WO2020047475A1

WO2020047475A1 - Drug screening for fgf23-fgfr4 inhibitors

Info

Publication number: WO2020047475A1
Application number: PCT/US2019/049161
Authority: WO
Inventors: Christian FAUL; Christopher YANUCIL
Original assignee: The Uab Research Foundation
Priority date: 2018-08-30
Filing date: 2019-08-30
Publication date: 2020-03-05

Abstract

The present disclosure provides a screening assay that detects the binding between FGF23 and FGFR4 in a very sensitive and quantitative manner that can be used to identify modulator compounds (i.e., inhibitor compounds and stimulator compounds) of FGF23-FGFR4 binding with high specificity. The disclosed methods can be used in a high-throughput format to screen large numbers of candidate compounds, and is cost-effective. The present disclosure also provides for modulator compounds (i.e., inhibitor compounds and stimulator compounds) that are identified by the assays disclosed as well as pharmaceutical formulations comprising such identified modulator compounds. Further, the present disclosure provides for methods of using the identified modulator compounds in methods of treating a disease or condition in a subject.

Description

DRUG SCREENING FOR FGF23-FGFR4 INHIBITORS

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under (F31DK115074) awarded by the National Institutes of Health. The government has certain rights in the invention. 37 CFR 401.14(f)(4).

FIELD OF THE DISCLOSURE

The present disclosure relates to methods of screening for and/or identifying modulators of the binding of a ligand to a fibroblast growth factor receptor. More specifically, the present disclosure relates to methods of screening for and/or identifying modulators of FGF23-FGFR4 binding.

BACKGROUND OF THE DISCLOSURE

Fibroblast growth factor (FGF) 23 is a hormone that regulates certain functions of the kidney. FGF23 acts on the kidneys, at least in part, by decreasing the expression of NPT2, a sodium-phosphate cotransporter in the proximal tubule. Thus, FGF23 decreases the reabsorption of phosphate and increases excretion of phosphate. FGF23 may also suppress 1- alpha-hydroxylase, reducing its ability to activate vitamin D and subsequently impairing calcium absorption. In patients with chronic kidney disease (CKD), blood levels of FGF23 are increased. In animals, increased FGF23 levels have been shown to cause organ damage. This same organ damage is also observed in the aging and in many CKD patients, such as injury of the heart and increased systemic inflammation.

FGF23 binds FGF receptor (FGFR) in the presence of a co-receptor protein called a- klotho (hereinafter“klotho”). However, in certain cell types FGF23 can bind to one or more FGFR isoforms in the absence of klotho. The pathologic effect of FGF23 is mediated by FGFR4. In previous studies, inhibition of FGFR4 signaling using anti-FGFR4 antibodies was shown to be protective against organ damage and other injuries in animal models of CKD (Faul, C. et al., J Clin Invest, 12, 4393-4408, 2011). Although such antibodies are commonly used in chemotherapy and other forms of treatment, antibody administration is not feasible for the long-term treatments of chronic diseases, such as CKD. Rather than antibody based treatments, the use of small molecules drugs would be desirable. However, currently available tools to screen for such small molecules that modulate FGF23-FGFR4 interaction suffer from a number of drawbacks. For example, current techniques for screening such small molecule inhibitors identify drugs with low specificity for FGFR4 which could lead to significant side effects (off-target effects) in humans. Furthermore, many of the current assays are labor intensive and expensive, limiting their application in large scale screening.

Therefore, the art is in need to new methods to screen for compounds that modulate the binding of FGF23 and FGFR4 to address the shortcomings in the art. The present disclosure provides a method to address the shortcomings in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an overview of one embodiment of the FGF23-FGFR4 screening assay. FIG. 1B shows an overview of an alternate embodiment of the FGF23-FGFR4 screening assay FIG. 2 shows the results of a screening experiment using the methods described in the present disclosure.

FIG. 3A shows the amino acid sequence of SEQ ID NO: 1 (human FGFR4).

FIG. 3B shows the amino acid sequence of SEQ ID NO: 2 (mouse FGFR4).

FIG. 4A shows the amino acid sequence of SEQ ID NO: 3 (human FGF23).

FIG. 4B shows the amino acid sequence of SEQ ID NO: 4 (mouse FGF23).

DETAILED DESCRIPTION

Definitions

As used herein, the term“pharmaceutical composition” refers to a mixture of one or more of the compounds of the disclosure, with at least one additional component, such as physiologically/pharmaceutically acceptable carriers and/or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure, including salts, tautomeric forms, hydrates and/or solvates to a subject (including pharmaceutically acceptable forms of the foregoing).

As used herein, the term“pharmaceutically acceptable” when modifying an item (such as a composition, compound, chemical or the like) means that the recited item is safe and non toxic to a subject and is suitable for use in a composition for administration to a subject, particularly a human subject.

As used herein, the term“pharmaceutically acceptable salt” is intended to include salts derived from inorganic or organic acids including, for example hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids. Pharmaceutically acceptable salt forms may also include forms wherein the ratio of molecules comprising the salt is not 1 : 1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of compound of formula I. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of compound of formula I per molecule of tartaric acid. Salts may also exist as solvates or hydrates.

As used herein, the term“about” is used herein to mean approximately. When the term

“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent up or down (higher or lower). The term“therapeutically effective amount” denotes that amount of a compound of the disclosure that will elicit a therapeutic response of a subject that is being sought, including a clinical response/result. The actual dose which comprises the therapeutically effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated, and the like. The therapeutically effective amount may be sufficient, for example, to reduce or ameliorate the severity and/or duration of a disease or condition, or one or more symptoms thereof, prevent the advancement of a disease or condition, prevent the recurrence, development, or onset of one or more symptoms associated with a disease or condition, enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy for a disease or condition, or a combination of the foregoing. In certain embodiments, a therapeutically effective amount is an amount of the compound of the disclosure that avoids or substantially attenuates undesirable side effects.

As used herein, the term“binding domain” means a group capable of interaction with a binding partner. Suitable binding domains are described herein.

As used herein, the term“binding partner” means a structure, molecule or substrate which interacts with a binding domain. The binding partner is not required to form a chemical bond with the binding domain. As one non-limiting example, when a binding domain is a magnetic moiety (such as a magnetic microsphere), the binding partner may be a magnet or other magnetic substrate. As another non-limiting example, when the binding domain is a biotin group (including biotin groups with reduced affinity such as desthiobiotin), the binding partner may be an avidin or streptavidin molecule. The interaction between a binding domain and its binding partner may be reversible under defined conditions. The interaction between a binding domain and its binding partner may be irreversible or maintained under defined conditions As used herein, the terms“treating” or“treat” refer to improving a symptom of a disease or disorder and may comprise curing the disorder, substantially preventing the onset of the disorder, alleviating of one symptom or most of the symptoms resulting from that disorder, or improving the subject's condition. The terms refer to the full spectrum of treatments for a given disorder from which the subject is suffering.

As used herein, the term“in need of treatment” as used herein refers to a judgment made by a healthcare professional that a subject requires or will benefit from treatment (including, but not limited to, the administration of a compound or composition disclosed herein). This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method, compound, or composition of the disclosure. As an example, the term“in need of treatment” indicates a patient has been diagnosed with CKD or a condition that causes, at least in part, CKD.

As used herein, the term“chronic kidney disease” or“CKD” refers to conditions that damage the kidneys and decrease the ability of the kidneys to function normally. The kidneys function to clear waste products from the blood. When kidney function is impaired, the concentration of waste products increases, causing further damage to the kidneys, damage to other organs, and leads to the development of additional pathologies in the subject. Such additional pathologies include, but not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease. CKD may be caused, at least in part, by a number of conditions, including, but not limited to, diabetes (both Type I and Type II), high blood pressure, glomerulonephritis, interstitial nephritis, polycystic kidney disease, prolonged obstruction of the urinary tract (for example, from conditions such as enlarged prostate, kidney stones and some cancers), vesicoureteral reflux, recurrent kidney infections (pyelonephritis) and other disorders. When CKD progresses, it may eventually lead to kidney failure, which requires dialysis or a kidney transplant to maintain life.

As used herein, the terms“subject” or“patient” means any mammal, including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment and/or compound. Typically, the term“subject” or “patient” is used in reference to a human.

As used herein, the terms“percent identity” or“percent identical” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the“Compared Sequence”) with the described or claimed sequence (the“Reference Sequence”). The percent identity is then determined according to the following formula:

percent identity = l00[l-(C/R)],

wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid. If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.

As used herein, the term“at least 90% identical” includes sequences that range from

90 to 99.99% identity to the indicated sequences and includes all ranges in between. Thus, the term at least 90% identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93, 93.5. 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence.

The terms“inhibitor” (when used to describe a compound identified by the methods of the present disclosure) or“inhibitor compound” refer to a compound that decreases the binding of FGF23 and FGFR4 and/or that interferes with the signal transduction initiated by FGF23 and FGFR4 binding. In certain embodiments, the terms refer to a compound that decreases the binding of FGF23 to FGFR4 (as compared to binding of FGF23 to FGFR4 in the absence of the inhibitor). In certain embodiments, the terms refer to a compound that decreases the affinity of the FGF23-FGFR4 binding interaction (as compared to the affinity of binding of FGF23 to FGFR4 in the absence of the inhibitor). In certain embodiments, the terms refer to a compound that decreases the FGF23-induced activation of FGFR4 (as compared to the FGF23-induced activation of FGFR4 in the absence of the inhibitor). In certain embodiments, the terms refer to a compound that decreases the FGF23-induced tyrosine phosphorylation of FGFR4 in the cytoplasmic tail of FGFR4 (as compared to the FGF23-induced tyrosine phosphorylation of FGFR4 in the absence of the inhibitor). In certain embodiments, the terms refer to a compound that blocks signaling initiated by FGF23-FGFR4 binding (for example, by inhibiting phospholipases C gamma (PLCy) activation). Each of the foregoing effects recited above occur in the absence of klotho (i.e., in a klotho-independent manner). The terms“inhibitor(s) of FGF23-FGFR4 binding” and“inhibitor(s) of FGF23- FGFR4 interaction” have corresponding meanings.

The terms“stimulator” (when used to describe a compound identified by the methods of the present disclosure) and“stimulator compound” refer to a compound that increases the binding of FGF23 and FGFR4 and/or that augments or enhances the signal transduction initiated by FGF23 and FGFR4 binding. In certain embodiments, the terms refer to a compound that increases the binding of FGF23 to FGFR4 (as compared to binding of FGF23 to FGFR4 in the absence of the stimulator). In certain embodiments, the terms refer to a compound that increases the affinity of the FGF23-FGFR4 binding interaction (as compared to the affinity of binding of FGF23 to FGFR4 in the absence of the stimulator). In certain embodiments, the terms refer to a compound that decreases the FGF23-induced activation of FGFR4 (as compared to the FGF23 -induced activation of FGFR4 in the absence of the stimulator). In certain embodiments, the terms refer to a compound that increases the FGF23- induced tyrosine phosphorylation of FGFR4 in the cytoplasmic tail of FGFR4 (as compared to the FGF23-induced tyrosine phosphorylation of FGFR4 in the absence of the stimulator). In certain embodiments, the terms refer to a compound that augments or enhances signaling initiated by FGF23-FGFR4 binding (for example, by stimulating PLCy activation). Each of the foregoing effects recited above occur in the absence of klotho (i.e., in a klotho-independent manner). The terms“stimulator(s) of FGF23-FGFR4 binding” and“stimulator(s) of FGF23- FGFR4 interaction” have corresponding meanings.

The term“klotho” as used herein refers specifically to a-klotho unless otherwise indicated.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of certain embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of“1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, for example 1 to 6.1, and ending with a maximum value of 10 or less, for example, 5.5 to 10.

It is further noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

Summary of the Disclosure

Based on biochemical characterization of FGF23 binding to FGFR4, a screening assay to identify modulators (i.e., inhibitors and stimulators) of FGF23-FGFR4 binding has been developed. Compared to currently available methods, the screening assay disclosed is more sensitive and is suitable for use in a high-throughput format to screen large numbers of candidate compounds. Finally, the screening assay is cost-effective, allowing the routine use of the assay to identify novel modulators of FGF23-FGFR4 binding. The disclosed screening assay can be used for a variety of purposes, including, but not limited to, the identification of inhibitors of FGF23-FGFR4 binding, the identification of stimulators of FGF23-FGFR4 binding, as well as determining the impact of such inhibitor and stimulator compounds on one or more binding characteristics of FGF23-FGFR4 binding (such as but not limited to, binding affinity). The present disclosure also provides for modulator compounds that are identified by the methods disclosed as well as pharmaceutical formulations comprising such identified modulator compounds.

The present disclosure also provides for use of the identified compounds in methods of treating a human subject. In one embodiment, the present disclosure provides methods for treating a subject having a disease or condition characterized by high FGF23 levels, increased FGFR4 levels, or increased FGF23 activation of FGFR4 (such as, but not limited to, aging and CKD). In one embodiment, such methods comprise administering to a subject an inhibitor compound identified by the disclosed screening assays. In one embodiment, the present disclosure provides methods for treating a subject having a disease or condition characterized by low FGF23 levels, decreased FGFR4 levels, or decreased FGF23 activation of FGFR4 (such as, but not limited to, metabolic syndrome, increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and hypercholesterolemia). In one embodiment, such methods comprise administering to a subject a stimulator compound identified by the disclosed screening assays.

Introduction

The family of FGFs consists of 22 members in humans, with a broad range of biological functions, including the regulation of embryonic development, organogenesis, and metabolism. FGFs are divided into seven subfamilies based on phylogenic analyses and overlapping structures. Members of the FGF19 subfamily, consisting of FGF19, FGF21, and FGF23, function as circulating hormones and are, therefore, termed endocrine FGFs. Unlike paracrine FGFs, such as FGF1 or FGF2, endocrine FGFs share a characteristic structure and lack the heparin-binding domain in their C-terminus which enables their secretion, circulation, and action on distant target organs. FGF23 is a phosphatunc hormone whose physiologic actions on target tissues are mediated by an FGFR, either in the presence or absence of kiotho. By stimulating FGFRl/klotho complexes in the kidney and parathyroid gland, FGF23 reduces renal phosphate uptake and secretion of parathyroid hormone, respectively, thereby acting as a key regulator of phosphate metabolism. When present, kiotho functions as a co-receptor that increases the binding affinity of FGF23 for FGFRs. Recently, it has been shown that FGF23 can also target cell types that lack kiotho. This klotho-independent signaling occurs in an FGFR-dependent manner and involves downstream signaling pathways different from the signaling pathways seen in klotho-dependent FGFR signaling. Klotho-independent signaling mechanisms are generally activated in the presence of high FGF23 concentrations and result in pathologic cellular changes. Therefore, high concentrations of FGF23 have been postulated to mediate the pathologies observed in diseases and conditions characterized by high levels of FGF23, such as, but not limited to, CKD and aging. Such pathologies include, but are not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac hypertrophy, fibrosis, and blood vessel disease.

The mammalian genome encodes four different FGFR isoforms (i.e., FGFR1-4) (Omitz DM, et al, Genome Biol (2001) 2(3)). FGFRs are receptor tyrosine kinases which are composed of an extracellular domain consisting of three immunoglobulin-like domains and containing the ligand-binding site, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. Upon FGF binding, FGFRs form dimers and auto-phosphorylate each other at specific tyrosine residues within their cytoplasmic tails which then initiates subsequent downstream signaling events. The FGFR isoforms differ in their affinity for particular FGF ligands. FGFR signaling is transduced by the cytoplasmic adaptors, PLCy and FGF receptor substrate 2a (FRS2a) (Omitz DM, et al, Genome Biol (2001) 2(3); Turner N, et al, Nat Rev Cancer (2010) 10(2): 116-29). Following ligand-induced auto-phosphorylation of FGFR, PLCy binds directly to one specific phosphorylated tyrosine residue within the FGFR cytoplasmic tail. Subsequent tyrosine phosphorylation of PLCy results in PLCy activation by the FGFR. Downstream signal transduction is mediated by PLCy-catalyzed production of diacylglycerol and inositol 1,4, 5-triphosphate that can increase cytoplasmic calcium levels thereby inducing the activation of several calcium-sensing signal mediators, including the protein phosphatase calcineurin. The dephosphorylation of the transcription factor, nuclear factor of activated T cells (NFAT), by activated calcineurin causes the translocation of NFAT into the nucleus to modulate the expression of specific target genes.

FGFR signaling can also be transduced via the activation of FRS2a by FGFR-mediated tyrosine phosphorylation. In contrast to PLC'y. FRS2a is constitutively bound to FGFR independently of the receptor’s activation state. FRS2a-mediated signaling results in the activation of Ras/mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling.

While FGFR1 acts as the main FGF23 receptor in“classic” klotho-expressing FGF23 target organs, klotho-independent effects of FGF23 appear to be mediated by FGFR4. Klotho- independent signaling via FGFR4 occurs through the PLCy/calcineurin/NFAT pathway whereas klotho-dependent signaling occurs through the FRS2a/Ras/MAPK pathway.

Levels of FGF23 in plasma of healthy subjects has been reported to be from 1 to 30 pg/ml, while levels of FGF23 in CKD stage 2, 3, 4 and end stage renal disease were reported to be between 30 to 80 pg/ml, 40 to 120 pg/ml, 110 to 230 pg/ml, and 4,000 to 60,000 pg/ml (Wolf, Kidney Int, 82(7), 737-747, 2012). For the purpose of the present disclosure a high FGF23 concentration is defined as a FGF23 concentration over 50 pg/ml as determined using the intact FGF23 ELISA assay (Eagle Biosciences, cat. No. 63278). For the purpose of the present disclosure a low FGF23 concentration is defined as a FGF23 concentration under 15 pg/ml as determined using the intact FGF23 ELISA assay (preferably under 1 pg/ml).

Since FGF23 stimulates normal (or physiological) signal transduction in the kidney via FGFR1 in the presence of klotho (particularly normal/physiological concentrations of FGF23), but requires FGFR4 for activation of signal transduction in the kidney and other cell types in the absence of klotho (particularly at high FGF23 concentrations), the pharmacologic blockade of FGFR4 is expected to interfere with FGF23's pathological actions while not interfering with FGF23’s normal/physiologic actions mediated by binding to FGFR1-4 in the presence of klotho. As a result, modulator compounds targeting the interaction of FGFR4 and FGF23 in the absence of klotho are expected to have minimal side effects after administration to a subject, including administration of a therapeutically effective amount. This view is supported by the finding that mice with global FGFR4 deletion do not develop an obvious phenotype or display a predisposition to develop any particular disease or condition.

As the expression and/or activity of FGFR isoforms, including FGFR4, is elevated in certain types of tumors, inhibitor compounds specific for FGFR4 may also be used as anti cancer agents and for treatment of cancer.

Standard approaches to screen for small molecule FGFR inhibitors focus on increasing or decreasing the tyrosine kinase activity of the cytoplasmic tyrosine kinase domain of FGFRs. However, the tyrosine kinase domain of the FGFRs, including its ATP binding pocket, is conserved among all FGFRs as well as other receptor tyrosine kinases. As a result, the identification of FGFR isoform-specific inhibitors, including specific inhibitors of FGFR4, is difficult using approaches that focus on inhibiting or stimulating tyrosine kinase activity.

Other approaches have focused on the development of blocking antibodies which specifically target the FGFR ectodomain and inhibit ligand binding to the receptor or inhibit receptor dimerization. Such antibodies appear to have a higher specificity for FGFR isoforms, including FGFR4. The present inventors have previously reported that an anti-FGFR4 antibody from U3 Pharma that is currently in clinical trials or cancer treatment reduced cardiac injury and inflammation in an animal model of CKD (Faul et al, The Journal of Clinical Investigations, 121(11), 4393-4408, 2011). However, the use of blocking antibodies as pharmacological agents for the treatment of chronic diseases, such as CKD, is not feasible, and the development and administration of small molecule inhibitors is desirable.

The use of a reliable, sensitive and quantitative assay to detect FGF23-FGFR4 binding and to identify small molecule inhibitors that disrupt or enhance FGF23-FGFR4 binding allows for the identification of inhibitor compounds that can specifically inhibit FGF23- induced FGFR4 activation and stimulator compounds that can specifically enhance FGF23- induced FGFR4 activation (i.e., modulator compounds). The present disclosure provides such as screening assay to identify modulator compounds. The inhibitor compounds identified by the methods of the present disclosure are candidate therapeutic agents for use in treating a variety of disease and conditions in subjects caused by high FGF23 levels, increased FGFR4 levels, or increased FGF23 activation of FGFR4. Such diseases and conditions include, but are not limited to, aging, CKD and their associated pathologies. Such associated pathologies include, but are not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy , fibrosis, and blood vessel disease. In a specific embodiment, the disease or condition is one disclosed in Schnedl et al. (Disease Markers, 2015:358086; doi 10.1155/2015/358086). The stimulator compounds identified by the methods of the present disclosure are candidate therapeutic agents for use in treating a variety of disease and conditions in subjects mediated by insufficient FGF23 activation of FGFR4. Such diseases and conditions include, but are not limited to, metabolic syndrome, increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and hypercholesterolemia.

Methods of Screening

FGFR4 has been identified as a klotho-independent FGF23 receptor ln co- immunoprecipitations assays using purified recombinant proteins, the present disclosure demonstrates that FGF23 can specifically bind FGFR4 in the absence of klotho. Based on these biochemical findings, an assay has been developed that can detect the FGF23-FGFR4 interaction in a sensitive and quantitative manner in the absence of klotho. Furthermore, this assay can be used to identify modulator compounds that can modulate the binding of FGF23 to FGFR4 in the absence of klotho, for example by promoting FGF23-FGFR4 interaction or inhibiting FGF23- FGFR4 interaction. In a specific embodiment, the disclosed assay may be used to identify a compound that interferes with the FGF23-FGFR4 interaction in the absence of klotho (an inhibitor compound). Such an inhibitor compound may further inhibit FGF23-induced activation of FGFR4 (such as, but not limited to, inhibiting tyrosine phosphorylation of the cytoplasmic portion of FGFR4), inhibit downstream signaling events triggered by FGFR4 activation (such, as, but not limited to, the PLCy/caicineurin/NFAT pathway), inhibit cellular effects mediated by FGFR4 activation, or any combination of the foregoing. In a further specific embodiment, the disclosed assay may be used to identify a compound that stimulates the FGF23-FGFR4 interaction in the absence of klotho (a stimulator compound). Such stimulator compound may stimulate FGF23- induced activation of FGFR4 (such as, but not limited to, stimulating tyrosine phosphorylation of the cytoplasmic portion of FGFR4), stimulate downstream signaling events triggered by FGFR4 activation (such, as, but not limited to, the PLCy/caicineurin/NFAT pathway), stimulate cellular effects mediated by FGFR4 activation, or any combination of the foregoing. In a specific aspect of this embodiment, a stimulator compound is not klotho or soluble klotho. Exemplary compound classes that may be screened include, but are not limited to, small molecules/small organic molecules, an antibody or a fragment thereof, a polypeptide, a recombinant polypeptide (with either wild-type or containing a specific mutation). Such compounds may be obtained from a library of pre-existing compounds.

In general, the method of screening involves providing a FGF23 ligand and a FGFR4 ligand, incubating the FGF23 ligand and the FGFR.4 ligand together in the presence and/or absence of a candidate modulator compound (for example, a candidate inhibitor compound or a candidate stimulator compound) and determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the presence and/or absence of the candidate compound. In such a method, the level of binding of the FGF23 ligand to the FGFR4 ligand or the binding characteristic of the FGF23 ligand/FGFR4 complex in the absence of the candidate compound represents a control value. If the level of binding of the FGF23 ligand to the FGFR4 ligand is decreased as compared to the control value or if a binding characteristic of the FGF23 ligand/FGFR4 ligand complex is decreased as compared to the control value, the candidate compound is an inhibitor compound. If the level of binding of the FGF23 ligand to the FGFR4 ligand is increased as compared to the control value or if a binding characteristic of the FGF23 ligand/FGFR4 ligand complex is increased as compared to the control value, the candidate compound is stimulator compound.

In a first embodiment, the method of screening described herein comprises: i) providing a FGF23 ligand; ii) providing a FGFR4 ligand and incubating the FGF23 ligand and the FGFR4 ligand for a period of time; iii) adding a candidate compound or a control solution; iv) incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound or the control solution for a period of time; v) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the absence of the candidate compound to determine a control value; vi) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic of the FGF23 ligand/FGFR4 ligand complex in the presence of the candidate compound to determine a test value; and vii) comparing the control value to the test value.

In a second embodiment, the method of screening described herein comprises: i) providing a FGF23 ligand; ii) adding a candidate compound or a control solution and incubating the candidate compound or the control solution with the FGF23 ligand for a period of time; iii) providing a FGFR4 ligand; iv) incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound for a period of time; v) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the absence of the candidate compound to determine a control value; vi) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic of the FGF23 ligand'F^'GFRd ligand complex in the presence of the candidate compound to determine a test value; and vii) comparing the control value to the test value.

In a third embodiment, the method of screening described herein comprises: i) providing a FGFR4 ligand; ii) adding a candidate compound adding a candidate compound or a control solution and incubating the candidate compound or the control solution with the FGFR4 ligand for a period of time; iii) providing a FGF23 ligand; iv) incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound for a period of time; v) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and'or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the absence of the candidate compound to determine a control value; vi) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic of the FGF23 ligand'FGFR4 ligand complex in the presence of the candidate compound to determine a test value; and vii) comparing the control value to the test value.

In any of the embodiments described herein, the FGF23 ligand, the FGFR4 ligand, or both the FGF23 and FGFR4 ligands may be bound to a solid support.

In a fourth embodiment, the method of screening described herein comprises: i) providing a FGF23 ligand, wherein the FGF23 ligand is bound to a solid support; ii) adding a candidate compound and incubating the candidate compound with the FGF23 ligand for a period of time; iii) providing a FGFR4 ligand; iv) incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound for a period of time; v) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the absence of the candidate compound to determine a control value; vi) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic of the FGF23 ligand/FGFR4 ligand complex in the presence of the candidate compound to determine a test value; and vii) comparing the control value to the test value. As would be obvious to the skilled person, steps ii) and iii) may be reversed if desired.

In an exemplary application of this embodiment, an ELISA-based assay is used. FGF23 ligand (for example, FGF23; SEQ ID NO: 3) is immobilized in the wells of a 96 well plate. Non-specific binding sites are blocked by incubation for 1 hour at room temperature with a blocking solution (such as PBS with 5% BSA, 0.1% Tween 20, and 0.25% nonidet-P40). The plate is washed with washing solution (such as 10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, and 0.25% nonidet-P40). The candidate compound is added to the wells of the plate at a desired concentration or range of concentrations and incubated for 30 minutes at room temperature. The plate is washed with washing solution (either with or without detergents). The FGFR4 ligand (such as human FGFR4 linked to a binding domain) is added and the plate incubated for 1 hour at room temperature. The plate is washed with washing solution and a detectable label that is a binding partner of the binding domain (for example, anti -human IgG conjugated to horseradish peroxidase, HRP) is added in blocking buffer and the plate is incubated for 1 hour at room temperature. The plate is washed with PBS and the substrate for the detectable label (such as TMB, 3,3',5,5'-tetramethylbenzidine when the detectable label utilizes HRP) is added and the plate incubated for 30 minutes at room temperature. The reaction is stopped by the addition of stopping solution (such as sulfuric acid for TMB/HRP) and absorbance determined using a standard plate reader via absorption at an appropriate wavelength (450 nM with TMB). Negative control wells (for example, an anti- FGF23 antibody known to disrupt FGF23-FGFR4 binding) and positive control wells (control solution/vehicle for the candidate compound only; total binding) may also be used. Absorbance values below the positive control value indicate the candidate compound is an inhibitor compound. Absorbance values above the positive control value indicate the candidate compound is a stimulator compound.

In a fifth embodiment, the method of screening described herein comprises: i) providing a FGFR4 ligand bound to a solid support; ii) adding a candidate compound and incubating the candidate compound with the FGFR4 ligand for a period of time; iii) providing a FGF23 ligand; iv) incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound for a period of time; v) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic (for example, a binding affinity of the FGF23 ligand to the FGFR4 ligand or vice versa) of the FGF23 ligand/FGFR4 ligand complex in the absence of the candidate compound to determine a control value; vi) determining the level of binding of the FGF23 ligand to the FGFR4 ligand and/or determining a binding characteristic of the FGF23 ligand/FGFR4 ligand complex in the presence of the candidate compound to determine a test value; and vii) comparing the control value to the test value. As would be obvious to the skilled person, steps ii) and iii) may be reversed if desired.

In an exemplary application of this embodiment, a flow cytometry protein interaction assay is used. This method can be used to evaluate protein-protein binding, calculate protein- protein interaction affinities and/or to perform competition experiments with unlabeled binding partners or small molecules. Beads are prepared (for example, LumAvidin beads) by washing three times in 1 mL of bead coupling buffer (BCB, PBS pH 7.4 supplemented with 1% bovine serum albumin) and resuspend in BCB. Biotinylated FGFR4 ligand (for example, 50-200 pmol of human FGFR4; SEQ ID NO: 1) is added to the bead solution, mixed, and incubated for 30 minutes at room temperature. The beads are washed three times with 1 mL of BCB and resuspend in 2 mL of flow buffer (FB, 50 mM HEPES pH 8.0, 100 mM NaCl, 0.1% Lubrol

PX, supplemented with 1% BSA). 50 mΐ of FB is added to all wells on the plate. Candidate compound is diluted to 2 ^c the final desired highest concentration in an appropriate buffer and incubated for 15 minutes at room temperature. Fluorescently labeled FGF23 ligand (for example, 10 pmol of human FGF23, SEQ ID NO: 3, labeled with AlexaFluor532) is added to each well and incubated for 30 minutes at room temperature. Negative control wells (for example, an anti-FGF23 antibody known to disrupt FGF23-FGFR4 binding and positive control wells (control solution/vehicle for the candidate compound only; total binding) may also be used. The results are analyzed on an appropriate flow cytometer (for example, a Luminex 200™ flow cytometer) to determine bead associated fluorescence collecting at least 100 events/well. Fluorescence values below the positive control value indicate the candidate compound is an inhibitor compound. Fluorescence values above the positive control value indicate the candidate compound is a stimulator compound. In any of the embodiments described herein, the FGF23 ligand may be FGF23 or an FGFR4 ligand binding portion of FGF23. In any of the embodiments described herein, the FGFR4 ligand may be FGFR4 or an FGF23 ligand binding portion of FGFR4.

In any of the embodiments described herein, the level of binding of the FGF23 ligand to the FGFR4 ligand and/or the binding characteristic of the FGF23 ligand/FGFR4 complex may be determined in the absence of the candidate compound to determine a control value. In any of the embodiments described herein, the level of binding of the FGF23 ligand to the FGFR4 ligand and/or the binding characteristic of the FGF23 ligand/FGFR4 complex may be determined in the presence of the candidate compound to determine a test value. If the test value is decreased as compared to the control value, the candidate compound is an inhibitor compound. If the test value is decreased as compared to the control value, the candidate compound is a stimulator compound.

In any of the embodiments described herein, the FGF23 ligand and the FGFR4 ligand bind to one another in the absence of any other protein or factor, specifically klotho or soluble klotho.

In any of the embodiments described herein, the FGF23 and FGFR4 ligands are each from the same species. In any of the embodiments described herein, the FGF23 and FGFR4 ligands are each human sequences. In any of the embodiments described herein, the FGF23 and FGFR4 ligands are each mouse sequences.

In any of the embodiments described herein, the FGF23 ligand is human FGF23 and the FGFR4 ligand is human FGFR4. In any of the embodiments described herein, the FGF23 ligand is human FGF23 and the FGFR4 ligand is a FGF23 ligand binding portion of human FGFR4. In any of the embodiments described herein, the FGF23 ligand is a FGFR4 ligand binding portion of human FGF23 and the FGFR4 ligand is human FGFR4. In any of the embodiments described herein, the FGF23 ligand is a FGFR4 ligand binding portion of human FGF23 and the FGFR4 ligand is a FGF23 ligand binding portion of human FGFR4. In any of the embodiments described herein, the FGF23 ligand is mouse FGF23 and the FGFR4 ligand is mouse FGFR4. In any of the embodiments described herein, the FGF23 ligand is mouse FGF23 and the FGFR4 ligand is a FGF23 ligand binding portion of mouse FGFR4. In any of the embodiments described herein, the FGF23 ligand is a FGFR4 ligand binding portion of mouse FGF23 and the FGFR4 ligand is mouse FGFR4. In any of the embodiments described herein, the FGF23 ligand is a FGFR4 ligand binding portion of mouse FGF23 and the FGFR4 ligand is a FGF23 ligand binding portion of mouse FGFR4.

In any of the embodiments described herein, at least one of the FGF23 or FGFR4 ligands is bound to a solid support and lacks a detectable label and/or a binding domain (such as to bind a detectable label) and the other of the FGF23 or FGFR4 ligands comprises a detectable label or a binding domain that binds a detectable label. In a preferred aspect of the fourth embodiment, the FGF23 ligand is bound to a solid support and lacks a detectable label and/or a binding domain and the FGFR4 ligand comprises a detectable label or a binding domain that binds a detectable label (for example, an enzyme conjugated antibody). In such an aspect, the binding of FGF23-FGFR4 may be detected using the detectable label on the FGFR4 ligand. Exemplary detectable labels are described herein. In a preferred aspect of the fifth embodiment, the FGFR4 ligand is bound to a solid support and lacks a detectable label and/or a binding domain and the FGF23 ligand comprises a detectable label or a binding domain that binds a detectable label (for example, an enzyme conjugated antibody). In such an aspect, the binding of FGF23-FGFR4 may be detected using the detectable label on the FGF23 ligand. Exemplary detectable labels are described herein.

In any of the embodiments described herein, the FGF23 and/or FGFR4 ligands may be linked to a binding domain that is capable of interacting with a binding partner. The binding domain may be used to bind a detectable label or to aid in the isolation of a complex of which the FGF23 and/or FGFR4 ligand is a member. A number of suitable binding domains are known in the art and may be used in conjunction with the FGF23 and/or FGFR4 ligands. Exemplary binding domains and binding partners include, but are not limited to, a magnetic moiety and a magnetic substrate, biotin/ desthiobiotin and avidin/streptavidin, protein (such as, but not limited to, immunoglobulin) domains and antibodies, histidine and nickel, FITC and anti- FITC, strep-tag II and strep-tactin, and digoxigenin and anti-digoxigenin. When the binding partner is used in detection, the binding partner may further comprises a detectable label.

A“detectable label” refers to molecule, protein, or compound which may be detected either directly or indirectly through the use of a suitable substrate or detection device. A “substrate” for a detectable label refers to a composition providing conditions suitable for detecting a detectable label. Such compositions may, for example, allow the generation and/or observation of a detectable signal, such as, but not limited to, a colorimetric, fluorescent, or chemiluminescent signal, when the detectable label is contacted with the substrate. Suitably detectable labels include, but are not limited to, a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, and a fluorescent protein. For example, the FGF23 and/or FGFR4 ligands may be biotinylated to bind a binding partner, such as a streptavidin-enzyme conjugate.

In the methods described herein, the FGF23 and/or FGFR4 ligands may be a fusion protein comprising the binding domain. In a particular aspect, the binding domain is a defined protein domain. In a particular aspect, the binding domain is an immunoglobulin domain, such as, but not limited to, an immunoglobulin domain comprising a constant portion of a light chain or heavy chain immunoglobulin region. In a particular aspect, the immunoglobulin domain comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain; a hinge domain, a CH2 domain, and a CH3 domain; a CH2 domain and a CH3 domain; a CH2 domain; or a CH3 domain from a light chain or heavy chain immunoglobulin. An exemplary fusion protein comprises a FGF23 ligand binding portion of FGFR4 (for example, amino acids 1 to 369 of SEQ ID NO: 1) linked to a heavy chain constant region from human IgGl comprising all or a portion of the hinge domain, the CH2 domain, and the CH3 domain. For example, the FGF23 and/or FGFR4 ligands may contain an immunoglobulin domain that binds a binding partner, such as an antibody-enzyme conjugate.

In the methods described herein, at least one of the FGF23 and/or FGFR4 ligands comprises a detectable label or a binding domain capable of binding a detectable label (i.e., the binding partner). In a particular aspect, the FGF23 or FGFR4 ligand that is not bound to a solid support comprises the detectable label or a binding domain capable of binding a detectable label. For example, at least one of the FGF23 and/or FGFR4 ligands may be directly labeled with an enzyme (such as alkaline phosphatase or HRP) or a fluorophore (such as AlexaFluor532). In one embodiment, at least one of the FGF23 and/or FGFR4 ligands may be provided as a fusion protein with a binding domain or be modified to contain a binding domain. When the binding domain is used to bind a detectable label, additional steps of the method may comprise incubating the FGF23 and/or FGFR4 ligand with a detectable label that binds the binding domain for a period of time, removing unbound detectable label, adding a substrate for the detectable label, and measuring the detectable signal (for example, obtaining an absorbance reading). Preferably, the detectable signal detected is directly proportional to the formation of a FGF23-FGFR4 complex.

In any of the embodiments described herein, at least one of the FGF23 ligand or FGFR4 ligand is provided linked to a solid support. In any of the embodiments described herein, each of the FGF23 ligand and FGFR4 ligand is provided linked to a solid support. In any of the embodiments described herein, the FGFR4 ligand is provided linked to a solid support. In any of the embodiments described herein, the FGF23 ligand is provided linked to a solid support.

In any of the embodiments described herein, the level of binding of the FGF23 ligand to the FGFR4 ligand in the presence of an inhibitor compound is 90% or less than the control value, 80% or less than the control value, 70% or less than the control value, 60% or less than the control value, 50% or less than the control value, 40% or less than the control value, 30% or less than the control value, 20% or less than the control value, 10% or less than the control value, or 5% or less than the control value.

In any of the embodiments described herein, the level of binding of the FGF23 ligand to the FGFR4 ligand in the presence of stimulator compound is 90% or greater than the control value, 80% or greater than the control value, 70% or greater than the control value, 60% or greater than the control value, 50% or greater than the control value, 40% or greater than the control value, 30% or greater than the control value, 20% or greater than the control value, 10% or greater than the control value, or 5% or greater than the control value.

In any of the embodiments described herein, the candidate modulator compound is added to the assay prior to the formati on of a binding complex the FGF23 ligand and the FGFR4 ligand. In any of the embodiments described herein, the candidate modulator compound is added to the assay after the formation of a binding complex the FGF23 ligand and the FGFR4 ligand.

In any of the embodiments described herein, the method may further comprise a washing step between one or more of steps of the method.

In any of the embodiments described herein, the method is conducted in solution. In any of the embodiments described herein, the method are performed ex vivo (for example, in vitro).

In any of the embodiments described herein, the period of time for the incubation steps is independently from 5 minutes to 5 hours, preferably 15 minutes to 1 hour.

In any of the embodiments utilizing a solid support, the solid support may be any solid support used in the art for the detection of proteins. In certain aspects, the solid support is manufactured from glass nitrocellulose, polyvinylidene difluoride, polyamide, polycarbonate, polyether, polymethyl methacrylate, nitrocellulose, nylon, polystyrene or polypropylene. Examples of solid support include, but are not limited to, plastic beads, agarose beads, magnetic beads, an antigen microarray or a microwell plate. Antigen microarray is a form of protein microarray, which is also known as a protein chip. A microarray is a solid support (typically glass) on which thousands of different proteins (for example, FGF23 ligand) are immobilized in discrete spatial locations, forming a high density protein dot matrix. A microwell plate is a flat plate with multiple "wells", where each well is used for one specific sample. The microwell plate is a standard tool in clinical diagnostic testing laboratories. A very common usage is in the enzyme-linked immunosorbent assay (ELISA). In certain aspects, the solid support is a bead. In certain aspects, the solid support is fluorescent bead. In certain aspects, the solid support is a chromatography resin.

The described method of detection provides significant advances over the prior art. The assay has high sensitivity, is applicable to a high-throughput format, and is cost-effective. Based on these advantages, the disclosed screening assay can be easily used to screen large numbers of candidate compounds.

FGF23 and FGFR4 Ligands

In certain aspects of the method of screening, the F GF23 ligand is full length F GF23. Such full length FGF23 may be from any species. In certain preferred aspects of the method of screening, the full length FGF23 has the sequence of SEQ ID NO: 3 (human) or SEQ ID NO: 4 (mouse). In additional preferred aspects of the method of screening, the FGF23 has a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ 1D NOS: 3 or 4, respectively, preferably 90% or greater or 95% or greater sequence identity.

In certain aspects of the method of screening, the FGF23 ligand is an FGFR4 ligand binding portion of FGF23. In a particular aspect of the method of screening described herein, the FGFR4 ligand binding portion of FGF23 is derived from human FGF23. In certain aspects, the human FGF23 has the sequence of SEQ ID NO: 3. In certain aspects, the FGFR4 ligand binding portion of FGF23 does not contain the signal peptide (amino acids 1-24 of SEQ ID NO: 3). In certain embodiments, the FGFR4 ligand binding portion of FGF23 is a portion of human FGF23 selected from Tables 1 A or 1B, or a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 1 A, preferably 90% or greater or 95% or greater sequence identity. In Table 1B, the 1 to 20 amino acids added to the amino terminal and/or carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the wild type polypeptide. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the method of screening described herein, the FGFR4 ligand binding portion of FGF23 is derived from mouse FGF23. In certain aspects, the mouse FGF23 has the sequence of SEQ ID NO: 4. In certain aspects, the FGFR4 ligand binding portion of FGF23 does not contain the signal peptide (amino acids 1-24 of SEQ ID NO: 4). In certain embodiments, the FGFR4 ligand binding portion of FGF23 is a portion of mouse FGF23 selected from Table 2A or 2B, or a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 2A, preferably 90% or greater or 95% or greater sequence identity. In Table 2B, the 1 to 20 amino acids added to the amino terminal and/or carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the wild type polypeptide. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In certain aspects of the method of screening, the FGFR4 ligand is full length FGFR4. Such full length FGFR4 may be from any species. In certain preferred aspects of the method of screening, the full length FGFR4 has the sequence of SEQ ID NO: 1 (human) or SEQ ID NO: 2 (mouse). In additional preferred aspects of the method of screening, the FGFR4 has a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NOS: 1 or 2, respectively, preferably 90% or greater or 95% or greater sequence identity⁷.

In certain aspects of the method of screening, the FGFR4 ligand is an FGF23 ligand binding portion of FGFR4. In a particular aspect of the method of screening described herein, the FGF23 ligand binding portion of FGFR4 is derived from human FGFR4. In certain aspects, the human FGFR4 has the sequence of SEQ ID NO: 1. In certain aspects, the FGF23 ligand binding portion of FGFR4 is an ectodomain portion of FGFR4 containing all or a portion of Ig G domains I-III (amino acids 22-349 of SEQ ID NO: 1 ) or IgG domains I-II (amino acids 22-240 of SEQ ID NO: 1). In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 1). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 370-390 of SEQ ID NO: 1).

In certain aspects, the ectodomain portion compri ses a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the FGF23 ligand binding portion of FGFR4 is a portion of human FGFR4 selected from Tables 3A and 3B, or a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 3 A, preferably 90% or greater or 95% or greater sequence identity. In Table 3B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the method of screening described herein, the FGF23 ligand binding portion of FGFR4 is derived from mouse FGFR4. In certain aspects, the mouse FGFR4 has the sequence of SEQ ID NO: 2. In certain aspects, the FGF23 ligand binding portion of FGFR4 is an ectodomain portion of FGFR4 containing all or a portion of IgG domains I-III or IgG domains l-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-16 of SEQ ID NO: 2). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 367-387 of SEQ ID NO: 2).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to

20 amino acids added to the amino and carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence.

ln certain aspects, the FGF23 ligand binding portion of FGFR4 is a portion of mouse FGFR4 selected from Tables 4A and 4B, or a sequence that shares 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 4 A, preferably 90% or greater or 95% or greater sequence identity. In Table 4B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a specific embodiment the following methodology is used to carry out the methods of the present disclosure. This embodiment is meant to illustrate the operation of the methods of screening disclosed herein only and not to be limiting to the materials and other limitations disclosed.

Briefly, a solid support (commercially available ELISA plates from Maxisorp) are coated with 5 pg/ml of recombinant FGF23 protein (R&D Systems). Non-specific binding sites are blocked by incubation with blocking buffer (for example, PBS containing 5% BSA and 0.1% Tween 20). The plate is washed with washing solution (such as 10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, and 0.25% nonidet-P40). Wells are incubated for one hour with 1.5 pg/ml of recombinant FGFR4-Fc (R&D Systems; SEQ ID NO: 5). FGFR4-Fc binds to FGF23 in the absence of klotho. After incubation with FGFR4-Fc, the wells are washed and incubated with 270 ng/ml of anti-human IgG conjugated to horseradish peroxidase (HRP; Jackson Labs). Wells are washed again and incubated with HRP substrate for about 30 minutes and the reaction stopped with PI2SO4. Each well is analyzed on a standard plate reader via absorptions at 450 nM to determine a control value. To screen for modulators of the FGF23-FGFR4 interaction, wells are incubated with a candidate modulator compound i) for 30 minutes prior addition of FGFR4-Fc; or ii) for 30 minutes after addition of FGFR4-Fc, and each well is analyzed on a standard plate reader via absorptions at 450 nM to determine a test value.

After a modulator compound (i.e., an inhibitor compound or a stimulator compound) is identified through the methods of the present disclosure, the identified modulator compound can be further examined. In one embodiment, the identified modulator compound is examined in an established in vitro or in vivo model system to determine the effects of the identified modulator compound the cellular effects of FGF23 activation of the FGFR4. Such in vitro or in vivo model systems include, but are not limited to, those model systems described in Faul et al. (J Clin Invest, 121(11), 4393-4408, 2011). Furthermore, the effect of the modulator compound of FGFR4 mediated signal transduction may also be examined in a suitable cell line (preferably one that does not express klotho, such as cardiomyocytes). Such effects include, but are not limited to, activation/inhibition of PLC'y. ealcineurin, and NFAT and increase/decrease in the production of inflammatory cytokines.

The described method of screening provides significant advances over the prior art. It aims to identify molecules that interfere with FGF23-FGFR4 binding, and not with FGFR kinase activity. As FGFR ectodomains are less conserved among receptor tyrosine kinases than their kinase domains, the method of screening will identify FGFR4 inhibitors with high isoform specificity. Furthermore, the method of screening has high sensitivity, provides quantitative read outs, is applicable to a high-throughput format, and is cost-effective. Based on these advantages, the method of screening can be easily used to screen large compounds libraries.

Polypeptide Derivatives

As discussed herein, polypeptide disclosed herein may be polypeptide derivatives containing one or more deletions, additions, and/or substitutions as compared to the corresponding wild-type sequence of the polypeptide. The deletions, additions and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired polypeptide derivative. With regard to deletions, a polypeptide derivative may be generated by deleting one or more amino acids from the n-terminal portion and/or the c- terminal portion of the amino acid sequence. With regard to substitutions, conservative substitutions or substitutions of amino acids with similar properties are expected to be well tolerated. Conservative modifications to the amino acid sequence is expected to produce a polypeptide derivative having functional and chemical characteristics similar to those of the wild-type polypeptide. For example, a“conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine.

Naturally occurring amino acid residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. For example, non conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol., 157: 105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +1-2 may be used; in an alternate embodiment, the hydropathic indices are with +/- 1; in yet another alternate embodiment, the hydropathic indices are within +/-0.5.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+- .1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (- 0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +1-2 may be used; in an alternate embodiment, the hydrophilicity values are with +/- 1; in yet another alternate embodiment, the hydrophilicity values are within +/-0.5.

Exemplary amino acid substitutions are set forth below.

Numerous scientific publications have been devoted to the prediction of secondary structure from analyses of amino acid sequences (see Chou et al, Biochemistry, 13 (2): 222-245, 1974; Chou et al, Biochemistry, 113(2):211-222, 1974; Chou et al, Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148, 1978; Chou et al, Ann. Rev. Biochem, 47:251-276, 1979; and Chou et al., Biophys. J., 26:367-384, 1979). Moreover, computer programs are currently available to assist with predicting secondary structure of polypeptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson et al, Comput. Appl. Biosci., 4(1): 181-186, 1998; and Wolf et al, Comput. Appl. Biosci., 4(1): 187-191 ; 1988), the program PepPlot.RTM. (Brutlag et al., CABS, 6:237-245, 1990; and Weinberger et al, Science, 228:740-742, 1985), and other new programs for protein tertiary structure prediction (Fetrow. et al., Biotechnology, 11 :479-483, 1993). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity or greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure (see Holm et al, Nucl. Acid. Res., 27(l):244-247, 1999). Modulator Compounds The present disclosure also provides for any modulator compound that is identified by the methods of screening of the present disclosure, as well as pharmaceutically acceptable forms, such as, but not limited to, pharmaceutically acceptable salts, of such modulator compound. Such modulator compounds may be polypeptides, antibodies, fragments of antibodies, recombinant polypeptides (either wild-type or containing a specific mutation), or small molecules, including organic small molecules. Such modulator compounds may be identified from a library of pre-existing compounds.

Methods of Treatment

The present disclosure provides for the use of the modulator compounds identified by the methods of the present disclosure in various methods of treatment. In one embodiment, the present disclosure provides for the use of an inhibitor compound identified by the methods of screening of the present disclosure in a method of treating a disease or condition associated with high FGF23 levels, increased FGFR4 levels, or increased FGF23 activation of FGFR4, the methods comprising administering an amount of an inhibitor compound identified by the screening methods of the present disclosure to a subject. Such diseases and conditions include, but are not limited to, aging, CKD and associated pathologies of CKD and aging, such as, but not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease. In a specific embodiment, the disease or condition is one disclosed in Schnedl et al. (Disease Markers, 2015:358086; doi 10.1155/2015/358086).

In one embodiment, the present disclosure provides for the use of a stimulator compound identified by the methods of screening of the present disclosure in a method of treating a disease or condition associated with low FGF23 levels, decreased FGFR4 levels or decreased FGF23 activation of FGFR4, the methods comprising administering an amount of a stimulator compound identified by the screening methods of the present disclosure to a subject. Such diseases and conditions include, but are not limited to, metabolic syndrome, increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and hypercholesterolemia.

In one embodiment, the present disclosure provides a method of treating CKD in a subject. Such method comprises the step of administering to the subject an amount of an inhibitor compound identified by the screening methods of the present disclosure.

In one embodiment, the present disclosure provides a method of treating aging in a subject. Such method comprises the step of administering to the subject an amount of an inhibitor compound identified by the screening methods of the present disclosure.

In another embodiment, the present disclosure provides method of treating a pathology or condition associated with CKD and/or aging in a subject (such as, but not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease). Such method comprises the step of administering to the subject an amount of an inhibitor compound identified by the screening methods of the present disclosure. In such an embodiment, the inhibitor compound may be administered prior to a formal diagnosis of CKD.

In one embodiment, the present disclosure provides a method of treating metabolic syndrome, increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, or hypercholesterolemia in a subject. Such method comprises the step of administering to the subject an amount of a stimulator compound identified by the screening methods of the present disclosure. In one embodiment, a method of treating a condition that arises from or is caused by an increased concentration of FGF23 is disclosed. In one embodiment, a method of treating a condition that arises from or is caused by an increased FGFR4 levels in a cell/tissue of interest is disclosed. In one embodiment, a method of treating a condition that arises from or is caused by an increased FGF23 activation of FGFR4 is disclosed. Such methods comprise the step of administering to the subject an amount of an inhibitor compound identified by the assays and/or methods of the present disclosure.

In one embodiment, a method of treating a condition that arises from or is caused by a decreased concentration of FGF23 is disclosed. In one embodiment, a method of treating a condition that arises from or is caused by a decreased FGFR4 levels in a cell/tissue of interest is disclosed. In one embodiment, a method of treating a condition that arises from or is caused by a decreased FGF23 activation of FGFR4 is disclosed. Such methods comprise the step of administering to the subject an amount of a stimulator compound identified by the assays and/or methods of the present disclosure.

In one embodiment, a method of treating a condition that arises from or is caused by a

FGFR4 polypeptide that comprises a mutation which leads to constitutive expression or overexpression is disclosed. Such method comprises the step of administering to the subject an amount of an inhibitor compound identified by the screening methods of the present disclosure.

In any of the foregoing methods of treatment, the modulator compound is administered in a therapeutically effective amount.

In any of the foregoing methods of treatment, the modulator compound is a small molecule/small organic molecule. In certain embodiments of the foregoing methods, the modulator compound is an antibody or a fragment thereof. In any of the foregoing methods of treatment, the modulator compound is a polypeptide. In any of the foregoing methods of treatment, the modulator compound comprises a recombinant polypeptide (either wild-type or containing a specific mutation). In any of the foregoing methods of treatment, the modulator compound is administered as a part of a pharmaceutical composition.

In certain embodiments of the foregoing methods, the modulator compound is administered alone or as a part of a pharmaceutical composition as described herein. A single modulator compound may be administered to a subject or a combination of modulator compounds may be administered to a subject. In certain embodiments of the foregoing methods, the subject is determined to be in need of such treatment prior to administration.

In certain embodiments of the foregoing methods, the subject may be a mammal. In certain embodiments of the foregoing methods, the subject is a human.

In any of the foregoing methods of treatment, a modulator compound, either alone or as a part of a pharmaceutical composition, may be administered to a subject by any route, including, but not limited to, intravenously, intraperitoneally, parenterally, intramuscularly or orally. In some embodiments, a modulator compound, either alone or as a part of a pharmaceutical composition, is administered intravenously. In some embodiments, a modulator compound, either alone or as a part of a pharmaceutical composition, is administered intraperitoneally. In some embodiments, a modulator compound, either alone or as a part of a pharmaceutical composition, is administered parenterally. In some embodiments, a modulator compound, either alone or as a part of a pharmaceutical composition, is administered intramuscularly. In some embodiments, a modulator compound, either alone or as a part of a pharmaceutical composition, is administered orally.

In any of the foregoing methods of treatment, the subjects treated can be further treated with one or more additional active agents that is known for the treatment of disease or condition (for example, CKD). The one or more additional active agents and the modulator compound(s) described herein or pharmaceutically acceptable salts or prodrugs thereof, can be administered together in a single composition or in separate compositions in any order, including simultaneous administration, as well as temporally spaced order of minutes to several days apart. The methods can also include more than a single administration of the one or more additional active agents and/or the modulator compound(s) described herein or pharmaceutically acceptable salts or prodrugs thereof. The administration of the one or more additional agents and the modulator compound(s) described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially.

Pharmaceutical Compositions

The modulator compounds described herein may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. The present disclosure provides a pharmaceutical composition comprising a modulator compound as described herein in admixture with a pharmaceutically acceptable carrier. The pharmaceutically-acceptable carrier must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically-acceptable carriers employed herein may be selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations and which are incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles and viscosity-increasing agents. Pharmaceutical additives, such as antioxidants, aromatics, colorants, flavor-improving agents, preservatives, and sweeteners, may also be added. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others. In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Often, the pharmaceutically acceptable carrier is chemically inert toward the active compounds and is non-toxic under the conditions of use. Examples of pharmaceutically acceptable carriers may include, for example, water or saline solution, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids, or alcohols. In some embodiments, the carrier is saline or water. In some embodiments, the carrier is saline. In some embodiments, the carrier is water.

Dosage and Administration

In accordance with the methods of the present disclosure, the compounds of the disclosure are administered to the subject (or are contacted with cells of the subject) in a therapeutically effective amount. This amount is readily determined by the skilled artisan, based upon known procedures, including analysis of titration curves established in vivo and methods and assays disclosed herein.

In certain embodiments, the therapeutically effective amount of a modulator compound as described herein ranges from about 0.01 mg/kg/day to about 500 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 400 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 300 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 200 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 100 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 50 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 25 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 20 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 15 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 10 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 5 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 2.5 mg/kg/day. In some embodiments, the therapeutically effective amount ranges from about 5 mg/kg/day to about 100 mg/kg/day. In some embodiments, the effective amount ranges from about 5 mg/kg/day to about 50 mg/kg/day. In some embodiments, the therapeutically effective amount ranges from about 5 mg/kg/day to about 30 mg/kg/day. In some embodiments, the therapeutically effective amount ranges from about 5 mg/kg/day to about 10 mg/kg/day.

In some embodiments, the therapeutically effective amount of a modulator compound as described herein is between about 0.1 mg/kg/day and about 50 mg/kg/day. In some embodiments, the therapeutically effective amount is between about 0.1 mg/kg/day and about 40 mg/kg/day. In some embodiments, the therapeutically effective amount is between about

0.1 mg/kg/day and about 30 mg/kg/day. In some embodiments, the therapeutically effective amount is between about 0.1 mg/kg/day and about 20 mg/kg/day. In some embodiments, the therapeutically effective amount is between about 0.1 mg/kg/day and about 10 mg/kg/day. In some embodiments, the therapeutically effective amount is between about 0.1 mg/kg/day and about 5 mg/kg/day.

In certain embodiments, the therapeutically effective amount is administered in one or more doses according to a course of treatment (where a dose refers to an amount of a compound of the invention administered in a single day). In certain embodiments, the dose is administered q.d. (1 time/administration per day). In certain embodiments, the dose is administered b.i.d. (2 times/administrations per day; for example, one-half of the therapeutically effective amount in two administrations a day). In certain embodiments, the dose is administered t.i.d. (three times/administrations per day; for example, one-third of the therapeutically effective amount in two administrations a day). When a dose is divided into multiple administrations per day, the dose may be divided equally or the dose may be divided unequally at each administration. Any given dose may be delivered in a single dosage form or more than one dosage form (for example, a tablet).

Kits

The present disclosure provides a kit comprising, consisting essentially of or consisting of at least one of a FGF23 ligand and'or a FGFR4 ligand, and one or more of the following: packaging material and instructions for carrying out an assay to identify a modulator (i.e., an inhibitor compound or a stimulator compound) of FGF23-FGFR4 binding.

The present disclosure further provides a kit comprising, consisting essentially of or consisting of FGF23 and FGFR4, and one or more of the following: packaging material and instructions for carrying out an assay to identify modulator (i.e., an inhibitor compound or a stimulator compound) FGF23-FGFR4 binding.

The present disclosure further provides a kit comprising, consisting essentially of or consisting of FGF23 and a FGF23 ligand binding portion of FGFR4, and one or more of the following: packaging material and instructions for carrying out an assay to identify a modulator (i.e., an inhibitor compound or a stimulator compound) of FGF23-FGFR4 binding.

The present disclosure provides a kit comprising, consisting essentially of or consisting of an FGFR4 ligand binding portion of FGF23 and FGFR4, and one or more of the following: packaging material and instructions for carrying out an assay to identify a modulator (i.e., an inhibitor compound or a stimulator compound) of FGF23-FGFR4 binding.

In any of the kits described above, one or more of the following may also apply: i) the kit further comprises a solid support; ii) the kit further comprises an additional reagent to determine the level of binding (such as, but not limited to, a detectable label and/or a substrate for the detectable label); iii) a positive control compound; iv) a negative control compound; or v) a combination of any of the foregoing.

In any of the kits described above, the FGF23, FGFR4, FGFR4 ligand binding portion of FGF23, and FGF23 ligand binding portion of FGFR4 may be any of the forms described in the present disclosure.

EXAMPLES

Example 1- Identification of Modulator Compounds

Preliminary data using FGFR4-specific blocking antibodies (compounds B and C in FIG. 2) as candidate inhibitor compounds support the sensitive and quantitative nature of the assay, and its ability to identify inhibitory actions of candidate modulator compounds on FGF23-FGFR4 binding.

The screening assay was conducted as follows. Commercially available ELISA plates (from Maxisorp) were coated with 5 pg/ml of recombinant FGF23 protein (R&D Systems). Non- specific binding sites were blocked by incubation with PBS containing 5% BSA and 0.1% Tween 20. Wells were incubated for one hour with 1.5 g/ml of recombinant FGFR4-Fc (R&D Systems SEQ ID NO: 5). After incubation with FGFR4-Fc, the w¾lls were washed and incubated with 270 ng/ml of anti-human IgG conjugated to horseradish peroxidase (HRP; Jackson Labs). FGFR4-Fc binds to FGF23 in the absence of klotho, and the formation of this dimeric complex is then measured by detecting the presence of the Fc domain that is coupled to FGFR4-Fc. Wells were washed again and incubated with HRP substrate for about 30 minutes and the reaction stopped with H2SO4. Each well was analyzed on a standard plate reader via absorptions at 450 nM. Compounds A and B (as well as two antibodies known to anti-FGFR4 antibodies, 6C9 and 3B6) were added to the assay 30 minutes prior addition of FGFR4-Fc. The results are presented in F1G. 2. As can be seen, the tested compounds decreased the interaction between FGF23-FGFR4 in comparison to control as indicated by a decrease in absorbance. As such, the compounds have been identified as inhibitor compounds.

Claims

CLAIMS What is claimed:

1. A method of identifying a modulator compound of fibroblast growth factor (FGF) 23 and fibroblast growth factor receptor (FGFR) 4 binding, the method comprising: a. providing a FGF23 ligand;

b. providing a FGFR4 ligand and incubating the FGF23 ligand and the FGFR4 ligand for a period of time;

c. adding a candidate compound or a control solution after step a) or step b) and incubating the FGF23 ligand, the FGFR4 ligand and the candidate compound or the control solution for a period of time;

d. determining a level of binding of the FGF23 ligand to the FGFR4 ligand, determining a binding characteristi c of a FGF23 ligand-FGFR4 ligand complex, or both, in the absence of the candidate compound to determine a control value; e. determining a level of binding of the FGF23 ligand to the FGFR4 ligand, determining a binding characteristi c of the FGF23 ligand-FGFR4 ligand complex, or both, in the presence of the candidate compound to determine a test value; and f. comparing the control value to the test value.

2. The method of claim 1, wherein the binding characteristic is a binding affinity of the FGF23 ligand for the FGFR4 ligand or a binding affinity of the FGFR4 ligand to the FGF23 ligand.

3. The method of claim 1, wherein the candidate compound is an inhibitor compound if the test value is less than the control value or the candidate compound is a stimulator compound if the test value is greater than the control value.

4. The method of claim 1 wherein the FGF23 ligand is bound to a solid support and the FGFR4 ligand comprises a detectable label.

5. The method of claim 4, wherein the determining steps are accomplished by detecting the detectable label.

6. The method of claim 1, wherein the FGF23 ligand is bound to a solid support and the FGFR4 ligand comprises a binding domain capable of binding a detectable label.

7. The method of claim 6, wherein the method further comprises adding the detectable label and he determining steps are accomplished by detecting the detectable label.

8. The method of claim 4, wherein the detectable label is a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, or a fluorescent protein.

9. The method of any one of claims 1 to 8, wherein the FGF23 ligand is FGF23 or a FGFR4 ligand binding portion of FGF23.

10. The method of claim 9, wherein the FGF23 ligand has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

11. The method of claim 9, wherein the FGF23 ligand has a sequence of:

a. amino acids 1 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

b. amino acids 25 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

c. amino acids 1 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

d. amino acids 25 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

e. amino acids 180 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4, or

a sequence at least 90% identical to any of the foregoing sequences.

12. The method of claim 9, wherein the FGF23 ligand has a sequence:

a. at least 90% identical to amino acids 25 to 251 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 25, 1 to 20 amino acids c-terminal to amino acid 251, or both 1 to 20 amino acid residues n-terminal to amino acid 25 and 1 to 20 amino acids c-terminal to amino acid 251;

b. at least 90% identical to amino acids 25 to 179 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 25, 1 to 20 amino acids c-terminal to amino acid 179, or both 1 to 20 amino acid residues n-terminal to amino acid 25 and 1 to 20 amino acids c-terminal to amino acid 179; or

c. at least 90% identical to amino acids 180 to 251 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 180,

1 to 20 amino acids c-terminal to amino acid 251, or both 1 to 20 amino acid residues n-terminal to amino acid 180 and 1 to 20 amino acids c-terminal to amino acid 251.

13. The method of any one of claims 1 to 8, wherein the FGFR4 ligand is FGFR4 or a FGF23 ligand binding portion of FGFR4.

14. The method of claim 13, wherein the FGFR4 ligand has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

15. The method of claim 13, wherein the FGFR4 ligand is an ectodomain portion of FGFR4 containing IgG domains I-III or IgG domains I-II.

16. The method of claim 13, wherein the FGFR4 ligand has the amino acid sequence of: a. amino acids 1 to 369 of SEQ ID NO: 1;

b. amino acids 22 to 369 of SEQ ID NO: 1 ;

c. amino acids 1 to 240 of SEQ ID NO: 1;

d. amino acids 22 to 240 of SEQ ID NO: 1 ;

e. amino acids 1 to 349 of SEQ ID NO: 1;

f. amino acids 22 to 349 of SEQ ID NO: 1

g. amino acids 1 to 366 of SEQ ID NO: 2

h. amino acids 17 to 366 of SEQ ID NO: 2;

i. amino acids 1 to 237 of SEQ ID NO: 2;

j. amino acids 17 to 237 of SEQ ID NO: 2

k. amino acids 1 to 346 of SEQ ID NO: 2;

l. amino acids 17 to 346 of SEQ ID NO: 2; or

or a sequence at least 90% identical to any of the foregoing sequences.

17. The method of claim 13, wherein the FGFR4 ligand has the amino acid sequence: a. at least 90% identical to amino acids 22 to 369 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 369, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 369; b. a sequence at least 90% identical to amino acids 22 to 349 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 349, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 349; c. a sequence at least 90% identical to amino acids 22 to 240 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 240, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 240; d. at least 90% identical to amino acids 17 to 366 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 366, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 366; e. at least 90% identical to amino acids 17 to 237 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n- terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 237, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 237; or f. at least 90% identical to amino acids 17 to 346 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1-20 amino acids c-terminal to amino acid 346, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1-20 amino acids c-terminal to amino acid 346.

18. A method of identifying a modulator compound of fibroblast growth factor (FGF) 23 and fibroblast growth factor receptor (FGFR) 4 binding, the method comprising: a. providing a FGFR4 ligand;

b. providing a FGF23 ligand and incubating the FGF23 ligand and the FGFR4 ligand for a period of time;

d. determining a level of binding of the FGF23 ligand to the FGFR4 ligand, determining a binding characteristic of a FGF23 ligand-FGFR4 ligand complex, or both, in the absence of the candidate compound to determine a control value; e. determining a level of binding of the FGF23 ligand to the FGFR4 ligand, determining a binding characteristic of the FGF23 ligand-FGFR4 ligand complex, or both, in the presence of the candidate compound to determine a test value; and f. comparing the control value to the test value.

19. The method of claim 18, wherein the binding characteristic is a binding affinity of the FGF23 ligand for the FGFR4 ligand or a binding affinity of the FGFR4 ligand to the FGF23 ligand.

20. The method of claim 18, wherein the candidate compound is an inhibitor compound if the test value is less than the control value or the candidate compound is a stimulator compound if the test value is greater than the control value.

21. The method of claim 18 wherein the FGFR4 ligand is bound to a solid support and the FGF23 ligand comprises a detectable label.

22. The method of claim 21, wherein the determining step is accomplished by detecting the detectable label.

23. The method of claim 18, wherein the FGFR4 ligand is bound to a solid support and the FGF23 ligand comprises a binding domain capable of binding a detectable label.

24. The method of claim 23, wherein the method further comprises adding the detectable label and the determining step is accomplished by detecting the detectable label.

25. The method of claim 21, wherein the detectable label is a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, or a fluorescent protein.

26. The method of any one of claims 18 to 25 wherein the FGF23 ligand is FGF23 or a FGFR4 ligand binding portion of FGF23.

27. The method of claim 26, wherein the FGF23 ligand has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

28. The method of claim 26, wherein the FGF23 ligand has a sequence of:

a. amino acids 1 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

b. amino acids 25 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

c. amino acids 1 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

d. amino acids 25 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

e. amino acids 180 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4, or

a sequence at least 90% identical to any of the foregoing sequences.

29. The method of claim 26, wherein the FGF23 ligand has a sequence:

a. at least 90% identical to amino acids 25 to 251 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 25, 1 to 20 amino acids c-terminal to amino acid 251, or both 1 to 20 amino acid residues n-terminal to amino acid 25 and 1 to 20 amino acids c-terminal to amino acid 251 ;

b. at least 90% identical to amino acids 25 to 179 of SEQ ID NOS: 3 or 4,

c. at least 90% identical to amino acids 180 to 251 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 180,

30. The method any one of claims 18 to 25, wherein the FGFR4 ligand is FGFR4 or a FGF23 ligand binding portion of FGFR4.

31. The method of claim 30, wherein the FGFR4 ligand has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

32. The method of claim 30, wherein the FGFR4 ligand is an ectodomain portion of FGFR4 containing IgG domains I-II1 or IgG domains I-II.

33. The method of claim 30, wherein the FGFR4 ligand has the amino acid sequence of: a. amino acids 1 to 369 of SEQ ID NO: 1;

b. amino acids 22 to 369 of SEQ ID NO: 1 ;

c. amino acids 1 to 240 of SEQ ID NO: 1;

d. amino acids 22 to 240 of SEQ ID NO: 1 ;

e. amino acids 1 to 349 of SEQ ID NO: 1;

f. amino acids 22 to 349 of SEQ ID NO: 1

g. amino acids 1 to 366 of SEQ ID NO: 2

h. amino acids 17 to 366 of SEQ ID NO: 2;

i. amino acids 1 to 237 of SEQ ID NO: 2;

j. amino acids 17 to 237 of SEQ ID NO: 2

k. amino acids 1 to 346 of SEQ ID NO: 2;

l. amino acids 17 to 346 of SEQ ID NO: 2; or

or a sequence at least 90% identical to any of the foregoing sequences.

34. The method of claim 30, wherein the FGFR4 ligand has the amino acid sequence: a. at least 90% identical to amino acids 22 to 369 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n- terminal to amino acid 22, 1 to 20 amino acids c-terminai to amino acid 369, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 369; b. a sequence at least 90% identical to amino acids 22 to 349 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 349, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 349; c. a sequence at least 90% identical to amino acids 22 to 240 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 240, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 240; d. at least 90% identical to amino acids 17 to 366 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n- terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 366, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 366; e. at least 90% identical to amino acids 17 to 237 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 237, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 237; or f. at least 90% identical to amino acids 17 to 346 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1-20 amino acids c-terminal to amino acid 346, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1-20 amino acids c-terminal to amino acid 346.

35. A method of using a stimulator compound identified by the method of claim 1 for treating a disease or condition associated with low FGF23 levels, decreased FGFR4 levels, or decreased FGF23 activation of FGFR4, the methods comprising administering an amount of the stimulator compound to a subject.

36. The method of claim 35, wherein the disease or condition is metabolic syndrome, increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, or hypercholesterolemia.

37. A method of using an inhibitor compound identified by the method of claim 1 for treating a disease or condition associated with high FGF23 levels, increased FGFR4 levels, or increased FGF23 activation of FGFR4, the methods comprising administering an amount of the inhibitor compound to a subject.

38. The method of claim 38, wherein the disease or condition is chronic kidney disease or aging.

39. The method of claim 38, wherein the disease or condition is high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, or blood vessel disease.

40. A kit comprising, aFGF23 ligand and aFGFR4 ligand, and one or more of the following: packaging material and instructions for carrying out an assay to identify a modulator compound of FGF23-FGFR4 binding.

41. The kit of claim 40, wherein the FGF23 ligand is FGF23 or a FGFR4 ligand binding portion of FGF23.

42. The kit of claim 40, wherein the FGF23 ligand has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

43. The kit of claim 40, wherein the FGF23 ligand has a sequence of:

a. amino acids 1 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

b. amino acids 25 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4;

c. amino acids 1 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

d. amino acids 25 to 179 of SEQ ID NO: 3 or SEQ ID NO: 4;

e. amino acids 180 to 251 of SEQ ID NO: 3 or SEQ ID NO: 4, or

a sequence at least 90% identical to any of the foregoing sequences.

44. The kit of claim 40, wherein the FGF23 ligand has a sequence:

a. at least 90% identical to amino acids 25 to 251 of SEQ ID NOS: 3 or 4,

b. at least 90% identical to amino acids 25 to 179 of SEQ ID NOS: 3 or 4,

c. at least 90% identical to amino acids 180 to 251 of SEQ ID NOS: 3 or 4,

optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 180,

45. The kit of claim 40, wherein the FGFR4 ligand is FGFR4 or a FGF23 ligand binding portion of FGFR4.

46. The kit of claim 40, wherein the FGFR4 ligand has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

47. The kit of claim 40, wherein the FGFR4 ligand is an ectodomain portion of FGFR4 containing IgG domains I-III or IgG domains I-II.

48. The kit of claim 40, wherein the FGFR4 ligand has the amino acid sequence of:

a. amino acids 1 to 369 of SEQ ID NO: 1;

b. amino acids 22 to 369 of SEQ ID NO: 1 ;

c. amino acids 1 to 240 of SEQ ID NO: 1;

d. amino acids 22 to 240 of SEQ ID NO: 1 ;

e. amino acids 1 to 349 of SEQ ID NO: 1;

f. amino acids 22 to 349 of SEQ ID NO: 1

g. amino acids 1 to 366 of SEQ ID NO: 2

h. amino acids 17 to 366 of SEQ ID NO: 2;

i. amino acids 1 to 237 of SEQ ID NO: 2;

j. amino acids 17 to 237 of SEQ ID NO: 2

k. amino acids 1 to 346 of SEQ ID NO: 2;

l. amino acids 17 to 346 of SEQ ID NO: 2; or

or a sequence at least 90% identical to any of the foregoing sequences.

49. The kit of claim 40, wherein the FGFR4 ligand has the amino acid sequence:

a. at least 90% identical to amino acids 22 to 369 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 369, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 369; b. a sequence at least 90% identical to amino acids 22 to 349 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 349, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 349; c. a sequence at least 90% identical to amino acids 22 to 240 of SEQ ID NO: 1, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 240, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 240; d. at least 90% identical to amino acids 17 to 366 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 366, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 366; e. at least 90% identical to amino acids 17 to 237 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n- terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 237, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 237; or f. at least 90% identical to amino acids 17 to 346 of SEQ ID NO: 2, optionally comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1-20 amino acids c-terminal to amino acid 346, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1-20 amino acids c-terminal to amino acid 346.

50. The kit of any one of claims 40 to 49, where the kit further comprises at least one of a solid support, a detectable label, a substrate for the detectable label, a positive control compound, or a negative control compound.

51. The method of claim 23, wherein the detectable label is a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, or a fluorescent protein.

52. The method of claim 6, wherein the detectable label is a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, or a fluorescent protein.