the reagents and/or methods of the present invention.
- One skilled in the art will understand that the present invention provides important research tools to develop an effective model of osteoporosis, to increase understanding of bone mass and lipid modulation, and to modulate bone mass and lipid metabolism.
- HBM-1 autosomal dominant trait
- Affected individuals have achieved a balance in bone turnover at a density that is significantly greater than necessary for normal skeletal stresses.
- the bones most affected are load-bearing bones which are subjected to the greatest mechanical and gravitational stresses (spine and hip). These are the most important bones to target fir therapeutic interventions in osteoporosis.
- the gene identified as being responsible for this phenotype, Zmax or LRP5 was not previously associated with bone physiology. The fact that modification of this gene, such as that produced by the polymorphism leading to the autosomal dominant inheritance of the HBM family phenotype, identifies Zmax/LRP5 and the pathway by which it is regulated, including Dkk Wnt pathways discussed above, as an important target for developing modulators of bone density.
- Polypeptides contemplated for use in this invention include those which modulate Dkk and Dkk interacting protein activities.
- Preferred polypeptides and peptides include those which modulate the Wnt pathway.
- Examples of preferred sequences include the Y2H baits exemplified in Figure 2, peptide aptamers of Figure 3 (SEQ ID NOs:171-188) and Figure 4 (SEQ ID NOs:189-192), the polypeptides of the Dkk-1 interacting proteins identified in Figure 5, those polypeptides shown in Figure 6, the LRP binding domain of Dkk (amino acids 138-266 of hDkkl ), the cysteine- ch domain 2 (a. a. 183-245 of hDkk-1 ), the cysteine-rich domain 1 (a. a. 97- 138 of hDkk), and LRP5 binding aptamers of Figure 13 (including SEQ ID NOs:204-
- Dkk-1 is exemplified, the other Dkk proteins contain substantially similar regions and may also be used according to the present invention.
- the baits depicted in Figure 2 were used in a yeast two hybrid (Y2H) screen.
- the Y2H screen was performed as described in Example 2 to determine the minimum required binding domain for Dkk-1 to bind LRP5.
- the minimum binding domain constructs i.e., residues 139-266 in bold below and residues 97-245 which are underlined, of Dkk-1 ) include the second cysteine rich domain which has sequence homology to a colipase fold.
- peptide aptamers A set of peptide aptamers was identified from a library of random peptides constrained and presented in a thioredoxin A (trxA) scaffold as described in Example 3.
- Peptide aptamers are powerful new tools for molecular medicine as reviewed by Hoppe-Seyler & Butz, J. Mol. Med., 78:426-430 (2000); Brody and Gold, Rev. Mol. Biotech., 74:5-13 (2000); and Colas, Curr. Opin. in Chem. Biol. 4:54-9 (2000) and the references cited therein. Briefly, peptide aptamers have been shown to be highly specific reagents capable of binding in vivo.
- peptide aptamers provide a method of modulating the function of a protein and may serve as a substitute for conventional knock-out methods, knock-down or complete loss of function.
- Peptide aptamers are also useful reagents for the validation of targets for drug development and may be used as therapeutic compounds directly or provide the necessary foundation for drug design. Once identified, the peptide insert may be synthesized and used directly or incorporated into another carrier molecule. References reviewed and cited by Brody and Gold (2000, supra) describe demonstrated therapeutic and diagnostic applications of peptide aptamers and would be known to the skilled artisan.
- the peptide aptamers of the present invention are useful reagents in the binding of Dkk-1 to its ligands and thereby modulation of the Wnt pathway and may be used to prevent Dkk-1 from inhibiting LRP5 modulation or Dkk-1 interacting protein modulation of the Wnt pathway.
- the sequence of these peptide aptamers is shown in Figure 3 (SEQ ID NOs: 171 -188).
- the peptide aptamers refers to the peptide constrained by the thioredoxin scaffold.
- the aptamers are also contemplated as therapeutic agents to treat Dkk-1 mediated diseases and conditions. Such aptamers are useful structural guides to chemists, for the design of mimetic compounds of the aptamers.
- Peptide aptamers were likewise developed to the LRP5 ligand binding domain (LBD) bait sequences.
- LBD LRP5 ligand binding domain
- the sequences of these peptide aptamers is shown in Figure 4 (SEQ ID NOs: 189-192).
- SEQ ID NOs: 189-192 These are useful reagents which may be used to disrupt the Dkk-1 /LRP5 binding interface while leaving Dkk-1 undisturbed. These can be used as comparative controls for Wnt signaling, thus, a control is provided for the specificity of any drug or therapy screened.
- the aptamers are also useful therapeutic agents to treat LRP mediated diseases and conditions. Such aptamers may also be used as structural guides to chemists, for the design of mimetic compounds of the aptamers.
- Dkk-1 interacting proteins Thirty proteins were identified which interact with Dkk-1 , Dkk-1 interacting proteins, were identified in a yeast-two-hybrid screen using the Dkk-1 bait and are shown in Figure 5. It was noted that these results suggest an interaction of Dkk-1 with Notch-2. It has been suggested that cross-talk exists between the Wnt and Notch signaling pathways. For instance, Presenilinl (Ps1 ) is required for Notch processing and inhibits the downstream Wnt pathway. The extracellular domain of Notch is thought to interact with Wnt. Furthermore, the Notch intracellular domain is thought to interact with disheveled and in signal induced processing, the intracellular domain is thought to interact with presenilin. (Soriano et al., J. Cell Biol.
- the BMP signaling pathway has an established role in bone and connective tissue development, repair and homeostasis (review in Rosen and Wozney "Bone Morphogenetic Proteins” In: Principles of Bone Biology, 2 nd Edition, Eds. J. Bilezikian, L. Raisz and G. Rodan, Academic Press, pp. 919-28 (2002)).
- Chordin is an important molecule during development which also modulates BMP signaling in adults by sequestering BMPs in latent complexes (Piccolo et al, Cell 86:589-98 (1996) reviewed in Reddi, Arthritis Res. 3:1-5 (2001 ); DeRobertis et al, Int. J. Dev. Biol 45:189-97 (2001 )). It may be that Dkk effects bone mass modulation through both the Wnt signaling pathway via LRP and the BMP pathway via chordin.
- Dkk-1 interacting proteins a number of putative growth factors, growth factor related proteins, and extracellular matrix proteins have been identified as Dkk-1 interacting proteins. Additional information regarding Dkk-1 interacting proteins identified in the Y2H assay may be obtained from publicly available databases such as PubMed via the use of the accession numbers provided in the present application.
- the amino acid sequences of these Dkk-1 interacting proteins or biologically active fragments thereof be used to modulate Dkk, Dkk-1 , LRP5, LRP6, HBM, or Wnt activity. Although these proteins were identified as interacting with Dkk-1 , due to the substantial homology between the various Dkk proteins, such interacting proteins are contemplated to interact with the other Dkk family members.
- the present invention further provides for mimetics of Dkk, particularly Dkk-1 , and LRP5 peptide aptamers.
- Such aptamers may serve as structural guides to chemists for the design of mimetic compounds of the aptamers.
- the aptamers and their mimetics are useful as therapeutic agents to treat LRP- or Dkk-mediated diseases and conditions.
- the present invention further provides nucleic acid molecules that encode polypeptides and proteins which interact with Dkk and Dkk interacting proteins, and/or LRP5 (also LRP6 and HBM) to modulate biological activities of these proteins.
- Preferred embodiments provide nucleic acids encoding for fragments of Dkk-1 protein, including the nucleic acids of Figure 7, the Dkk-1 interacting proteins listed in Figure 5, polypeptide aptamers of Dkk-1 ( Figure 3 - SEQ ID NOs:171-188), LRP5 ( Figure 4 - SEQ ID NOs:189-192), Figure 13 peptide aptamers (including SEQ ID NO:204-214) encoded by Figure 12 polynucleotides (including SEQ ID NO:193- 203), LRP6 and HBM and the related fusion proteins herein described, preferably in isolated or purified form.
- nucleic acid is defined as RNA, DNA, or cDNA that encodes a peptide as defined above, or is complementary to a nucleic acid sequence encoding such peptides, or hybridizes to either the sense or antisense strands of the nucleic acid and remains stably bound to it under appropriate stringency conditions.
- the nucleic acid may encode a polypeptide sharing at least about 75% sequence identity, preferably at least about 80%, and more preferably at least about 85%, with the peptide sequences; at least about 90%, 95%, 96%, 97%, 98%, and 99% or greater are also contemplated.
- genomic DNA e.g., genomic DNA
- cDNA e.g., cDNA
- mRNA e.g., ribozymes
- hybridizing or complementary nucleic acids are defined further as being novel and nonobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to a nucleic acid encoding a protein according to the present invention.
- hybridization hybridizing
- specificity specific for in the context of nucleotide sequences
- the ability of two nucleotide sequences to hybridize to each other is based upon the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the greater the degree of hybridization of one to the other.
- the degree of hybridization also depends on the conditions of stringency which include temperature, solvent ratios, salt concentrations, and the like.
- selective hybridization pertains to conditions in which the degree of hybridization of a polynucleotide of the invention to its target would require complete or nearly complete complementarity.
- the complementarity must be sufficiently high so as to assure that the polynucleotide of the invention will bind specifically to the target nucleotide sequence relative to the binding of other nucleic acids present in the hybridization medium.
- complementarity will be about 90-100%, preferably about 95-100%, more preferably about 100%.
- “Stringent conditions” are those that (1 ) employ low ionic strength and high temperature for washing, for example: 0.015 M NaCI, 0.0015 M sodium titrate, 0.1% SDS at 50°C; or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42°C.
- formamide for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42°C.
- Another example is use of 50% formamide, 5X SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2X SSC and 0.1 % SDS.
- 5X SSC 0.75 M NaCI, 0.075 M sodium citrate
- 50 mM sodium phosphate pH 6.8
- 0.1 % sodium pyrophosphate 5X Denhardt's solution
- sonicated salmon sperm DNA 50 ⁇ g/ml
- 0.1 % SDS 10% dextran sulfate at 42°C
- nucleic acid molecule is said to be "isolated” or “purified” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
- Isolated or purified is also meant to include nucleic acids which encode Dkk or fragments thereof which lack surrounding genomic sequences that flank the Dkk gene.
- Isolated or purified is further intended to include nucleic acids which encode Dkk interacting proteins or biologically active fragments thereof which lack surrounding genomic sequences that flank the Dkk interacting protein genes.
- the present invention further provides fragments of the encoding nucleic acid molecule.
- a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence.
- the size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.
- Fragments of the encoding nucleic acid molecules of the present invention i.e., synthetic oligonucleotides
- PCR polymerase chain reaction
- Fragments of the encoding nucleic acid molecules of the present invention can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191 (1981 )) or using automated synthesis methods.
- larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.
- polypeptide encoding nucleic acid molecules of the present invention may further be modified to contain a detectable label for diagnostic and probe purposes.
- a detectable label for diagnostic and probe purposes.
- a variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule.
- Antisense molecules corresponding to the polypeptide coding or complementary sequence may be prepared. Methods of making antisense molecules which bind to mRNA, form triple helices or are enzymatically active and cleave TSG RNA and single stranded DNA (ssDNA) are known in the art. See, e.g., Antisense and Ribozyme Methodology aboratory Companion (Ian Gibson, ed., Chapman & Hall, 1997) and Ribozvme Protocols: Methods in Molecular Biology (Phillip C. Turner, ed., Humana Press, Clifton, NJ, 1997).
- RNA interference also contemplated is the use of compounds which mediate postranscriptional gene silencing (PTGS), quelling and RNA interference (RNAi). These compounds typically are about 21 to about 25 nucleotides and are also known as short interfering RNAs or short inhibitory RNAs (siRNAs).
- the siRNAs are produced from an initiating double stranded RNA (dsRNA). Although the full mechanism by which the siRNAs function is not fully elucidated, it is known that these siRNAs transform the target mRNA into dsRNA, which is then degraded.
- Preferred forms are 5' phosphorylated siRNAs, however, hydroxylated forms may also be utilized.
- siRNAs for additional background regarding the preparation and mechanism of siRNAs generally, see, e.g., Lipardi et al, Cell 107(3): 297-307 (2001 ); Boutla et al, Curr. Biol. 11(22): 1776-80 (2001 ); Djikeng et al, RNA 7(11 ): 1522-30 (2001 ); Elbashir et al., EMBO J. 20(23): 6877-88 (2001 ); Harborth et al, J. Cell. Sci. 114(Pt.
- shRNAs short hairpin RNAs
- shRNAs are a modification of the siRNA method described above. Instead of transfecting exogenously synthesized dsRNA into a cell, sequence-specific silencing can be achieved by stabling expressing siRNA from a DNA template as a fold-back stem- loop, or hairpin. This approach is known as shRNA.
- This method permits the analysis of loss of function phenotypes due to sequence-specific gene silencing in mammalian cells by avoiding many of the problems associated with siRNAs, such as RNase degradation of the reagents, expensive chemical synthesis, etc.
- siRNAs such as RNase degradation of the reagents, expensive chemical synthesis, etc.
- shRNAs For additional background regarding the preparation and mechanism of shRNAs generally, see, e.g., Yu et al, PNAS 99:6047-6052 (2002); Paddison et al, Genes and Devel. 16:948-58 (2002); and Brummelkamp et al, Science 296:550-553 (2002).
- Tuschl Nature Biotech. 20:446-448 (2002) (and references therein).
- the siRNA or shRNA is directed to a Dkk encoding mRNA, wherein a preferred Dkk is Dkk-1.
- the siRNA or shRNA is directed towards a protein which binds to and modulates the activity of or is modulated by a Dkk; these proteins include LRP5, LRP6 and HBM as well as other members of the Wnt pathway.
- nucleic acid molecule of Dkk allows a skilled artisan to isolate nucleic acid molecules that encode other members of the Dkk family (see, Krupnik et al, 1999). Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode Dkk-1 -like proteins, in addition to Dkk-1. The presently disclosed Dkk-1 interacting proteins and their corresponding nucleic acid molecules allows a skilled artisan to further isolate other related protein family members which interact with Dkk-1.
- a skilled artisan can readily use the amino acid sequence of Dkk and Dkk interacting proteins to generate antibody probes to screen expression libraries prepared from appropriate cells.
- polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as a human macrophage library, to obtain the appropriate coding sequence for other members of the protein family.
- the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the desired protein.
- a portion of the coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism.
- Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
- pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule.
- a PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.
- degenerate primers can be utilized to obtain sequences related to Dkk-1 or Dkk-1 interacting proteins.
- Primers can be designed that are not perfectly complementary and can still hybridize to a portion of a target sequence or flanking sequence and thereby provide for amplification of all or a portion of a target sequence.
- Primers of about 20 to 30 nucleotides have up to about 30% mismatches and can still hybridize to a target sequence.
- Hybridization conditions for primers with mismatch can be determined by the method described in Maniatis et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982) or by reference to known methods. The ability of the primer to hybridize to a sequence of either Dkk-1 , a Dkk-1 interacting protein, or a related sequence under varying conditions can be determined using this method. Because a target sequence is known, the effect of mismatches can be determined by methods known to those of skill in the art. Degenerate primers would be based on putative conserved amino acid sequences of the Dkk-1 and Dkk-1 interacting protein genes.
- the present invention further provides recombinant DNA molecules (rDNAs) that contain a polypeptide coding sequence.
- a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Clonin ⁇ : A Laboratory Manual (Cold Spring Harbor Laboratory, Cold
- a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
- vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
- a vector contemplated by the present invention is at least capable of directing the replication and/or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.
- Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
- the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
- Preferred promoters include yeast promoters, which include promoter regions for metallothionein, 3- phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.
- yeast promoters include promoter regions for metallothionein, 3- phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.
- Vectors and promoters suitable for use in yeast expression are further described in EP 73.675A.
- Non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al, Nature, 273:113 (1978)) or promoters derived from Moloney murine leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma.
- the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
- DHFR e.g., DHFR
- Preferred bone related promoters include CMVbActin or type I collagen promoters to drive expression of the human HBM, Zmaxl /LRP5 or LRP6 cDNA.
- Other preferred promoters for mammalian expression are from cytomegalovirus (CMV), Rous sarcoma virus (RSV), Simian virus 40 (SV40), and EF-1a (human elongation factor 1a-subunit).
- the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
- a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
- a prokaryotic host cell such as a bacterial host cell, transformed therewith.
- vectors with a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
- Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
- Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli.
- a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
- Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), and pPL and pKK223 available from Pharmacia (Piscataway, NJ).
- Expression vectors compatible with eukaryotic cells can also be used to form a rDNA molecule that contains a coding sequence.
- Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of a desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), vector systems that include Histidine Tags and periplasmic secretion, or other vectors described in the art.
- Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
- a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene (Southern et al, J. Mol. Anal. Genet. 1 :327-341 (1982)).
- the selectable marker can be present on a separate plasmid, and the two vectors introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
- the present invention further provides host cells transformed with a nucleic acid molecule that encodes a polypeptide or protein of the present invention.
- the host cell can be either prokaryotic or eukaryotic.
- Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
- Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line but also can include invertebrates with, for example, cartilage.
- Preferred eukaryotic host cells include but are not limited to Chinese hamster ovary (CHO) cells (ATCC No. CCL61 ), NIH Swiss mouse embryo cells NIH/3T3 (ATCC No. CRL 1658), baby hamster kidney cells (BHK), HOB-03-CE6 osteoblast cells, and other like eukaryotic tissue culture cell lines.
- Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention.
- a preferred prokaryotic host is E. coli.
- Transformation of appropriate cell hosts with a recombinant DNA (rDNA) molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed.
- electroporation and salt treatment methods are typically employed; see, for example, Cohen et al, Proc. Natl. Acad. Sci. USA 69: 2110 (1972); Maniatis et al. (1982); and Sambrook et al. (1989).
- electroporation, cationic lipid or salt treatment methods are typically employed; see, for example, Graham et al. , Virol. 52: 456 (1973); Wigler et al. , Proc. Nat Acad. Sci. USA 76: 1373- 76 (1979).
- Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
- cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol. 98: 503 (1975), or Berent et al, Biotech. 3: 208 (1985).
- the cells can be cultured to produce the proteins encoded by the rDNA and the proteins harvested and assayed, using for example, any suitable immunological method.
- Recombinant DNA can also be utilized to analyze the function of coding and non-coding sequences. Sequences that modulate the translation of the mRNA can be utilized in an affinity matrix system to purify proteins obtained from cell lysates that associate with the Dkk-1 or Dkk-1 interacting protein or expression control sequence. Synthetic oligonucleotides would be coupled to the beads and probed with the lysates, as is commonly known in the art. Associated proteins could then be separated using, for example, a two dimensional SDS-PAGE system. Proteins thus isolated could be further identified using mass spectroscopy or protein sequencing. Additional methods would be apparent to the skilled artisan.
- the invention also relates to nucleic acid molecules which encode a Dkk protein and polypeptide fragments thereof, and proteins and polypeptides which bind to Dkk (e.g., LRP5, LRP6 and HBM, Dkk interacting proteins such as the proteins of Figure 5) and molecular analogues.
- Dkk e.g., LRP5, LRP6 and HBM, Dkk interacting proteins such as the proteins of Figure 5
- the polypeptides of the present invention include the full length Dkk and polypeptide fragments thereof, Dkk binding proteins and polypeptides thereof.
- these proteins are mammalian proteins, and most preferably human proteins and biologically active fragments thereof.
- Alternative embodiments include nucleic acid molecules encoding polypeptide fragments having a consecutive amino acid sequence of at least about 3, 5, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 amino acid residues from a common polypeptide sequence; amino acid sequence variants of a common polypeptide sequence wherein an amino acid residue has been inserted N- or C-terminal to, or within, the polypeptide sequence or its fragments; and amino acid sequence variants of the common polypeptide sequence or its fragments, which have been substituted by another conserved residue.
- Recombinant nucleic acid molecules which encode polypeptides include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and recombinant Dkk proteins or polypeptide fragments of other animal species, including but not limited to vertebrates (e.g., rabbit, rat, murine, porcine, camelid, reptilian, caprine, avian, fish, bovine, ovine, equine and non-human primate species) as well as invertebrates, and alleles or other naturally occurring variants and homologs of Dkk binding proteins of the foregoing species and of human sequences.
- vertebrates e.g., rabbit, rat, murine, porcine, camelid, reptilian, caprine, avian, fish, bovine, ovine, equine and non-human primate species
- Dkk, Dkk interacting proteins, or fragments thereof are derivatives of the commonly known Dkk, Dkk interacting proteins, or fragments thereof, wherein Dkk, Dkk interacting proteins, or their fragments have been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope) and soluble forms of Dkk.
- a detectable moiety such as an enzyme or radioisotope
- the present invention also includes nucleic acids with silent mutations which will hybridize to the endogenous sequence and which will still encode the same polypeptide.
- the nucleic acid molecules encoding Dkk binding proteins, the LRP5 binding domain fragment of Dkk, or other polypeptides of the present invention are preferably those which share a common biological activity (e.g., mediate Dkk activity such as its interaction with LRP5, HBM or LRP6).
- the polypeptides of the present invention include those encoded by a nucleic acid molecule with silent mutations, as well as those nucleic acids encoding a biologically active protein with conservative amino acid substitutions, allelic variants, and other variants of the disclosed polypeptides which maintain at least one Dkk activity.
- the amino acid compounds of the invention are polypeptides which are partially defined in terms of amino acid residues of designated classes.
- Polypeptide homologs would include conservative amino acid substitutions within the amino acid classes described below.
- Amino acid residues can be generally sub-classified into four major subclasses as follows: Acidic: The residue has a negative charge due to loss of H + ion at physiological pH, and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium, at physiological pH.
- Basic The residue has a positive charge due to association with H + ion at physiological pH, and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
- Neutral/non-polar The residues are not charged at physiological pH, but the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. These residues are also designated "hydrophobic.”
- Neutral/polar The residues are not charged at physiological pH, but the residue is attracted by aqueous solution so as to seek the outer positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
- Amino acid residues can be further subclassified as cyclic or noncyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side chain substituent groups of the residues, and as small or large.
- the residue is considered small if it contains a total of 4 carbon atoms or less, inclusive of the carboxyl carbon. Small residues are, of course, always nonaromatic.
- the gene-encoded secondary amino acid proline although technically within the group neutral/nonpolar/large/cyclic and nonaromatic, is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in this defined group.
- Other amino acid substitutions of those encoded in the gene can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme according to their structure.
- All of the compounds of the invention may be in the form of the pharmaceutically acceptable salts or esters.
- Salts may be, for example, Na + , K + , Ca +2 , Mg +2 and the like; the esters are generally those of alcohols of 1 -6 carbons.
- the present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described.
- the production of a recombinant form of a protein typically involves the following steps.
- a nucleic acid molecule is obtained that encodes Dkk, such as a nucleic acid molecule encoding human Dkk or any other Dkk sequence, or that encodes a Dkk binding protein, a Dkk aptamer or a biologically active fragment thereof.
- Dkk binding peptides the nucleotides encoding the peptide are incorporated into a nucleic acid in the form of an in-frame fusion, insertion into or appended to a thioredoxin coding sequence.
- the coding sequence (ORF) is directly suitable for expression in any host, as it is not interrupted by introns.
- Eukaryotic hosts include mammalian cells and vertebrate (e.g., osteoblasts, osteosarcoma cell lines, Drosophila S2 cells, hepatocytes, tumor cell lines and other bone cells of any mammal, as well as insect cells, such as Sf9 cells using recombinant baculovirus).
- a DNA expressing an open reading frame (ORF) under control of a type I collagen promoter, or such osteoblast promoters as osteocalcin histone, type I collagen, TGF ⁇ l , MSX2, cfos/cJun and Cbfal can be used to regulate the Dkk in animal cells.
- ORF open reading frame
- the nucleic acid can be placed downstream from an inducible promoter, which can then be placed into vertebrate or invertebrate cells or be used in creating a transgenic animal model.
- proteins and polypeptides of the present invention can be expressed in an heterologous system.
- the human cell line GM637, SV-40 transformed human fibroblasts can be transfected, with a plasmid containing a Dkk ligand binding domain coding sequence under the control of the chicken actin promoter (Reis et al, EMBO J. 11 : 185-193 (1992)). Such transfected cells could be used as a source of Dkk binding domain in functional assays.
- polypeptides encoding only a portion of Dkk or any of the disclosed Dkk binding peptides Dkk aptamers or a polypeptide encoding a Dkk interacting protein can be expressed alone or in the form of a fusion protein.
- Dkk derived peptides can be expressed in bacteria (e.g., E. coli) as GST- or His-Tag fusion proteins. These fusion proteins are then purified and can be used to generate polyclonal antibodies or can be used to identify other Dkk ligands.
- the nucleic acid coding sequence is preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein encoding open reading frame.
- the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
- the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
- the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
- expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
- the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
- Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
- a skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein. 10. Methods to Identify Binding Partners
- Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of Dkk or Dkk interacting proteins.
- Dkk or a Dkk interacting protein or a polypeptide fragment thereof can be mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with Dkk or with Dkk interacting proteins.
- the peptides, polypeptides, proteins or other molecules that have become associated with Dkk or a Dkk interacting protein are separated from the mixture.
- the binding partner that bound to the polypeptide then can be purified and further analyzed.
- Determination of binding partners of Dkk and Dkk interacting proteins as well as agents which prevent the interaction of Dkk with one of its interacting proteins can be performed using a variety of different competition assays as are known in the art.
- the minimal sequence of Dkk, as described herein can be used to identify antibodies which compete with LRP5 (or LRP6, HBM or other ligand binding partners) for binding to Dkk- 1 and vice versa.
- the minimal Dkk sequence can be bound to the bottom of a 96-well plate (or other solid substrate), and antibodies or other potential binding agents (e.g., polypeptides, mimetics, homologs, antibody fragments and the like) can be screened in a competition assay to identify agents with binding affinities, for example, greater than the natural ligand binding partner of Dkk.
- antibodies or other potential binding agents e.g., polypeptides, mimetics, homologs, antibody fragments and the like
- suitable cells are used for preparing assays, for the expression of a LRP and/or Dkk or proteins that interact therewith.
- the cells may be made or derived from mammals, yeast, fungi, or viruses.
- a suitable cell for the purposes of this invention is one that includes but is not limited to a cell that can exhibit a detectable Dkk-LRP (or HBM) interaction, and preferably, the differential interaction between Dkk-1 -LRP5 and Dkk-1 -HBM.
- the cell type may vary.
- bone cells are preferred, for example, a human osteoblast cell (e.g. hOB-03-CE6) or osteosarcoma cell (e.g. U20S).
- hOB- 03-C5 hOB-02-02
- hOB-01- C1-PS-09 cells which are deposited with American Type Culture Collection in Manassas, Va. with the designation PTA-785
- Examples of osteosarcoma cells would include SaoS2, MG63 and HOS TE85 Immortalized refers to a substantially continuous and permanently established cell culture with substantially unlimited cell division potential. That is, the cells can be cultured substantially indefinitely, i.e., for at least about 6 months under rapid conditions of growth, preferably much longer under slower growth conditions, and can be propagated rapidly and continually using routine cell culture techniques.
- preferred cells can be cultured for at least about 100, 150 or 200 population doublings. These cells produce a complement of proteins characteristic of normal human osteoblastic cells and are capable of osteoblastic differentiation. They can be used in cell culture studies of osteoblastic cell sensitivity to various agents, such as hormones, cytokines, and growth factors, or in tissue therapy. Certain non bone cells such as HEK 293 cells that exhibit detectable Dkk-LRP (or HBM) interaction are also be useful for the assays of this invention. To identify and isolate a binding partner, the entire Dkk protein (e.g., human Dkk-1 , GenBank Accession No. BAA34651 ) or a Dkk interacting protein (Genbank
- Dkk-1 interacting proteins are given in Figure 5.
- a polypeptide fragment of the protein can be used. Suitable fragments of the protein include at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150 or more contiguous amino acid residues of any Dkk or Dkk interactor sequence.
- Preferable sequences of Dkk include portions or all of one or both of the cysteine rich domains (e.g., Cys-1 and Cys-2 of Dkk-1 ) or the conserved sequences at the amino terminus of Dkk-1 (See Krupnik et al, Gene 238: 301-313 (1999)).
- portions of LRP5, LRP6, HBM and other Dkk interacting proteins such as those in Figure 5 that interact with Dkk-1 can be used to identify and isolate agents which modulate Dkk activity.
- peptide aptamers of LRP5, LRP6, HBM, Dkk and other Dkk interacting proteins such as those in Figure 5 that interact with Dkk-1 can be used to identify and isolate agents which modulate Dkk activity.
- a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.
- a variety of methods can be used to obtain cell extracts.
- Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
- the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur.
- conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
- Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
- the bound complex is separated from the mixture.
- a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
- a protein of the invention is expressed with an affinity tag such as a His tag. The His labeled protein and any bound molecule may be retained and selectively eluted from a Ni-NTA column.
- the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
- the protein of the invention can be immobilized on a solid support.
- the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract.
- the identified binding partners can be either a single protein or a complex made up of two or more proteins.
- the nucleic acid molecules of the invention can be used in a Y2H system. The Y2H system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described. Methods of performing and using Y2H systems are known.
- Dkk-1 interacting proteins identified in Figure 5 were identified using the Y2H interacting system using Dkk-1 as bait.
- One preferred in vitro binding assay for Dkk modulators would comprise a mixture of a LRP binding domain of Dkk and one or more candidate binding targets or substrates.
- one or more of the components usually comprises or is coupled to a label.
- the label may provide for direct detection, such as radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc.
- direct detection such as radioactivity, luminescence, optical or electron density, etc.
- indirect detection such as an epitope tag, an enzyme, etc.
- a variety of methods may be employed to detect the label depending on the nature of the label and other assay components.
- the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and the label thereafter detected. Fluorescence resonance energy transfer may be utilized to monitor the interaction of two labeled molecules.
- a fluorescence label on Dkk and another label on LRP5 or a soluble fragment thereof such as the extracellular domain will exchange fluorescence resonance energy when in close proximity indicating that the two molecules are bound.
- a preferred binding partner for Dkk will increase or decrease the affinity between Dkk and LRP5 which will be readily observable in a fluorescence spectrometer.
- an instrument such as a surface plasmon resonance detector manufactured by BIAcore (Uppsala, Sweden), may be used to observe interactions with a fixed target.
- BIAcore Uppsala, Sweden
- the present invention provides methods for screening candidates including polypeptides of the present invention for activity which identifies these candidates as valuable drug leads.
- Other suitable methods are also known in the art and are suitable for use herein, including Xenopus oocyte injection studies and TCF luciferase assays.
- Additional assays can be used to identify the activity of Dkk and Dkk interacting proteins in the Wnt pathway, as well as the impact of modulators of Dkk and Dkk interacting proteins on the Wnt pathway. These include, for example, a Xenopus embryo assay and a TCF-luciferase reporter gene assay to monitor Wnt signaling modulation.
- Xenopus embryos are an informative in vivo assay system to evaluate the modulation of Wnt signaling. Ectopic expression of certain Wnts or other activators of the Wnt signaling pathway results in a bifurcation of the anterior neural plate. This bifurcation results in a duplicated body axis, which suggests a role for Wnt signaling during embryonic development (McMahon et al., Cell 58: 1075-84 (1989); Sokol et al., Cell 67: 741-52 (1991 )). Since these original observations, the Xenopus embryo assay has been extensively used as an assay system for evaluating modulation of the Wnt signaling pathway. One preferred embodiment of the present invention is demonstrated in Example 6.
- Constructs for Xenopus expression can be prepared as would be known in the art.
- a variety of cDNAs have been engineered into the vector pCS2+ (Turner et al, Genes Devel. 8: 1434-1447 (1994)) to facilitate the in vitro generation of mRNA for use in Xenopus embryo injection experiments.
- DNA inserts are subcloned in the sense orientation with respect to the vector SP6 promoter. Downstream of the insert, the vector provides an SV40 virus polyadenlylation signal and a T3 promoter sequence (i.e., for the generation of antisense mRNA).
- Constructs can be generated for various Dkk family members, LRP5, LRP6, HBM, Dkk-1 interactors, etc.
- Constructs could be generated in pCS2 + that contain the nucleic acid sequence encoding for the peptide aptamers that were identified in yeast screens. These sequences would be fused to a 5' synthetic translation initiation sequence followed by a canonical signal sequence to ensure that the peptide aptamer would be translated and secreted from the cell.
- RNA can be synthesized and injected into Xenopus oocytes.
- mRNA for microinjection into Xenopus embryos is generated by in vitro transcription using the cDNA constructs in the pCS2 + vector described above as template.
- Various amounts of RNA can be injected into the ventral blastomere of the 4- or 8-cell Xenopus embryo substantially as described in Moon et al., Technique-J. of Methods in Cell and Mol. Biol. 1 : 76-89 (1989), and Peng, Meth. Cell. Biol. 36: 657-62 (1991 ).
- Lineage tracing experiments can be performed where a marker gene such as green fluorescent protein (GFP) or LacZ is co-injected with the experimental RNAs. Detecting marker gene expression would identify the targeted cells of the microinjection and aid in elucidating the mechanism of action.
- GFP green fluorescent protein
- the point at which HBM acts upon the Wnt pathway can also be analyzed. This can be done by co-injections of various dominant-negative constructs. For example, a dominant negative TCF-3 construct would be useful to demonstrate that the observed axis duplication (and Wnt activation) is mediated via the ⁇ -catenin-TCF response. If so, such a construct would be expected to abolish the observed duplicated axis phenotype.
- the marker genes of the injected Xenopus embryos can be analyzed as follows. Representative embryos are collected at stage 10.5 (11 hours post fertilization) for marker gene analysis. RNA is extracted and purified from the embryos following standard protocols (Sambrook et al, 1989 at 7.16).
- Marker genes could include the following: Siamois (i.e., Wnt responsive gene), Xnr3 (i.e., Wnt responsive gene), slug (i.e., neural crest marker), Xbra (i.e., early mesoderm marker), HNK-1 (i.e., ectodermal/neural marker), endodermin (i.e., endoderm), Xlhbox ⁇ (i.e., pancreatic), BMP2 and BMP4 (i.e., early mesoderm), XLRP6 (i.e., maternal and zygotic expression, it is also the LRP6 homolog in the frog), EF-1 (i.e., control) and ODC (i.e., control). Induction of marker genes is analyzed and quantitated by RT-PCR/TaqMan®.
- This type of marker analysis is excellent to monitor changes in gene expression that result very early in the embryo as a direct result of signaling perturbation.
- Other experiments could be designed that would monitor changes in gene expression in a more tissue or spatially-restricted fashion. Examples would include the generation of a transgenic Xenopus model. For example, Zmax/LRP5 and HBM expression could be under the control of the brachyury or cardiac-actin promoters directing gene expression transiently in the mesoderm or in the somites, respectively.
- Phenotype analyses of these transgenic Xenopus animals would include marker gene analysis/transcriptional profiling (from a restricted tissue source) and histologic examination of the tissue.
- a TCF-luciferase assay system such as that described in Example 7 can also be used to monitor Wnt signaling activity, Dkk activity and Dkk interacting protein activity.
- Constructs for the TCF-luciferase assays can be prepared as would be known in the art. For example, Dkk and Dkk interacting protein peptides, LRP5/LRP6, among others, can be expressed in pcDNA3.1 , using Kozak and signal sequences to target peptides for secretion.
- constructs Once constructs have been prepared, cells such as osteoblasts and HEK293 cells are seeded in well plates and transfected with construct DNA, CMV beta- galactosidase plasmid DNA, and TCF-luciferase reporter DNA. The cells are then lysed and assayed for beta-galactosidase and luciferase activity to determine whether Dkk, Dkk interacting proteins, or other molecules such as antibodies affect Wnt signaling.
- Additional assays for monitoring Wnt signaling activity, Dkk activity, and Dkk interacting protein activity include:
- Modulation of another Wnt-responsive transcription factor, LEF as visualized by a reporter gene activity.
- LEF Wnt-responsive transcription factor
- One example includes the activation of the LEF1 promoter region fused to the luciferase reporter gene (Hsu et al, Mol. Cell. Biol. 18: 4807-18 (1999)).
- Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding Dkk. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of Dkk, if it is capable of up- or down-regulating expression of the nucleic acid in a cell (e.g., mRNA).
- a cell e.g., mRNA
- cell lines that contain reporter gene fusions between the nucleic acid encoding Dkk (or proteins which modulate the activity of Dkk) and any assayable fusion partner may be prepared.
- Numerous assayable fusion partners are known and readily available, including but not limited to the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al, Anal. Biochem. 188: 245-254 (1990)).
- Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time.
- Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding Dkk or other protein which modulates Dkk activity.
- Such assays can similarly be used to determine whether LRP5 and even LRP6 activity is modulated by regulating Dkk activity.
- Additional assay formats may be used to monitor the ability of the agent(s) to modulate the expression of a nucleic acid encoding Dkk, alone or Dkk and LRP5, and/or Dkk interacting proteins such as those identified in Figure 5. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention.
- RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (1989); Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Co., NY, 1995); Maniatis et al, Molecular Clonin ⁇ : A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982); and Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (Frederick M. Ausubel ef al, April 1999).
- Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and potential probe:non-target hybrids. Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available. See for example, Sambrook et al. (1989) or Ausubel er al. (Current Protocols in Molecular Biology. Greene Publishing Co., NY, 1995).
- Hybridization conditions are modified using known methods, such as those described by Sambrook et al. (1989) and Ausubel et al. (1995), as suitable for each probe.
- Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format.
- total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the nucleic acid sequences of the invention under conditions in which the probe will specifically hybridize.
- nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a porous glass wafer.
- the glass or silica wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
- Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755).
- Beattie WO 95/11755
- agents which up- or down- regulate the expression of a nucleic acid encoding Dkk, a Dkk interacting protein, and/or LRP5 can be identified.
- Microarray technology and transcriptional profiling are examples of methods which can be used to analyze the impact of putative Dkk or Dkk interacting protein modulating compounds.
- a potential Dkk modulating agent such as the Dkk interacting proteins identified in the present invention (e.g., those identified in Figure 5)
- agents which modulate Dkk interacting proteins, and mRNA from the same type of cells that were not exposed to the agent could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with the agent. If, for example a putative Dkk modulating agent down-regulates the expression of Dkk in the cells, then use of the agent may be undesirable in certain patient populations.
- Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of Dkk, Dkk interacting proteins, and/or LRP5/LRP6/HBM proteins or preferably which specifically modulate an activity of a Dkk Dkk interacting protein complex or an LRP5(or LRP6/HBM)/Dkk complex, or a biologically active fragment of Dkk (e.g., comprising the domain which binds LRP5/LRP6/HBM) or a Dkk interacting protein complex.
- Such methods or assays may utilize any means of monitoring or detecting the desired activity as would be known in the art (See, e.g., Wu et al, Curr. Biol. 10:1611-4 (2000); Fedi etal, J. Biol. Chem. 274:19465-72 (1991); Grotewold et al, Mech. Dev. 89:151-3 (1999); Shibata et al, Mech. Dev. 96:243-6 (2000); Wang et al., Oncogene 19:1843-8 (2000); and Glinka et al, Nature 391 :357-62 (1998)).
- Potential agents which modulate Dkk include, for example, p53, the tumor suppressor protein, which can induce Dkk-1.
- Dkk-1 Damage to DNA has also been observed to up-regulate Dkk-1 expression via a stabilization and activation of p53 (Wang er al, Oncogene 19:1843-48 (2000)); and, Shou et al, Oncogene 21 :878-89 (2002)). Additionally, Fedi et al. (1999) purportedly showed that Dkk-1 can block the Wnt2-induced oncogenic transformation of NIH-3T3 cells. Furthermore, it has been suggested that Dkk expression can be modulated by BMP signaling in the developing skeleton (Mukhopadhyay et al, Dev. Cell. 1 :423-34 (2001 ); and Grotewold et al, EMBO J. 21 :966-75 (2002)).
- Grotewold et al. additionally describe altered Dkk expression levels in response to stress signals including UV irradiation and other genotoxic stimuli. They propose that Dkk expression is pro- apoptotic. In animals expressing HBM constructs conferring high bone mass, a reduced osteoblast apoptosis effect was observed. Thus, HBM and HBM-like variants may control/alter Dkk's role in programmed cell death. Other agents which potentially modulate Dkk activity include the Dkk interacting proteins identified in Figure 5. In one embodiment, the relative amounts of Dkk or a Dkk interacting protein of a cell population that has been exposed to the agent to be tested is compared to an un- exposed control cell population. Antibodies can be used to monitor the differential expression of the protein in the different cell populations.
- Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
- Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe, as would be known in the art. See, e.g., Ed Harlow and David Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, NY, 1988) and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual (Cold Spring Harbor, NY 1998).
- N- and C- terminal fragments of Dkk can be expressed in bacteria and used to search for proteins which bind to these fragments.
- Fusion proteins such as His-tag or GST fusion to the N- or C-terminal regions of Dkk (or to biologically active domains of Dkk-1) or a whole Dkk protein can be prepared. These fusion proteins can be coupled to, for example, Talon or Glutathione-Sepharose beads and then probed with cell lysates to identify molecules which bind to Dkk. Prior to lysis, the cells may be treated with purified Wnt proteins, RNA, or drugs which may modulate Wnt signaling or proteins that interact with downstream elements of the Wnt pathway. Lysate proteins binding to the fusion proteins can be resolved by SDS-PAGE, isolated and identified by, for example protein sequencing or mass spectroscopy, as is known in the art. See, e.g..
- the activity of Dkk, a Dkk interacting protein, or a complex of Dkk with LRP5/LRP6/HBM may be affected by compounds which modulate the interaction between Dkk and a Dkk interacting protein (such as those shown in Figure 5) and/or Dkk and LRP5/LRP6/HBM.
- the present invention provides methods and research tools for the discovery and characterization of these compounds.
- the interaction between Dkk and a Dkk interacting protein and/or Dkk and LRP5/6/HBM may be monitored in vivo and in vitro.
- Compounds which modulate the stability of a Dkk - LRP5/LRP6/HBM complex are potential therapeutic compounds.
- Example in vitro methods include: Binding LRP5/6/HBM, Dkk, or a Dkk interacting protein to a sensor chip designed for an instrument such are made by Biacore (Uppsala, Sweden) for the performance of an plasmon resonance spectroscopy observation.
- the chip with one of Dkk, a Dkk interacting protein, or LRP5/6 is first exposed to the other under conditions which permit them to form the complex.
- a test compound is then introduced and the output signal of the instrument provides an indication of any effect exerted by the test compound.
- Another, in vitro, method is exemplified by the SAR-by-NMR methods (Shuker et al, Science. 274:1531-4 (1996)).
- a Dkk-1 binding domain and/or LRP 5 or 6 LBD are expressed and purified as 15 N labeled protein by expression in labeled media.
- the labeled protein(s) are allowed to form the complex in solution in an NMR sample tube.
- the heteronuclear correlation spectrum in the presence and absence of a test compound provides data at the level of individual residues with regard to interactions with the test compound and changes at the protein-protein interface of the complex.
- affinity capillary electrophoresis e.g. affinity capillary electrophoresis (Okun et al.
- Nucleic acids according to the invention which encode the interacting Dkk and LRP LBD domains are incorporated into bait and prey plasmids.
- the Y2H protocol is performed in the presence of one or more test compounds.
- the modulation of the complex is observed by a change in expression of the complex activated gene.
- test compounds can be added to the assay directly or, in the case of proteins, can be coexpressed in the yeast with the bait and prey compounds.
- fusion proteins of Dkk and Dkk interacting proteins can also be used in a Y2H screen to identify other proteins which modulate the Dkk Dkk interacting protein complex.
- Assay protocols such as these may be used in methods to screen for compounds, drugs, treatments which modulate the Dkk/Dkk interacting protein and/or Dkk/LRP5/6 complex, whether such modulation occurs by competitive binding, or by altering the structure of either LRP 5/6 or Dkk at the binding site, or by stabilizing or destablizing the protein-protein interface. It may be anticipated that peptide aptamers may competitively bind, although induction of an altered binding site structure by steric effects is also possible.
- polypeptide immunogens can be conjugated to suitable carriers.
- suitable carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other carrier proteins are well known in the art (See, Harlow et al., 1988).
- hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
- Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.
- Anti-peptide antibodies can be generated using synthetic peptides, for example, the peptides derived from the sequence of any Dkk, including Dkk-1 , or LRP5/LRP6/HBM. Synthetic peptides can be as small as 2-3 amino acids in length, but are preferably at least 3, 5, 10, or 15 or more amino acid residues long. Such peptides can be determined using programs such as DNAStar. The peptides are coupled to KLH using standard methods and can be immunized into animals such as rabbits. Polyclonal anti-Dkk or anti-LRP5/LRP6/HBM peptide antibodies can then be purified, for example using Actigel beads containing the covalently bound peptide.
- Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known (See, e.g., Harlow et al, 1988 and 1998).
- the immortalized cell lines secreting the desired antibodies can be screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
- the cells can be cultured either in vitro or by production in ascites fluid.
- the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies which contain the immunologically significant portion can be used as agonists or antagonists of Dkk activity. Use of immunologically reactive fragments, such as the Fab, scFV, Fab', of F(ab') 2 fragments are often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
- the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of Dkk or LRP5/LRP6/HBM can also be produced in the context of chimeras with multiple species origin. Antibody reagents so created are contemplated for use diagnostically or as stimulants or inhibitors of Dkk activity.
- antibodies against Dkk bind Dkk with high affinity, i.e., ranging from 10 "5 to 10 9 M.
- the anti-Dkk antibody will comprise a chimeric, primate, Primatized®, human or humanized antibody.
- the invention embraces the use of antibody fragments, e.g., Fab's, Fv's, Fab's, F(ab) 2 , and aggregates thereof.
- Another embodiment contemplates chimeric antibodies which recognize Dkk or
- LRP5/LRP6/HBM LRP5/LRP6/HBM.
- a chimeric antibody is intended to refer to an antibody with non- human variable regions and human constant regions, most typically rodent variable regions and human constant regions.
- a “primatized® antibody” refers to an antibody with primate variable regions, e.g., CDR's, and human constant regions. Preferably, such primate variable regions are derived from an Old World monkey.
- humanized antibody refers to an antibody with substantially human framework and constant regions, and non-human complementarity-determining regions (CDRs). "Substantially” refers to the fact that humanized antibodies typically retain at least several donor framework residues (i.e., of non-human parent antibody from which CDRs are derived).
- the selection of human constant regions may be significant to the therapeutic efficacy of the subject anti-Dkk or LRP5/LRP6/HBM antibody.
- the subject anti-Dkk or LRP5/LRP6/HBM antibody will comprise human, gamma 1, or gamma 3 constant regions and, more preferably, human gamma 1 constant regions.
- Methods for making human antibodies include, by way of example, production in SCID mice, and in vitro immunization.
- the subject anti-Dkk or LRP5/LRP6/HBM antibodies can be administered by various routes of administration, typically parenteral. This is intended to include intravenous, intramuscular, subcutaneous, rectal, vaginal, and administration with intravenous infusion being preferred.
- the anti-Dkk or LRP5/LRP6/HBM antibody will be formulated for therapeutic usage by standard methods, e.g., by addition of pharmaceutically acceptable buffers, e.g., sterile saline, sterile buffered water, propylene glycol, and combinations thereof. Effective dosages will depend on the specific antibody, condition of the patient, age, weight, or any other treatments, among other factors. Typically effective dosages will range from about 0.001 to about 30 mg/kg body weight, more preferably from about 0.01 to 25 mg/kg body weight, and most preferably from about 0.1 to about 20 mg/kg body weight.
- pharmaceutically acceptable buffers e.g., sterile saline, sterile buffered water, propylene glycol, and combinations thereof.
- Effective dosages will depend on the specific antibody, condition of the patient, age, weight, or any other treatments, among other factors. Typically effective dosages will range from about 0.001 to about 30 mg/kg body weight, more preferably from about 0.01 to
- Such administration may be effected by various protocols, e.g., weekly, biweekly, or monthly, depending on the dosage administered and patient response. Also, it may be desirable to combine such administration with other treatments.
- Antibodies to Dkk-1 interacting proteins such as those identified in Figure 5, are also contemplated according to the present invention, and can be used similarly to the Dkk-1 antibodies mentioned in the above methodology.
- the antibodies of the present invention can be utilized in experimental screening, as diagnostic reagents, and in therapeutic compositions. 12.2 Chemical Libraries
- Agents that are assayed by these methods can be randomly selected or rationally selected or designed.
- an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of Dkk-1 alone, Dkk-1 interacting proteins alone, or with their associated substrates, binding partners, etc.
- An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism.
- the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
- DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems.
- the production of polypeptides using solid phase peptide synthesis is necessitated if non-nucleic acid-encoded amino acids are to be included.
- the proteins and nucleic acids of the invention such as the proteins or polypeptides containing an amino acid sequence of LRP5, Dkk, and Dkk interacting proteins are involved in bone mass modulation and lipid modulation of other Wnt pathway mediated activity.
- Agents that modulate (i.e., up and down-regulate) the expression of Dkk or Dkk interacting proteins, or agents, such as agonists and antagonists respectively, of at least one activity of Dkk or a Dkk interacting protein may be used to modulate biological and pathologic processes associated with the function and activity of Dkk or a Dkk interacting protein.
- a subject can be preferably any mammal, so long as the mammal is in need of modulation of a pathological or biological process modulated by a protein of the invention.
- mammal means an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
- a biological or pathological process modulated by Dkk or a Dkk interacting protein may include binding of Dkk to a Dkk interacting protein, Dkk to LRP5 or LRP6 or release therefrom, inhibiting or activating Dkk or a Dkk interacting protein mRNA synthesis or inhibiting Dkk or Dkk interacting protein modulated inhibition of LRP5 or LRP6 mediated Wnt signaling. Further bone-related markers may be observed such as alkaline phosphatase activity, osteocalcin production, or mineraiization.
- Pathological processes refer to a category of biological processes which produce a deleterious effect.
- expression or up-regulation of expression of LRP5 or LRP6 and/or Dkk and/or a Dkk interacting protein may be associated with certain diseases or pathological conditions.
- an agent is said to modulate a pathological process when the agent statistically significantly (p ⁇ 0.05) alters the process from its base level in the subject.
- the agent may reduce the degree or severity of the process mediated by that protein in the subject to which the agent was administered.
- a disease or pathological condition may be prevented, or disease progression modulated by the administration of agents which reduce or modulate in some way the expression or at least one activity of a protein of the invention.
- one embodiment of this invention is to use Dkk or Dkk interacting protein expression as a method of diagnosing a bone condition or disease.
- Certain markers are associated with specific Wnt signaling conditions (e.g., TCF/LEF activation).
- Diagnostic tests for bone conditions may include the steps of testing a sample or an extract thereof for the presence of Dkk or Dkk interacting protein nucleic acids (i.e., DNA or RNA), oligomers or fragments thereof or protein products of TCF/LEF regulated expression.
- standard in situ hybridization or other imaging techniques can be utilized to observe products of Wnt signaling.
- This invention also relates to methods of modulating bone development or bone loss conditions.
- Inhibition of bone loss may be achieved by inhibiting or modulating changes in the LRP5/6 mediated Wnt signaling pathway. For example, absence of LRP5 activity may be associated with low bone mass. Increased activity LRP5 may be associated with high bone mass. Therefore, modulation of LRP5 activity will in turn modulate bone development.
- Modulation of the Dkk/LRP5/6 or Dkk/Dkk interacting protein complex via agonists and antagonists is one embodiment of a method to regulate bone development. Such modulation of bone development can result from inhibition of the activity of, for example, a Dkk/LRP(5/6) protein complex, a Dkk/Dkk interacting protein complex, upregulated transcription of the LRP5 gene or inhibited translation of Dkk or Dkk interacting protein mRNA.
- the agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
- two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
- the agents of the present invention can be administered via parenteral, subcutaneous (sc), intravenous (iv), intramuscular (im), intraperitoneal (ip), transdermal or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
- the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
- the present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
- Typical dosages of the active agent which mediate Dkk or Dkk interacting protein activity comprise from about 0.0001 to about 50 mg/kg body weight.
- the preferred dosages comprise from about 0.001 to about 50 mg/kg body weight.
- the most preferred dosages comprise from about 0.1 to about 1 mg/kg body weight. In an average human of 70 kg, the range would be from about 7 ⁇ g to about 3.5 g, with a preferred range of about 0.5 mg to about 5 mg.
- compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
- suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
- suspensions of the active compounds as appropriate oily injection suspensions may be administered.
- Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, (e.g., ethyl oleate or triglycerides).
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
- the suspension may also contain stabilizers.
- Liposomes and other non-viral vectors can also be used to encapsulate the agent for delivery into the cell.
- the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral, or topical (top) administration.
- Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
- any compound which binds Dkk or a Dkk interacting protein or modulates the Dkk/LRP5 or Dkk/LRP6 or Dkk Dkk interacting protein complex may be a therapeutic compound.
- a peptide or nucleic acid aptamer according to the invention is used in a therapeutic composition.
- Such compositions may comprise an aptamer, or a LRP5 or LRP6 fragment unmodified or modified.
- the therapeutic compound comprises a Dkk-1 interacting protein, or biologically active fragment thereof.
- Nucleic acid aptamers have been used in compositions for example by chemical bonding to a carrier molecule such as polyethylene glycol (PEG) which may facilitate uptake or stabilize the aptamer.
- PEG polyethylene glycol
- a di-alkylgylcerol moiety attached to an RNA will embed the aptamer in liposomes, thus stabilizing the compound.
- Incorporating chemical substitutions i.e. changing the 2'OH group of ribose to a 2'NH in RNA confers ribonuclease resistance
- capping, etc. can prevent breakdown.
- Peptide aptamers may by used in therapeutic applications by the introduction of an expression vector directing aptamer expression into the affected tissue such as for example by retroviral delivery, by encapsulating the DNA in a delivery complex or simple by naked DNA injection. Or, the aptamer itself or a synthetic analog may be used directly as a drug. Encapsulation in polymers and lipids may assist in delivery.
- the use of peptide aptamers as therapeutic and diagnostic agents is reviewed by Hoppe-Syler and Butz (J. Mol. Med. 78:426-430 (2000)).
- the structure of a constrained peptide aptamer of the invention may be determined such as by NMR or X-ray crystallography. (Cavanagh et al., Protein NMR Spectroscopy: Principles and Practice. Academic Press, 1996; Drenth, Principles of Protein X-Rav Crystallography. Springer Verlag, 1999)
- the structure is determined in complex with the target protein.
- a small molecule analog is then designed according to the positions of functional elements of the 3D structure of the aptamer. (Guidebook on Molecular Modeling in Drug Design. Cohen, Ed., Academic Press, 1996; Molecular Modeling and Drug Design (Topics in Molecular and Structural Biology).
- the present invention provides a method for the design of effective and specific drugs which modulate the activity of Dkk, Dkk interacting proteins, Dkk/Dkk interacting protein complex and the Dkk/LRP complex.
- Small molecule mimetics of the peptide aptamers of the present invention are encompassed within the scope of the invention.
- the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
- the compounds of this invention may be co- administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
- the compounds of this invention can be administered in combination with other therapeutic agents for the treatment of bone loss.
- Bone loss mediating agents include bone resorption inhibitors such as bisphosphonates (e.g., alendronic acid, clodronic acid, etidronic acid, pamidronic acid, risedronic acid and tiludronic acid), vitamin D and vitamin D analogs, cathepsin K inhibitors, hormonal agents (e.g., calcitonin and estrogen), and selective estrogen receptor modulators or SERMs (e.g., raloxifene). And bone forming agents such as parathyroid hormone (PTH) and bone morphogenetic proteins (BMP).
- bisphosphonates e.g., alendronic acid, clodronic acid, etidronic acid, pamidronic acid, risedronic acid and tiludronic acid
- vitamin D and vitamin D analogs e.g., cathepsin K inhibitors
- hormonal agents e.g., calcitonin and estrogen
- SERMs selective estrogen receptor modulators or SERMs
- SERMs
- agents which regulate Dkk-1 and agents which regulate lipid levels
- agents which regulate lipid levels such as HMG-CoA reductase inhibitors (i.e., statins such as Mevacor®, Lipitor® and other inhibitors such as Baycol®, Lescol®, Pravachol® and Zocor®), bile acid sequestrants (e.g., Colestid® and Welchol®), fibric acid derivatives (Atromid-S®, Lopid®, Tricor®), and nicotinic acid.
- HMG-CoA reductase inhibitors i.e., statins such as Mevacor®, Lipitor® and other inhibitors such as Baycol®, Lescol®, Pravachol® and Zocor®
- bile acid sequestrants e.g., Colestid® and Welchol®
- fibric acid derivatives e.g.,romid-S®, Lopid®, Tricor®
- nicotinic acid nico
- Transgenic animal models can be created which conditionally express Dkk and/or LRP5 or LRP6 and/or Dkk interacting proteins, such as those shown in Figure 5. These animals can be used as research tools for the study of the physiological effects of the Dkk-1/Dkk-1 interacting protein interaction and/or the LRP5 / Dkk interaction.
- transgenic animals can be created which express a transgenic form of Dkk alone or in addition to a transgenic form of HBM or express Dkk interacting proteins alone or in addition to a transgenic form of Dkk.
- Transgenic animals expressing HBM or LRP5 can be crossed with transgenic animals expressing Dkk or Dkk interacting proteins to obtain heterozygote as well as homozygote animals which express both desired genes.
- Animal models may be created to directly modulate the Dkk/Dkk interacting protein or Dkk/ LRP5 interaction activity in vivo to serve as a research tool for determining the efficacy of candidate compounds which modulate the Dkk/Dkk interacting protein or LRP5 / Dkk interaction activity in vitro.
- Animals such as transgenic mice, can be created using the techniques employed to make transgenic mice that express for example, human Dkk or a Dkk interacting protein, or knockouts (KO), which may be conditional, of the gene encoding mouse Dkk or Dkk interacting protein.
- Knock-in animals include animals wherein genes have been introduced and animals wherein a gene that was previously knocked-out is reintroduced into the animal.
- transgenic animals can be created with inducible forms of Dkk or a Dkk interacting protein to study the effects of the gene on bone mass development and loss as well as lipid level regulation. These animals can also be used to study long term effects of Dkk or Dkk interacting protein modulation.
- Transgenic animals may be created to express peptide aptamers, or produce RNA aptamers.
- the transgenic vectors may direct expression in a tissue specific manner by the use of tissue specific promoters.
- a peptide aptamer fusion protein is expressed using a bone specific promoter.
- Such systems can provide a tissue specific knock-out of Dkk or Dkk interacting protein activity.
- Peptide aptamers are molecules in which a variable peptide domain is displayed from a scaffold protein.
- Thioredoxin A trxA
- the peptide insert destroys the catalytic site of trxA. It is recognized that numerous proteins may also be used as scaffolding proteins to constrain and/or present a peptide aptamer.
- scaffold proteins that could display a constrained peptide aptamer could include staphylococcal nuclease, the protease inhibitor eglin C, the Streptomyces tendea alpha- amylase inhibitor Tendamistat, Sp1 , and green fluorescent protein (GFP) (reviewed in Hoppe-Seyler et al, J. Steroid Biochem Mol. Biol. 78:105-11 (2001 )), and the S1 nuclease from Staphylococcus or M13 for phage display. Any molecule to which the aptamer could be anchored and presented in its bioactive conformation would be suitable.
- Aptamers can then specifically bind to a given target protein in vitro and in vivo and have the potential to selectively block the function of their target protein.
- Peptide aptamers are selected from randomized expression libraries on the basis of their in vivo binding capacity to the desired target protein. Briefly, a target protein (e.g., Dkk, a Dkk interacting protein, or LRP5/6) is linked to a heterologous DNA binding domain (BD) and expressed as bait in a yeast test strain. Concomitantly, a library coding for different peptides (e.g., 16-mers) of randomized sequence inserted in a scaffold protein sequence, which are linked to a heterologous transcriptional activation domain (AD) is expressed as prey.
- BD heterologous DNA binding domain
- AD heterologous transcriptional activation domain
- a functional transcription factor is reconstituted, in which the BD and AD are bridged together by interacting proteins. This transcription factor is then able to activate the promoter of a marker gene which can be monitored by colorimetric enzymatic assays or by growth selection. Additional variation, methods of preparing and screening methodologies are described in, for example, Hoppe-Seyler et al, J. Mol. Med. 78: 426-430 (2000). Nucleotide aptamers are described for example in Brody er al., Trends Mol. Biotechnol. 74: 5-13 (2000).
- SELEX Systematic Evolution of Ligands by Exponential Enrichment.
- SELEX is a process of isolating oligonucleotide ligands of a chosen target molecule (see Tuerk and Gold, Science 249:505-510 (1990); U.S. Pat. Nos. 5,475,096, 5,595,877, and 5,660,985).
- SELEX as described in Tuerk and Gold, involves admixing the target molecule with a pool of oligonucleotides (e.g., RNA) of diverse sequences; retaining complexes formed between the target and oligonucleotides; recovering the oligonucleotides bound to the target; reverse-transcribing the RNA into DNA; amplifying the DNA with polymerase chain reactions (PCR); transcribing the amplified DNA into a pool of oligonucleotides (e.g., RNA) of diverse sequences; retaining complexes formed between the target and oligonucleotides; recovering the oligonucleotides bound to the target; reverse-transcribing the RNA into DNA; amplifying the DNA with polymerase chain reactions (PCR); transcribing the amplified DNA into
- PCR polymerase chain reactions
- RNA reverse transcriptase
- RNA reverse transcriptase
- Three enzymatic reactions are required for each cycle. It usually takes 12-15 cycles to isolate aptamers of high affinity and specificity to the target.
- An aptamer is an oligonucleotide that is capable of binding to an intended target substance but not other molecules under the same conditions.
- Bock et al Nature 355:564-566 (1990), describe a different process from the SELEX method of Tuerk and Gold in that only one enzymatic reaction is required for each cycle (i.e., PCR) because the nucleic acid library in Bock's method is comprised of DNA instead of RNA.
- PCR enzymatic reaction
- the identification and isolation of aptamers of high specificity and affinity with the method of Bock et al. still requires repeated cycles in a chromatographic column.
- nucleotide aptamer methods include those described by Conrad et al, Meth. Enzymol 267:336-367 (1996).
- Conrad et al. describe a variety of methods for isolating aptamers, all of which employ repeated cycles to enrich target-bound ligands and require a large amount of purified target molecules.
- More recently described methods of making and using nucleotide aptamers include, but are not limited to those described in U.S. Patent Nos. 6,180,348; 6,051 ,388; 5,840,867; 5,780,610, 5,756,291 and 5,582,981.
- any compound which binds Dkk or a Dkk interacting protein or modulates the Dkk/Dkk interacting protein or Dkk/LRP5 or Dkk/LRP6 complex may be a therapeutic compound.
- a peptide or nucleic acid aptamer according to the invention is used in a therapeutic composition.
- Such compositions may comprise an aptamer, or a LRP5 or LRP6 fragment unmodified or modified.
- Nucleic acid aptamers have been used in compositions for example by chemical bonding to a carrier molecule such as polyethylene glycol (PEG) which may facilitate uptake or stabilize the aptamer.
- PEG polyethylene glycol
- a di-alkylglycerol moiety attached to an RNA will embed the aptamer in liposomes, thus stabilizing the compound.
- Incorporating chemical substitutions i.e., changing the 2'-OH group of ribose to a 2'-NH in RNA confers ribonuclease resistance
- capping, etc. can prevent breakdown.
- Peptide aptamers may by used in therapeutic applications by the introduction of an expression vector directing aptamer expression into the affected tissue such as for example by retroviral delivery, by encapsulating the DNA in a delivery complex or simple by naked DNA injection. Or, the aptamer itself or a synthetic analog may be used directly as a drug. Encapsulation in polymers and lipids may assist in delivery.
- the use of peptide aptamers as therapeutic and diagnostic agents is reviewed by Hoppe-Syler and Butz J. Mol. Med. 78:426-430 (2000).
- the structure of a constrained peptide aptamer of the invention may be determined such as by NMR or X-ray crystallography.
- the structure is determined in complex with the target protein.
- a small molecule analog is then designed according to the positions of functional elements of the 3D structure of the aptamer. (Guidebook on Molecular Modeling in Drug Design. Cohen, Ed., Academic Press, 1996; Molecular Modeling and Drug Design (Topics in Molecular and Structural Biology).
- variants comprising changed residues in structurally equivalent positions in other blades; as well as residues that are slightly more interior to the binding pocket, but still accessible to the surface, are important embodiments of the present invention for the study of bone mass modulation by LRP5/HBM, for the development of pharmaceuticals and treatments of bone mass disorders, and for other objectives of the present invention.
- E128V (a position equivalent to 171 in blade 3; glutamate is not conserved in other propellers),
- LRP5/Zmax1 has four beta-propeller structures; the first three beta-propeller modules conserve a glycine in the position corresponding to residue 171 in human LRP5/Zmax1. Therefore, variants bearing a valine in the equivalent positions in the other propellers are important embodiments of the present invention.
- the following variants are useful for the study of bone mass modulation by LRP5/HBM, for the development of pharmaceuticals and treatments of bone mass disorders, and for other objectives of the present invention: G479V, G781 V, and Q1087V.
- the G171V HBM polymorphism results in "occupied space" of the beta-propeller 1 , with the side-chain from the valine residue sticking out into an open binding pocket and potentially altering a ligand/protein interaction.
- the glycine residue is conserved in LRP5/Zmax1 propellers 1 , 2 and 3 but is a glutamine in propeller 4. Therefore, the following variants of LRP5/HBM are important embodiments of the present invention for the study of bone mass modulation by LRP5/HBM, for the development of pharmaceuticals and treatments of bone mass disorders, and for other objectives of the present invention:
- G171K (which introduces a charged side-chain)
- G171 F (which introduces a ringed side-chain)
- LRP6 is the closest homolog of LRP5/Zmax1.
- LRP6 has a beta- propeller structure predicted to be similar, if not identical to Zmaxl .
- the position corresponding to glycine 171 in human LRP5/Zmax1 is glycine 158 of human LRP6.
- corresponding variants of LRP6 are an important embodiment of the present invention for the study of the specificity of LRP5/Zmax1 versus its related family member, for the development of pharmaceuticals and treatments of bone mass disorders, and for other objectives of the present invention.
- a glycine to valine substitution at the structurally equivalent position, residue 158, of human LRP6 and similar variants of other species' LRP6 homologs represent important research tools.
- E128V AAGCTGTACTGGACGGACTCAGTGACCAACCGCATCGAGG (SEQ ID NO:
- G171 K ATGTACTGGACAGACTGGAAGGACGCCCCGGATTGAGCG
- G171 F ATGTACTGGACAGACTGGTTTGAGACGCCCCGGATTGAGCG (SEQ ID NO:134)
- G171 I ATGTACTGGACAGACTGGATTGAGACGCCCCGGATTGAGCG (SEQ ID NO:137) and
- G171 Q ATGTACTGGACAGACTGGCAGGAGACGCCCCGGATTGAGCG
- G199V CGGACATTTACTGGCCCAATGTACTGACCATCGACCTGGAGG
- M282V GAGTGCCCTCTACTCACCCGTGGACATCCAGGTGCTGAGCC (SEQ ID NO: 144)
- G479V CATGTACTGGACAGACTGGGTAGAGAACCCTAAAATCGAGTGTGC (SEQ ID NO:147) and
- G781V CATCTACTGGACCGAGTGGGTCGGCAAGCCGAGGATCGTGCG
- FIG 24 shows the effects of the G171 F mutation in propeller 1 of LRP5. This mutation is at the same position as HBM's G171V substitution. Expression of G171F results in an HBM effect. That is, in the presence of Wnt, G171 F is able to activate the TCF-luciferase reporter construct. In fact, it may activate the reporter to a greater extent than either LRP5 or HBM. Furthermore, in the presence of Dkkl and Wntl , G171F is less susceptible than LRP5 to modulation by Dkk. These data exemplify that the G171 F variant modulates Wnt signaling in a manner similar to HBM.
- FIG. 25 shows the effects of the M282V mutation in propeller 1 of LRP5.
- M282 expression results in an HBM-effect. That is, in the presence of Wnt, M282 is able to activate the TCF-luciferase reporter construct. Furthermore, in the presence of Dkkl and Wntl , M282V is less susceptible than LRP5 to modulation by Dkk. These data show that the M282V variant modulates Wnt signaling in a manner similar to HBM. In addition, this data confirms that modifications of other residues in propeller 1 of LRP5 can result in an HBM effect.
- Variants of LRP5 which have demonstrated HBM activity in assays include G171F, M282V, G171K, G171Q and A214V. Clearly, other variants may be contemplated within the scope of the present invention. Furthermore, wherever HBM is recited in the methods of the invention, it should be understood that any such alternative variant of LRP which demonstrates HBM biological activity is also encompassed by those claims.
- the two-hybrid system is extremely useful for studying protein: protein interactions. See, e.g., Chien et al, Proc. Natl Acad. Sci. USA 88:9578-82 (1991); Fields et al, Trends Genetics 10:286-92 (1994); Harper et al, Cell 75:805-16 (1993); Vojtek et al, Cell 74:205-14 (1993); Luban et al, Cell 73: 1067-78 (1993); Li et al, FASEB J. 7:957-63 (1993); Zang et al, Nature 364:308-13 (1993); Golemis et al, Mol. Cell. Biol.
- the success of the two-hybrid system relies upon the fact that the DNA binding and polymerase activation domains of many transcription factors, such as GAL4, can be separated and then rejoined to restore functionality (Morin et al, Nuc. Acids Res. 21 :2157-63 (1993)). While these examples describe two-hybrid screens in the yeast system, it is understood that a two-hybrid screen may be conducted in other systems such as mammalian cell lines. The invention is therefore not limited to the use of a yeast two-hybrid system, but encompasses such alternative systems.
- Yeast strains with integrated copies of various reporter gene cassettes such as for example GAL.fwdarw.LacZ, GAL.fwdarw.HIS3 or GAL.fwdarw.URA3 (Bartel, in Cellular Interactions and Development: A Practical Approach. 153-179 (1993); Harper et al, Cell 75:805-16 (1993); Fields et al., Trends Genetics 10:286-92 (1994)) are co- transformed with two plasmids, each expressing a different fusion protein.
- various reporter gene cassettes such as for example GAL.fwdarw.LacZ, GAL.fwdarw.HIS3 or GAL.fwdarw.URA3 (Bartel, in Cellular Interactions and Development: A Practical Approach. 153-179 (1993); Harper et al, Cell 75:805-16 (1993); Fields et al., Trends Genetics 10:286-92 (1994)
- One plasmid encodes a fusion between protein "X” and the DNA binding domain of, for example, the GAL4 yeast transcription activator (Brent et al, Cell 43:729-36 (1985); Ma et al, Cell 48:847-53 (1987); Keegan et al, Science 231 :699-704 (1986)), while the other plasmid encodes a fusion between protein "Y” and the RNA polymerase activation domain of GAL4 (Keegan et al, 1986).
- the plasmids are transformed into a strain of the yeast that contains a reporter gene, such as lacZ, whose regulatory region contains GAL4 binding sites.
- proteins X and Y interact, they reconstitute a functional GAL4 transcription activator protein by bringing the two GAL4 components into sufficient proximity to activate transcription. It is well understood that the role of bait and prey proteins may be alternatively switched and thus the embodiments of this invention contemplate and encompass both alternative arrangements.
- the reporter gene cassettes consist of minimal promoters that contain the GAL4 DNA recognition site (Johnson et al, Mol. Cell. Biol. 4:1440-8 (1984); Lorch et al, J. Mol. Biol. 186:821-824 (1984)) cloned 5' to their TATA box.
- Transcription activation is scored by measuring either the expression of ⁇ -galactosidase or the growth of the transformants on minimal medium lacking the specific nutrient that permits auxotrophic selection for the transcription product, e.g., URA3 (uracil selection) or HIS3 (histidine selection).
- auxotrophic selection e.g., URA3 (uracil selection) or HIS3 (histidine selection).
- URA3 uracil selection
- HIS3 histidine selection
- these methods include two proteins to be tested for interaction which are expressed as hybrids in the nucleus of a yeast cell.
- One of the proteins is fused to the DNA-binding domain (DBD) of a transcription factor and the other is fused to a transcription activation domain (AD). If the proteins interact, they reconstitute a functional transcription factor that activates one or more reporter genes that contain binding sites for the DBD.
- DBD DNA-binding domain
- AD transcription activation domain
- Exemplary two-hybrid assays which have been used for Dkk-1 or Dkk-1/LRP5 are presented in the Examples below.
- LRP5 Ligand Binding Domain Bait Sequences
- HOB03C5 a custom Gibco generated Y2H compatible cDNA library from a human osteoblast cell line as described by Bodine and Med, Bone 25:535-43 (1999)
- LRP5 ligand binding domain (LBD) baits used for this screen are depicted in Figures 2B and C.
- the basic protocol is as follows: An overnight culture of the yeast strain containing the bait of interest is grown in
- an aliquot of the frozen prey library is grown in 150 ml YAPD medium for 5 hours at 30°C.
- the number of diploids to be screened is typically ten times the number of clones originally present in the prey library of interest. Assuming a mating efficiency of 20% minimum, fifty times (i.e., ten times coverage multiplied by 20% mating efficiency) as many haploid cells containing the bait and as many cells containing the prey are used in any given mating event. The mixture is filtered over a 47 mm, 0.45 mm sterile Metricel filter membrane (Gelman).
- the filter is transferred onto a 100 mm 2 YAPD agar plate with the cell side up, removing all air bubbles underneath the filter. The plate is incubated overnight at room temperature.
- the filter is transferred into a 50 ml Falcon tube using sterile forceps and 10 ml SD medium containing 2% glucose are added to resuspend the cells.
- the filter once free of cells, is removed and the cell suspension is spun for 5 min. at 2,000 xg.
- the cells are resuspended in 10 ml SD medium containing 2% glucose. An aliquot of 100 ⁇ l is set aside for titration.
- the cells are plated onto large square plates containing appropriate selective media and incubated at 30 °C for three to five days.
- the 100 ⁇ l aliquot set aside for titration is diluted and plated onto different selective media.
- the mating efficiency is calculated by dividing the number of diploids/ml by the lowest number of haploids/ml, either bait or prey, and multiplied by 100. For example, if 2 million diploids were obtained by mating 10 million of haploids containing a bait and 12 million of haploids containing a prey, then the mating efficiency is calculated by dividing 2 million by 10 million, which equals 0.2 and multiplied by 100 which equals 20%. Typical mating efficiencies under the above conditions are within about 20 to about 40%.
- the total number of diploids screened in a mating event is obtained by multiplying the number of diploids/ml by the total number of ml plated, typically about 10. Isolation of colonies containing pairs of interacting proteins.
- Yeast colonies from the interaction selection (large square) plates are picked with a sterile toothpick and patched onto plates containing the appropriate selective media and incubated at 30°C for two days. To further ensure purity of the yeast, the plates are replicated onto another plate containing the same media and incubated at 30 °C for another two days.
- Yeast patches are scraped using a sterile toothpick and placed into a 96-well format plate containing 100 ⁇ l SD -L -W -H with 2% glucose liquid medium.
- Half the volume of the plate is transferred to a 96-well plate containing 50 ⁇ l of 40% glycerol for storage. The other half is set aside for replication and galactosidase- activity assay (see below).
- Cells are replicated onto a SD -L -W -H plate with 2% glucose plate to create a master plate, and incubated two days at 30°C.
- the master plate is replicated onto different selective media to score the strength of each interaction.
- Cells are also replicated onto media selecting for the prey vector only for colony
- Y2H yeast two hybrid
- PCR primers containing restriction sites suitable for cloning are designed to cover multiple sub-domains of the protein of interest (bait or prey).
- the methods involved in cloning, sequencing, yeast transformation, mating, and scoring of interactions are readily performed by one of ordinary skill in the art of molecular biology and genetic engineering.
- primers were designed for PCR of the Dkk-1 clone isolated by screening a primary osteoblast cell strain (HOB03C5) library with pooled Zmax1/LRP5 ligand binding domain (LBD) baits: LBD1 (Leu969-Pro1376) and LBD4 (Arg1070-Pro1376).
- LBD1 Leu969-Pro1376)
- LBD4 Arg1070-Pro1376
- PCR products were gel purified, digested with Sa/I/ Not ⁇ and ligated to pPC86 (Gibco/BRL) which had been linearized with Sa/l/ ⁇ /ofl. Clones were recovered and sequenced to ascertain that the structure was as expected and that the Gal4 activation domain and Dkk-1 were in-frame.
- the ORF of Dkk-1 was Met1-His266, as in human Dkk-1 (GenBank Accession No. XM_005730).
- the clones used were as follows: D5 (F1/R3: Asn34-His266), D4 (F1/R2: Asn34-Cys245), D3 (F1/R1 : Asn34-Lys182), D9 (F2/R3: Cys97-His266), D12 (F3/R3, val139-His266), D14 (F4/R3: Gly183-His266), D8 (F2/R2: Cys97-Cys245), and D11 (F3/R2: Val139-Cys245).
- F1 , F2, F3 and F4 refer respectively to Forward primers 1 , 2, 3 and 4.
- R1 , R2 and R3 refer respectively to reverse primers 1 , 2 and 3.
- Tpep a peptide aptamer library
- the Tpep library is a combinatorial aptamer library composed of constrained random peptides, expressed within the context of the disulfide loop of E. coli thioredoxin (trxA), and as C-termini fusion to the S. cerevisiae Gal4 activation domain.
- the Tpep library was generated using a restriction enzyme modified recombinant Y2H prey vector, pPC86 (Gibco), which contains the trxA scaffold protein.
- aptamer-encoding sequences were produced as follows. DNA encoding random stretches of approximately sixteen amino acids surrounded by appropriate restriction sites were generated by semi-random oligonucleotide synthesis. The synthetic oligonucleotides were PCR-amplified, restriction digested, and cloned into the permissive sites within the trxA scaffold protein. The cloning strategy was to insert the random oligonucleotide sequence is in-frame with the scaffold protein coding sequence, resulting in expression of a scaffold protein-aptamer chimera.
- the scaffold protein is itself in-frame with the activation domain of Gal4, within the pPC86 vector that is appropriate for the aptamer to be expressed and functional in a regular Y2H assay. Additional methods of preparing aptamers would be apparent to the skilled artisan.
- the E. coli trxA coding sequence was cloned into the Sa/I and ⁇ /o.l sites of the f?s/1l-modified pPC86. EcoRI and Spel sites were then introduced within the trxA site.
- the oligonucleotides encoding the peptide aptamers were cloned into the EcoRI and Spel sites of the resulting vector.
- a Dkk-1 bait sequence was utilized in a yeast two hybrid screen to identify Dkk-1 interacting proteins.
- the procedure for the Y2H was carried out similarly to that employed in Example 1 , except that the Dkk-1 bait from Figure 2C was used instead of LRP baits.
- the screen was performed using Hela and fetal brain libraries (Invitrogen Corporation, Carlsbad, CA). Multiple libraries were used to identify additional Dkk-1 interacting proteins and to confirm interactions found in other libraries.
- the list of Dkk-1 interacting proteins uncovered in these Y2H screens are listed in Figure 5.
- the interacting proteins identified in the Dkk-1 bait screen can be used in other
- Antibodies were generated to the following peptides to obtain antibodies which distinguish the HBM polymorphism versus wild-type LRP5/Zmax: MYWTDWVETPRIE (SEQ ID NO:123) (mutant peptide) and MYWTDWGETPRIE (SEQ ID NO:124) (wild- type peptide for negative selection). Immunofluorescence data confirmed that the antibody, after affinity purification, is specific for HBM and does not recognize LRP5 ( Figure 17).
- LRP5 monospecific polyclonal antibodies were generated to the following amino acid sequences of LRP5: Peptide 1 (a.a. 265-277) - KRTGGKRKEILSA (SEQ ID NO:125), Peptide 2 (a.a. 1178-1194) - ERVEKTTGDKRTRIQGR (SEQ ID N0.126), and Peptide 3 (a.a. 1352-1375) - KQQCDSFPDCIDGSDE (SEQ ID NO: 127). Immunofluorescence confirmed that the antibody generated detects LRP5.
- Dkk-1 monospecific polyclonal antibodies were generated to the following amino acid sequences of Dkk-1 : Peptide 1 (a.a. 71-85) - GNKYQTIDNYQPYPC (SEQ ID NO.118), Peptide 2 (a.a. 165-186) - LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO:119 ) prevail Peptide 3 (a.a. 246-266) - RIQKDHHQASNSSRLHTCQRH (SEQ ID NO.120), Peptide 4 (a.a.
- Figure 26 shows the location of the various peptides selected, their relationship to the Dkk-1 amino acid sequence and polyclonal antibodies generated.
- FIG. 27 shows Western blots using 500 ⁇ l of conditioned medium (CM) from non-transfected 293 cells or from 293 cells transfected with Dkk1-V5 that were immunoprecipitated by anti-V5 antibody. Bead elutes were separated by non-reducing SDS-PAGE (lanes #4, 5 of Figure 27). 20 ⁇ l of conditioned medium from both samples (lanes #2, 3 of Figure 27) and from Dkk1-AP transfected 293 cells (lane #6 of Figure 27) were additionally separated on the gel. The Western was performed using antibodies Anti ⁇ 5/AP (1 :10,000) and Ab#5521 (10 ⁇ g/ml). Ab#5521 detected Dkk1-V5 and Dkk1-AP from conditioned medium.
- CM conditioned medium
- Ab#5521 detected Dkk1-V5 and Dkk1-AP from conditioned medium.
- Figure 28 shows Western blot results using Ab#74397.
- Anti-V5/AP was tested at a 1 :4000 dilution and Ab#74397 was tested at a 1 :500 dilution.
- Ab#74397 was able to detect Dkk1-V5 in both conditioned medium and immunoprecipitated conditioned medium.
- Xenopus embryos are an informative and well-established in vivo assay system to evaluate the modulation of Wnt signaling (McMahon et al, Cell 58: 1075-84 (1989); Smith and Harland, 1991 ; reviewed in Wodarz and Nusse 1998) .
- Modification of the Wnt signaling pathway can be visualized by examining the embryos for a dorsalization phenotype (duplicated body axis) after RNA injection into the ventral blastomere at the 4- or 8-cell stage.
- phenotypes can be analyzed by looking for expression of various marker genes in stage 10.5 embryos.
- markers would include general endoderm, mesoderm, and ectoderm markers as well as a variety of tissue-specific transcripts. Analysis can be done by RT-PCR/TaqMan® and can be done on whole embryo tissue or in a more restricted fashion (microdissection).
- Constructs were prepared using the vector pCS2 + . DNA inserts were subcloned in the sense orientation with respect to the vector SP6 promoter.
- the pCS2 + vector contains an SV40 virus polyadenylation signal and T3 promoter sequence (for generation of antisense mRNA) downstream of the insert.
- Insert cDNA was isolated from the full length cDNA retrovirus constructs (with optimized Kozak sequences) by BglW- EcoRI digestion and subcloned into the SamHI-EcoRI sites of the pCS2 + vector.
- Full length XWnt ⁇ This cDNA was PCR amplified from a Xenopus embryo cDNA library using oligos 114484 (SEQ ID NO:162) (5'-
- CAGTGAATTCACCATGCAAAACACCACTTTGTTC-3' and 114487 (SEQ ID NO:163) (5'-CAGTTGCGGCCGCTCATCTCCGGTGGCCTCTG-3').
- the oligos were designed to amplify the ORF with a consensus Kozak sequence at the 5' end as determined from GenBank #X57234. PCR was carried out using the following conditions: 96°C, 45 sec; 63°C, 45 sec; 72°C, 2 min. for 30 cycles. The resulting PCR product was purified, subcloned into pCRII-TOPO (Invitrogen Corp.), sequence verified, and digested with Bam ⁇ IXho ⁇ . This insert was subcloned into the vector at the Bam ⁇ -Xho ⁇ sites.
- Full length Wnt5a A murine Wnt ⁇ a cDNA clone was purchased from Upstate Biotechnology (Lake Placid, NY) and subcloned into the EcoRI site of the vector. Sequencing confirmed insert orientation.
- Full length human Dkk-1 A human cDNA with GenBank accession number AF127563 was available in the public database. Using this sequence, PCR primers were designed to amplify the open reading frame with a consensus Kozak sequence immediately upstream of the initiating ATG. Oligos 117162 (SEQ ID NO:164) (5'- CAATAGTCGACGAATTCACCATGGCTCTGGGCGCAGCGG-3') and 117163 (SEQ ID NO:164) (5'- CAATAGTCGACGAATTCACCATGGCTCTGGGCGCAGCGG-3') and 117163 (SEQ ID NO:164
- Full length human Dkk-2 A full length cDNA encoding human Dkk-2 was isolated to investigate the specificity of the Zmax/LRP5/HBM interaction with the Dkk family of molecules.
- Dkk-1 was identified in yeast as a potential binding partner of Zmax/LRP5/HBM.
- Dkk-1 has also been shown in the literature to be an antagonist of the Wnt signaling pathway, while Dkk-2 is not (Krupnik et al, 1999).
- the Dkk-2 full length cDNA serves as a tool to discriminate the specificity and biological significance of Zmax/LRP5/HBM interactions with the Dkk family (e.g., Dkk-1 , Dkk-2, Dkk-3, Dkk-4, Soggy, their homologs and variant, etc.).
- a human cDNA sequence for Dkk-2 (GenBank Accession No. NM_014421) was available in the public database. Using this sequence, PCR primers were designed to amplify the open reading frame with a consensus Kozak sequence immediately upstream of the initiating ATG. Oligos 51409 (SEQ ID NO:166) (5'- CTAACGGATCCACCATGGCCGCGTTGATGCGG-3') and 51411 (SEQ ID NO:167) (5'-GATTCGAATTCTCAAATTTTCTGACACACATGG-3') were used to screen human embryo and brain cDNA libraries by PCR.
- the resulting PCR product was purified, subcloned into pCRII-TOPO, sequence verified, and digested with BamHI/EcoRI. This insert was subcloned into the pCS2 + vector at the SamHI-EcoRI sites.
- Full length LRP6 was isolated from the pED6dpc4 vector by Xho ⁇ -Xba ⁇ digestion. The full length cDNA was reassembled into the Xho ⁇ -Xba ⁇ sites of pCS2 + . Insert orientation was confirmed by DNA sequencing.
- mRNA Synthesis and Microinjection Protocol mRNA for microinjection into Xenopus embryos is generated by in vitro transcription using the cDNA constructs in the pCS2 + vector described above as template. RNA is synthesized using the Ambion mMessage mMachine high yield capped RNA transcription kit (Cat. #1340) following the manufacturer's specifications for the Sp6 polymerase reactions.
- RNA products were brought up to a final volume of 50 ⁇ l in sterile, glass-distilled water and purified over Quick Spin Columns for Radiolabelled RNA Purification G50-Sephadex (Roche, Cat. #1274015) following the manufacturer's specifications. The resulting eluate was finally extracted with phenol:chloroform:isoamyl alcohol and isopropanol precipitated using standard protocols (Sambrook et al, 1989). Final RNA volumes were approximately 50 ⁇ l. RNA concentration was determined by absorbance values at 260 nm and 280 nm.
- RNA integrity was visualized by ethidium bromide staining of denaturing (formaldehyde) agarose gel electrophoresis (Sambrook et al, 1989).
- denaturing (formaldehyde) agarose gel electrophoresis (Sambrook et al, 1989).
- Various amounts of RNA (2 pg to 1 ng) are injected into the ventral blastomere of the 4- or 8-cell Xenopus embryo.
- RNA is purified and injected into a ventral blasomere of the 4- or 8-cell Xenopus embryo (approx. 2 hours post-fertilization). At stage 10.5 (approx. 11 hours post-fertilization), the injected embryos are cultured for a total of 72 hours and then screened for the presence of a duplicated body axis (dorsalization) ( Figure 7). Using XWnt ⁇ -injected (2-10 pg) as a positive control (Christian et al.
- the HBM/Wnt5a embryos also appear to be more "anteriorized" than the Zmax(LRP5)/Wnt5a embryos, again suggestive of a gain-of-function mutation.
- Wnt activity can be antagonized by many proteins including secreted Frizzled related proteins (SFRPs), Cerberus, Wnt Inhibitory Factor-1 and Dkk-1 (Krupnik et al, 1999).
- the Dkk family of proteins consists of Dkk-1 -4 and Soggy, a Dkk-3-like protein. Dkk-1 and Dkk-4 have been shown to antagonize Wnt mediated Xenopus embryo development, whereas Dkk-2, Dkk-3, and Soggy do not.
- Dkk-1 acts by binding to two recently identified Wnt coreceptors, LRP5 and LRP6.
- Dkk-1 peptides were developed, including Dkk-1 peptides, constrained LRP5 peptide aptamers, constrained Dkk-1 peptide aptamers and polyclonal antibodies to Dkk-1 (in Example 5 above) to identify factors that mimic HBM mediated Wnt signaling.
- LRP5 constrained peptides were developed. Specifically, four peptides that interact with the LBD of LRP5 ( Figure 4,constructs OST259-262 in Figure 12) and three peptides that interact with the cytoplasmic domain of LRP5 (constructs OST266- OST26 ⁇ in Figure 12). In addition two Dkk-1 peptides were developed: constructs OST264 and OST265 in Figure 12, corresponding to Dkk-1 amino acids 139-266 and 96-245, containing the smallest region of Dkk-1 that interacts with LRP5 ( Figure 6).
- the cDNA clones encoding the LRP5 LBD interacting peptides and the Dkk-1 peptides were subcloned into pcDNA3.1 with the addition of a Kozak and signal sequence to target the peptide for secretion.
- the constructs encoding the three peptides interacting with the cytoplasmic domain of LRP5 were also subcloned into pcDNA3.1. However, these latter constructs do not contain a signal sequence.
- HOB-03-CE6 osteoblastic cells developed by Wyeth Ayerst (Philadelphia, PA) were seeded into 24-well plates at 150,000 cells per well in 1 ml of the growth media (D-MEM/F12 phenol red-free) containing 10% (v/v) heat-inactivated FBS, 1X penicillin streptomycin, and 1X Glutamax-1 , and incubated overnight at 34°C.
- D-MEM/F12 phenol red-free 10% (v/v) heat-inactivated FBS, 1X penicillin streptomycin, and 1X Glutamax-1
- the cells were transfected using Lipofectamine 2000® (as described by the manufacturer, Invitrogen) in OptiMEM (Invitrogen) with 0.35 ⁇ g /well of LRP5, HBM, or control plasmid DNA (empty vector pcDNA3.1) and either Wntl or Wnt3a plasmid DNA. Similar experiments were performed with LRP6 plasmid DNA (0.35 ⁇ g/well) or a control pEDdpc4 empty vector. Furthermore, each of these groups were then divided into three groups, those receiving 0.35 ⁇ g/well Dkk-1 , Dkk-2, or pcDNA3.1 control DNA.
- the extracts were assayed for beta-galactosidase activity (Galacto Reaction Buffer Diluent & Light Emission Accelerator, Tropix) using 5 ⁇ l extract + 50 ⁇ l beta- galactosidase diluent and luciferase activity (Luciferase Assay Reagent, Promega) using 20 ⁇ l extract.
- U20S human osteosarcoma cells were also utilized.
- U20S cells (ATCC) were seeded into 96-well plates at 30,000 cells per well in 200ul of the growth media (McCoy's 5A) containing 10% (v/v) heat-inactivated FBS, 1X penicillin streptomycin, and 1X Glutamax-1 , and incubated overnight at 37°C.
- nmedia was replaced with OptiMEM (Invitroge) and cells were transfected using Lipofectamine 2000® (as described by the manufacturer, Invitrogen) with 0.005 ⁇ g/well of LRP5, HBM, LRP6 or contol plasmid DNA (empty vector pcDNA3.1) and either Wntl (.0025ug/well) or Wnt3a (.0025ug/well) plasmid DNA.
- cells were co-transfected with 0.005 ⁇ g/well of LRP5, 0.0025ug/well of Wntl or Wnt3a (using 0.0025 ⁇ g/well of a control pcDNA3.1 ) with LRP5-interacting aptamers (0.05ug/well).
- Cells were cultured for an additional 18-20 hours at 37°C. Culture medium was removed.
- Cells were cultured for an additional 1 ⁇ -20 hours at 37°C. Culture medium was removed.
- Example 8 Yeast-2 Hybrid Interaction Trap Small molecule inhibitors (or partial inhibitors) of the Dkk-LRP interaction may be an excellent osteogenic therapeutic.
- Y2H techniques substantially as described above and as is well known in the art. Regions of LRP5, such as LRP5 LBD, have been found to functionally interact with Dkk. This interaction is quantitated using a reporter element known in the art, e.g., LacZ or luciferase, which is only activated when bait and prey interact.
- the Y2H assay is used to screen for compounds which modulate the LRP- Dkk interaction.
- the Y2H assay can be used as a high-throughput screening technique to identify compounds which disrupt or enhance Dkk interaction with LRP5/LRP6/HBM, which may serve as potential therapeutics.
- the Interaction Trap methodology can be used as follows.
- the LRP5 LBD for example, was fused with LexA and Dkk-1 was fused with either Gal4-AD or B42.
- the TCF-luciferase assay described in Example 7 was modified utilizing low level expression of endogenous LRP5/6 in U20S and HEK293 cells.
- U20S human osteosarcoma
- HEK293 HEK293
- Wnt3a alone by using endogenous LRP5/6, was able to stimulate TCF reporter gene activation.
- Dkk When Dkk, is co-transfected with Wnt3a/Wnt 1 and reporters (TCF-luci and tk-Renilla), Dkk represses reporter element activity.
- the TCF-luci signal is activated by Wnt3a/Wnt1 can be repressed by the addition of Dkk-enriched conditioned media to the cells containing Wnt3a/Wnt1 and reporters.
- the assay is further validated by the lack of TCF-reporter inhibition by a point mutant construct (C220A) of Dkkl .
- the Dkk-mediated repression of the reporter is dependent upon the concentration of transfected Dkk cDNA or on the amount of Dkk-conditioned media added.
- the Dkk-mediated reporter suppression can be altered by the co- transfection of LRP5, LRP6, and HBM cDNAs in the U20S or HEK293 cells.
- U20S cells show greater sensitivity to Dkk-mediated reporter suppression than that in HEK-293 cells.
- the transfection of LRP5/LRP6/HBM cDNA leads to moderate activation of TCF-luci in the absence of Wnt3a/Wnt1 transfection. This activation presumably utilizes the endogenous Wnts present in U20S cells.
- Dkkl can repress TCF-luci and shows a differential signal between LRP5 and HBM.
- Wnt3a/Wnt1 By co-transfecting Wnt3a/Wnt1 , there is a generalized increase in the TCF- luci signal in the assay.
- the assay can detect the functional impact of the LRP5 interacting peptide aptamers (Figure 4), Dkkl interacting aptamers and binding domains of Dkk-1 ( Figure 6; OST264 and OST265 of Figures 12 and 13).
- this system with a suppressed Wnt-TCF signal due to the presence of both Dkk and Wnt3a, one can screen for compounds that could alter Dkk modulation of Wnt signaling, by looking for compounds that activate or the TCF-luciferase reporter, and thereby relieve the Dkk-mediated repression of the Wnt pathway.
- Such compounds identified may potentially serve as HBM-mimetics and be useful, for example, as osteogenic therapeutics.
- Figure 19 shows that Dkkl represses Wnt3a- mediated signaling in U20S bone cells.
- Figure 20 demonstrates the functional differences between LRP5, LRP6, and HBM.
- Dkk-1 represses LRP6 and LRP5 but has little or no effect on HBM-generated Wntl signaling in U20S cells.
- Figure 21 demonstrates the differential effects of various Dkk family members and modified Dkks, including Dkk-1 , a mutated Dkk-1 (C220A), Dkk-1 -AP (modified with alkaline phosphatase), Dkk-3, and Soggy.
- LRP5 is immobilized to a solid surface, such as a tissue culture plate well.
- a solid surface such as a tissue culture plate well.
- supports such as a nylon or nitrocellulose membrane, a silicon chip, a glass slide, beads, etc.
- the form of LRP5 used is actually a fusion protein where the extracellular domain of LRP5 is fused to the Fc portion of human IgG.
- the LRP5-Fc fusion protein is produced in CHO cell extracts from stable cell lines.
- the LRP5-Fc fusion protein is immobilized on the solid surface via anti-human Fc antibody 5 or by Protein-A or Protein G-coated plates, for example. The plate is then washed to remove any non-bound protein.
- Conditioned media containing secreted Dkk protein or secreted Dkk-epitope tagged protein (or purified Dkk or purified Dkk-epitope tagged protein) is incubated in the wells and binding of Dkk to LRP is investigated using antibodies to either Dkk or to an epitope tag.
- Dkk-V ⁇ epitope tagged protein would be 0 detected using an alkaline phosphatase tagged anti-V ⁇ antibody.
- the Dkk protein could be directly fused to a detection marker, such as alkaline phosphatase.
- a detection marker such as alkaline phosphatase.
- the bound Dkk is detected in an alkaline phosphatase assay. If the Dkk-alkaline phosphatase fusion ⁇ protein is bound to the immobilized LRP ⁇ , alkaline phosphatase activity would be detected in a colorimetric readout. As a result, one can assay the ability of small molecule compounds to alter the binding of Dkk to LRP using this system.
- the assay can be calibrated by doing cold competition experiments with unlabeled Dkk or with a second type of epitope-tagged Dkk.
- Any small molecule that is able to modulate the Dkk-LRP interaction may be a suitable therapeutic candidate, more preferably an osteogenic therapeutic candidate.
- Example 11 Functional Evaluation of Peptide Aptamers in Xenopus
- the constrained peptide aptamers constructs OST2 ⁇ 8-263 (where 2 ⁇ contains the signal sequence by itself and 263 contains an irrelevant constrained peptide) 0 ( Figures 12 and 13) were used to generate RNA substantially as described in Example 7, except the vector was linearized by restriction endonuclease digestion and RNA was generated using T7 RNA polymerase.
- RNA was injected at 2 ⁇ 0 pg per blastomere using the protocol of Example 7. Wnt signaling was activated, as visualized by embryo dorsalization ⁇ (duplicated body axis) with aptamers 261 and, more strongly, 262. The results of this assay are shown in Figures 22 and 23. These results suggest that aptamers 261 and 262 are able to activate Wnt signaling possibly by binding to the LBD of LRP, thereby preventing the modulation of LRP-mediated signaling by Dkk.
- the aptamers of the present invention can serve as HBM-mimetics. In the 0 Xenopus system they are able to induce Wnt signaling all by themselves. They may also serve as tools for rational drug design by enhancing the understanding of how peptides are able to interact with LRP and modulate Wnt signaling at the specific amino acid level. Thus, one would be able to design small molecules to mimic their effects as therapeutics. In addition, the aptamers identified as positives in this assay may be used ⁇ as therapeutic molecules themselves.
- FRET Fluorescence Resonance Energy Transfer
- purified LRP protein, or portions or domains thereof, fused to CFP 0 and purified Dkk protein, or portions or domains thereof that interact with Dkk or LRP respectively, fused to YFP can be generated and purified using standard approaches. If LRP-CFP and Dkk-YFP are in close proximity, the transfer of energy from CFP to YFP will result in a reduction of CFP emission and an increase in YFP emission. Energy is supplied with an excitation wavelength of 4 ⁇ 0 nm and the energy transfer is ⁇ recorded at emission wavelengths of 480 nm and ⁇ 70 nm. The ratio of YFP emission to CFP emission provides a guage for changes in the interaction between LRP and Dkk.
- This system is amenable for screening small molecule compounds that may alter the Dkk-LRP protein-protein interaction. Compounds that disrupt the interaction would be identified by a decrease in the ratio of YFP emission to CFP emission. Such 0 compounds that modulate the LRP-Dkk interaction would then be considered candidate HBM mimetic molecules. Further characterization of the compounds can be done using the TCF-luciferase or Xenopus embryo assays to elucidate the effects of the compounds on Wnt signaling.
- LRP-CFP fusion protein can be expressed in cells.
- the Dkk-YFP fusion protein then could be added to the cells either as purified protein or as conditioned media. The jnteraction of LRP and Dkk is then monitored as described above.
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US8367822B2 (en) | 2003-09-22 | 2013-02-05 | Enzo Therapeutics, Inc. | Compositions and methods for bone formation and remodeling |
US9046537B2 (en) | 2003-09-22 | 2015-06-02 | Enzo Biochem, Inc. | Method for treating inflammation by administering a compound which binds LDL-receptor-related protein (LRP) ligand binding domain |
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WO2005049797A2 (en) | 2003-11-17 | 2005-06-02 | Merck & Co., Inc. | Cynomolgus monkey dickkopf-4, nucleotides encoding same, and uses thereof |
US7622553B2 (en) | 2003-11-17 | 2009-11-24 | Merck & Co., Inc. | Rhesus monkey dickkopf-1, nucleotides encoding same, and uses thereof |
US9052324B2 (en) | 2004-05-19 | 2015-06-09 | Enzo Biochem, Inc. | Compounds and assays for controlling Wnt activity |
EP2336177A1 (en) | 2004-08-04 | 2011-06-22 | Amgen, Inc | Antibodies to DKK-1 |
WO2006015497A1 (en) * | 2004-08-13 | 2006-02-16 | Val-Chum, S.E.C. | Methods of use of a dkk1 protein, immunogenic polypeptides thereof, nucleic acid encoding the dkk1 protein or polypeptides, or ligands thereof for detecting tumors; and for eliciting immune response against tumors |
CA2677356A1 (en) * | 2007-02-08 | 2008-08-14 | Zhiqiang An | Antibodies specific for dkk-1 |
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AR075989A1 (en) | 2009-04-10 | 2011-05-11 | Lilly Co Eli | ANTIBODY DKK -1 (DICKKOPF-1) HUMAN DESIGNED BY ENGINEERING |
WO2010129752A1 (en) * | 2009-05-07 | 2010-11-11 | Novartis Ag | Compositions and methods of use for binding molecules to dickkopf-1 or dickkopf-4 or both |
CA2761696A1 (en) | 2009-05-12 | 2010-11-18 | Pfizer Inc. | Blocking anti-dkk-1 antibodies and their uses |
WO2011103426A2 (en) | 2010-02-19 | 2011-08-25 | The Board Of Regents Of The University Of Oklahoma | Monoclonal antibodies that inhibit the wnt signaling pathway and methods of production and use thereof |
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JP2013527762A (en) | 2010-05-06 | 2013-07-04 | ノバルティス アーゲー | Therapeutic low density lipoprotein related protein 6 (LRP6) antibody compositions and methods of use |
PE20130206A1 (en) | 2010-05-06 | 2013-02-28 | Novartis Ag | MULTIVALENT ANTIBODIES ANTAGONISTS OF LRP6 (LOW DENSITY LIPOPROTEIN-RELATED PROTEIN 6) AND COMPOSITIONS |
ES2666856T3 (en) | 2011-11-04 | 2018-05-08 | Novartis Ag | Protein 6 related to low density lipoproteins (LRP6) - half-life extender constructs |
CN107760686B (en) * | 2016-08-23 | 2021-04-16 | 上海伯豪医学检验所有限公司 | Aptamer of DKK-1 protein and application thereof |
CN110709102A (en) * | 2017-03-24 | 2020-01-17 | 耶鲁大学 | Inhibition of low density lipoprotein receptor-related protein 5 suppresses tumor formation |
MY200744A (en) | 2017-05-31 | 2024-01-13 | Boehringer Ingelheim Int | Polypeptides antagonizing wnt signaling in tumor cells |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000052047A2 (en) * | 1999-03-05 | 2000-09-08 | Millennium Pharmaceuticals, Inc. | Human dickkopf-related protein and nucleic acid molecules and uses therefor |
WO2002066509A2 (en) * | 2001-02-16 | 2002-08-29 | Genentech, Inc. | Treatment involving dkk-1 or antagonists thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046743A1 (en) * | 1997-04-15 | 1998-10-22 | The Wellcome Trust Limited As Trustee To The Wellcome Trust | Novel ldl-receptor |
WO2001077327A1 (en) * | 2000-04-05 | 2001-10-18 | Genome Therapeutics Corporation | THE HIGH BONE MASS GENE OF 11q13.3 |
BR0111057A (en) * | 2000-05-26 | 2003-04-15 | Genome Therapeutics Corp | A method of identifying a molecule and a protein involved in lipid regulation, testing a substance as a therapeutic agent for modulating lipid levels, treating lipid-mediated disorders and a patient suffering from a lipid-mediated condition, lipid levels in a host, to treat or prevent a lipid-mediated disorder, to treat or prevent atherosclerosis or a condition associated with atherosclerosis, to screen for diagnosis of a genetic predisposition to atherosclerosis or a condition associated with atherosclerosis or a lipid-mediated disorder, expressing hbm protein in tissue and modulating lipid levels in a patient, diagnostic assay to determine a predisposition to a lipid-mediated disorder, composition for treating a lipid-mediated condition, and, Combination therapy to treat a patient suffering from a disease or lipid-mediated condition |
BR0209563A (en) * | 2001-05-11 | 2004-12-07 | Genome Theraupeutics Corp | nucleic acid, polypeptide, vector, cell, antibody, methods of diagnosing a phenotype and a patient as expressing a nucleic acid and of identifying agents that modulate the activity of a nucleic acid and a protein and lrp5, lrp6, or hbm and compounds that modulate the interaction of dkk with the wnt signaling path, composition to modulate bone mass and / or lipid levels in a patient, transgenic animal, embryo and animal model, and, agent |
-
2002
- 2002-05-17 JP JP2002588934A patent/JP2005512508A/en not_active Withdrawn
- 2002-05-17 EP EP02744162A patent/EP1395285A4/en not_active Withdrawn
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000052047A2 (en) * | 1999-03-05 | 2000-09-08 | Millennium Pharmaceuticals, Inc. | Human dickkopf-related protein and nucleic acid molecules and uses therefor |
WO2002066509A2 (en) * | 2001-02-16 | 2002-08-29 | Genentech, Inc. | Treatment involving dkk-1 or antagonists thereof |
Non-Patent Citations (3)
Title |
---|
DATABASE WPI Section Ch, Week 200312 Derwent Publications Ltd., London, GB; Class B04, AN 2003-129214 XP002304054 & WO 02/092000 A2 (null) 21 November 2002 (2002-11-21) * |
GLINKA ET AL: "Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 391, no. 6665, 22 January 1998 (1998-01-22), pages 357-362, XP002096088 ISSN: 0028-0836 * |
See also references of WO02092015A2 * |
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CA2446582A1 (en) | 2002-11-21 |
JP2005512508A (en) | 2005-05-12 |
JP2009142274A (en) | 2009-07-02 |
EP1395285A4 (en) | 2005-06-01 |
IL158750A0 (en) | 2004-05-12 |
AU2002342734B2 (en) | 2007-07-26 |
WO2002092015A3 (en) | 2003-10-23 |
WO2002092015A2 (en) | 2002-11-21 |
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