WO2009040512A2 - Modulation de rsk - Google Patents

Modulation de rsk Download PDF

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Publication number
WO2009040512A2
WO2009040512A2 PCT/GB2008/003221 GB2008003221W WO2009040512A2 WO 2009040512 A2 WO2009040512 A2 WO 2009040512A2 GB 2008003221 W GB2008003221 W GB 2008003221W WO 2009040512 A2 WO2009040512 A2 WO 2009040512A2
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rsk
pathway
cells
disease
cell
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PCT/GB2008/003221
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WO2009040512A3 (fr
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Colin Watts
Rossana Zaru
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University Court Of The University Of Dundee
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to specific compounds for use in controlling inflammatory cell function and their potential use in modulating an immune response or inflammation.
  • the present invention also relates to the identification of an alternative RSK pathway in, for example, dendritic cells and the development of assays for the identification of compounds capable of modulating the alternative RSK associated pathway in dendritic cells/macrophage cells and their potential use in enhancing, or controlling an immune response or in treating inflammation.
  • Microbial stimuli induce a set of responses in dendritic cells (DC) that enhance their performance as antigen presenting cells, stimulate their migration to lymphoid tissues and boost their ability to induce T cell activation 1A3 .
  • DC dendritic cells
  • TLR signalling induces enhanced acidification of antigen processing compartments, transport of class I and class II MHC molecules 5 ' 6 to the cell surface and the de-ubiquitination of the latter to ensure they remain stably expressed on the cell surface 7>8 .
  • TLR signalling also boosts antigen capture by both macropinocytosis and phagocytosis 9 ' 10 and influences protein turnover and storage of ubiquitinated proteins ⁇ .
  • DC vacuolar and cytoskeletal systems are also affected as well as protein synthesis and turnover.
  • the production of T cell polarising cytokines requires activation of the NFKB pathway, the IRF family of transcription factors and one or more of the major MAP kinase pathways 12 the signalling pathways that drive the internal DC maturation programme outlined above have not been defined.
  • the Erkl/2 and p38 MAP kinases are likely to be involved in most of these responses 9>10 but there is virtually no information on which downstream signalling pathways are involved.
  • MKs MAP kinase activated protein kinases
  • MSKl, MSK2 16 and MNKl 17 can be activated by both p38 and Erkl/2
  • MK2 and MK3 are exclusively activated by p38 and MNK2 17
  • RSK 1-3 16 ' 18 ' 19 are exclusively activated by Erkl/2
  • RSK4 appears to be constitutively active 2 .
  • the related MSK and RSK kinases have two distinct kinase domains: activation of the C-terminal domain (by upstream MAP kinases) is required for the N-terminal domain to be activated.
  • the C-terminal domain activates the N-terminal 16 ' 21 domain directly whereas in RSK the C-terminal domain phosphorylates a hydrophobic motif between the two domains creating a docking site for PDKl, a member of the AGC kinase family, which then activates the RSK N-terminal kinase domain 18 ' 22 .
  • MKs have been shown to control a broad range of cellular functions.
  • MK2 controls the stability and translation of TNF ⁇ mRNA " and the rearrangement of the actin cytoskeleton through hsp27 26 .
  • MSKl and 2 are involved in the regulation of transcription through CREB and ATFl I6 as well as in chromatin remodelling by phosphorylating histone H3 27 ' 28 .
  • MNKl and 2 regulate mRNA translation by phosphorylating the initiation factor eIF4E 29 .
  • RSK has been proposed to activate a wide variety of substrates implicated in the control of transcription, for example through the activation of c-fos and nur77, cell proliferation, cytoskeleton rearrangement, glycogen metabolism and cell survival (reviewed in ).
  • MKs are likely to be key agents in the propagation of p38- and Erkl/2-dependent responses in DC yet to date, their role has not been investigated. A more detailed picture of their contribution to TLR-signalled responses might permit selective modulation of the ability of DC to deliver particular immune responses.
  • the inventors recently described an acute response to TLR (toll like receptor) signalling in murine dendritic cells which involved a transient increase in actin- dependent antigen endocytosis (macropinocytosis) .
  • Phagocytosis is similarly transiently boosted by TLR ligation 9 .
  • This response serves to maximise antigen capture at the time of pathogen sensing and antigen presentation was boosted when antigen and a TLR stimulus were present simultaneously rather than sequentially 10 .
  • the response was observed when several different TLRs were stimulated and was extinguished by the commonly used inhibitors of MAP kinase signalling SB203580 and PD184352 which respectively block the p38 and Erkl/2 signalling pathway.
  • the inventors show that these combined inhibitors were exerting their effect by blocking two different pathways of activation of the downstream kinase Rsk.
  • Rsk emerges as a key mediator of TLR signalling and dendritic cell activation.
  • the present invention is based on the discovery that agents which activate dendritic cells and macrophages and which induce pronounced effects on the cells including the stimulation of macropinocytosis and the production of inflammatory cytokines, do so by activating the Rsk kinases. Since dendritic cell and/or macrophage activation is crucial to initiate pro-inflammatory immune responses, activation of such cells under conditions where Rsk is inhibited can either suppress an inflammatory response and/or divert the response into more desirable directions.
  • the present invention is also based in part on the identification by the inventors of a novel RSK associated pathway in dendritic cells and the potential use of certain inhibitory compounds in treating inflammatory disease.
  • the present inventors have identified compounds which display RSK inhibitory activity and block toll like receptor induced macropinocytosis and production of inflammatory cytokines.
  • Ri is H or OH
  • R 2 , R 3 , R 4 , R 5 are independently selected at each position from the group consisting of H, OH, C 1 -C 4 alkyl -OCOR 5 , -COR 6 , Ci-C 4 alkoxy, -O-glucoside and -O-rhamnoside
  • R 6 is H or -CH 3 for the manufacture of a medicament for treating a disease or condition associated with undesirable RSK activity in cells associated with the production of an inflammatory or immune response.
  • R 1 is OH and R 2 , R 3 and R 4 is independently selected from the group consisting of hydroxy and -OCOCH 3 and R 5 is CH 3 .
  • the compound is as represented by formula
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently selected, at each position, from C 1 -C 6 alkyl, OH, H, NH 2 , NO 2 , H, halo; and R 7 is a substituted or unsubstituted aromatic ring, when substituted, the ring may be independently substituted at one or more positions by a Ci-C 6 alkyl, OH, NH 2 , NO 2 or halo.
  • X is O.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently selected, at each position, from H, or Ci-C 6 alkyl. More preferably Ri, R 4 and R 6 are H and R 2 , R 3 and R 5 are C 1 -C 6 alkyl. More preferably Ri, R 4 and R 6 are H and R 2 and R 3 are methyl and R 5 is isopentyl.
  • R 7 is phenyl substituted by OH and/or halo. More preferably R 7 is phenyl substituted by F at positions 3 and 5 of the ring and by OH at position 4.
  • the abovementioned compounds find particular application in treating diseases associated with an inflammatory response or associated with an immune response, such as Rheumatoid arthritis, multiple sclerosis, osteoarthritis, chronic obstructive pulmonary (lung) disease, inflammatory bowel disease, psoriasis, arthrosclerosis, pelvic inflammatory disease, allergy, graft vs host disease, autoimmune disease.
  • diseases associated with an inflammatory response or associated with an immune response such as Rheumatoid arthritis, multiple sclerosis, osteoarthritis, chronic obstructive pulmonary (lung) disease, inflammatory bowel disease, psoriasis, arthrosclerosis, pelvic inflammatory disease, allergy, graft vs host disease, autoimmune disease.
  • two representative compounds of the present invention were tested for their RSK inhibition activity and showed significant potency. These compounds can therefore efficiently serve for treating diseases or disorders in which inhibiting the activity of RSK, would be beneficial, for example inflammation and/or a disease associated with an aberrant immune response.
  • an RSK related disease or disorder such as inflammation or a disease associated with undesirable inflammation, or a disease/condition associated with an undesirable immune response
  • uses and methods of treating an RSK related disease or disorder comprising the step of administering an RSK inhibitor compound or cells treated with an RKS inhibitor compound to a subject.
  • the cells may be treated with the inhibitor alone or may additionally be activated e.g by a Toll-like receptor ligand or other activator prior to administration to a patient (see for example Signalling to NFKb by Toll like receptors T. Kawai and S. Akira, Trends in Molecular Medicine 13: 460-469 (2007); Toll like receptor signalling S. Akira & K.
  • the compound may be one of the compounds identified herein, for example.
  • the method according to this aspect of the present invention is effected by administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention, as described hereinabove, either per se, or, more preferably, as a part of a pharmaceutical composition, mixed with, for example, a pharmaceutically acceptable carrier, as is detailed hereinunder.
  • a pharmaceutically acceptable carrier as is detailed hereinunder.
  • the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • administering refers to a method for bringing a compound of the present invention and a target kinase together in such a manner that the compound can affect the enzyme activity of the kinase either directly; i.e., by interacting with the kinase itself or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
  • administration can be accomplished either in vitro, i.e. in a test tube, or in vivo, i.e., in cells or tissues of a living organism.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease or disorder, substantially ameliorating clinical symptoms of a disease or disorder or substantially preventing the appearance of clinical symptoms of a disease or disorder.
  • preventing refers to a method for barring an organism from acquiring a disorder or disease in the first place.
  • terapéuticaally effective amount refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease or disorder being treated.
  • a therapeutically effective amount can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 or the ICJ OO as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data. Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans.
  • toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 and the ED 50 .
  • the dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell cultures assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, chapter 1, page 1).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for oral administration range from about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and most preferably from about 250-500 mg/kg/day.
  • therapeutically effective serum levels will be achieved by administering multiple doses each day.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • RSK related disease or disorder refers to a disease or disorder characterized by inappropriate RSK activity or over-activity of RSK or one of the enzymes of the alternative RSK pathway in DCs as identified herein.
  • Inappropriate activity refers to either; (i) kinase expression in cells which normally do not express said kinase; (ii) increased kinase expression leading to unwanted cell proliferation, differentiation and/or growth; (iii) decreased kinase expression leading to unwanted reductions in cell proliferation, differentiation and/or growth, or (iv) kinase expression is generally normal, but some other factor leads to aberrant kinase activity (v) kinase expression and activity are normal but some other factor makes this level of activity disease causing, e.g an increase in expression of one or more kinase substrates.
  • Over-activity of kinase refers to either amplification of the gene encoding a particular kinase or production of a level of kinase activity, which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the kinase increases, the severity of one or more of the symptoms of the cellular disorder increases).
  • Over activity can also be the result of ligand independent or constitutive activation as a result of mutations such as deletions of a fragment of a kinase responsible for ligand binding.
  • RSK activators may have potential application in augmenting an immune response and as such RSK activators (including other enzymes of the alternative pathway as identified herein) may be of use as an adjuvant during immunisation.
  • mixing of a vaccine and RSK activator according to the present invention may increase the immunogenicity of the vaccine.
  • the outcome of DC antigen presentation is the activation of T cells and their differentiation along various pathways (THl, TH2, TH17, Tregulatory etc). This 'T cell polarisation' dependes crucially on the cytokines being made by the DC in contact with the T cell which in turns depends on the conditions under which the DC was activated. Since the present invention enables the selective modulation of DC cytokine production by inhibition of Rsk, it becomes possible to modulate T cell polarisation by activated DC through the inhibition of Rsk.
  • the compounds or physiologically acceptable salt, ester or other physiologically functional derivative thereof, described herein may be presented as a pharmaceutical formulation, comprising the compounds or physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic and/or prophylactic ingredients.
  • the carrier (s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation.
  • the formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent.
  • Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored.
  • Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner.
  • Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope.
  • An active compound may also be formulated as dispersable granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
  • Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.
  • Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art.
  • the suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
  • compositions suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
  • Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use.
  • an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
  • An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly.
  • Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
  • Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
  • such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.
  • suitable liquid propellants include propane and the chlorofluorocarbons
  • suitable gaseous propellants include carbon dioxide.
  • Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.
  • Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
  • an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
  • Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
  • the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
  • a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
  • Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer.
  • a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.
  • a method of treating a patient suffering from a disease associated with inflammation comprising the step of administering to the subject an effective amount of an RSK inhibitor compound according to formula I, II, or III, or IV.
  • a method of augmenting an immune response to an immunogenic agent comprising the step of administering to the subject an effective amount of a RSK activating compound.
  • the present invention further provides use of an RSK inhibitory compound for the manufacture of medicaments for the treatment of diseases where it is desirable to inhibit RSK function, such as inflammation and immune diseases/conditions.
  • the present invention further provides use of compounds capable of activating or enhancing RSK activity in the manufacture of medicaments for enhancing immune function.
  • the invention provides for the preparation of a medicament comprising dendritic cells that have been activated or matured under conditions of Rsk inhibition for treating a disease or condition associated with an undesirable inflammatory or immune response.
  • the present inventors have also observed a novel pathway in dendritic cells and possibly other cells associated with inflammation/immune response, such as macrophages. Such a novel pathway allows for identification of modulators of the pathway.
  • a method of screening for modulators of RSK of an alternative RKS associated pathway in cells which produce an inflammatory response or are associated with an immune response comprising the steps of: a) providing an inflammatory/immune response cell in which an Erkl/2 associated RSK activation pathway has been inhibited; b) contacting a test compound with said cell; and c) detecting if a RSK of said alternative pathway displays a modulation in activity in response to the addition of the test compound.
  • the cells may be dendritic or macrophage cells.
  • DCs Dendritic cells
  • APCs potent antigen presenting cells
  • They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface.
  • DCs posses a heterogeneous haemopoietic lineage, in that subsets from different tissues have been shown to posses a differential morphology, phenotype and function.
  • the ability to stimulate na ⁇ ve T cell proliferation appears to be shared between these various DC subsets. It has been suggested that the so-called myeloid and lymphoid- derived subsets of DCs perform specific stimulatory or tolerogenic function, respectively.
  • DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens (Ags), and their subsequent presentation on the cell surface linked to major histocompatibility (MHC) molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present antigen to T cells and induce an immune response. DCs are of clinical and research interest due to their key role in anti-cancer host responses and potential use as biological adjuvants in vaccines, as well as their involvement in the immunobiology of tolerance and autoimmunity.
  • antigens Ags
  • MHC major histocompatibility
  • DC have been administered to patients in a number of clinical trials in attempts to 'kick-start' immune responses to cancer or otherwise modulate the immune response in man (see for example Palucka et al J.Immnunotherapy.29: 545- (2006); O'Rouke et al Cancer Immunol. Immunother. 52: 387 (2003).
  • the identification of agents which are capable of modulating Rsk activity by direct inhibition or the activity of one or both of Rsk's upstream activating pathways finds potential application in a variety of therapeutic aspects, such as in anti-cancer treatment, or immune stimulation, autoimmune treatment, including allergy and the like.
  • the method may further comprise contacting the test agent with a cell having functional Erkl/2 associated RSK activation pathway, in order to ascertain the specificity or otherwise of the agent for the alternative RSK activation pathway,
  • Figure 6 hereinafter discloses the "conventional" Erkl/2 associated RSK activation pathway and the alternative pathway identified by the present inventors.
  • Dendritic cells in which the Erkl/2 associated pathway has been inhibited may be provided by way of inhibiting the Erkl/2 pathway, using, for example, appropriate inhibitors (including small molecule chemicals and antibodies which may interfere with enzyme action), inhibiting expression of one or more of the enzymes in the Erkl/2 pathway, using RNAi or antisense technologies, or knocking out or down expression of one or more of the Erkl/2 pathway genes encoding for said Erkl/2 pathway enzymes (e.g upstream kinases), using appropriate gene knock-down/knockout techniques known in the art.
  • appropriate inhibitors including small molecule chemicals and antibodies which may interfere with enzyme action
  • RNAi or antisense technologies or knocking out or down expression of one or more of the Erkl/2 pathway genes encoding for said Erkl/2 pathway enzymes (e.g upstream kinases)
  • the compound may, for example, be added to the culture medium in which the DCs are suspended, or the cells themselves may be added to a solution comprising the test compound.
  • Other ways of contacting the test compound with the DCs may be envisaged by the skilled addressee.
  • Suitable compounds may be small molecule chemicals, peptide mimetics, or fragments of proteins of the alternative pathway, which may modulate enzyme activity.
  • Antibodies or antibody fragments against enzymes of the alternative pathway may also find application.
  • the detection step may simply be detecting whether or not the test compound binds to RSK of the alternative pathway, or has an effect on kinase activity (i.e. ability to phosphorylate a substrate).
  • a test compound may for example bind to the kinase itself and interfere with its activity, or bind to the substrate for the kinase and affect activity.
  • the detecting may be carried out via a method such as capillary electrophoresis, Western blot, means spectroscopy, ELISA, immunochromatography, or immunohistochemistry.
  • Binding of test compounds to the kinases can be tested by measuring or observing changes in kinase activity or by, e.g., changes in spectroscopic characteristics or in chromatographic or solubility properties. Binding of test compounds can also be ascertained in competitive binding assays, for example, by ascertaining whether unlabeled test compounds prevent the interaction between the kinase and a biotinylated or fluorescent derivative of a reference compound.
  • the assays that form an aspect of this invention may be designed to screen large chemical libraries for inhibition of one or more of the kinases using automated assay steps, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).
  • high throughput screening methods are used that involve providing a combinatorial chemical or other library containing a large number of potential inhibitory compounds. Such libraries are then screened in one or more assays, as described herein, to identify those library members (either particular chemical species or subclasses) that display the described activity.
  • a positive assay result need not indicate that particular test agent is a good pharmaceutical.
  • test agent can be used to inhibit activity of a kinase and/or can also serve as a lead compound in the development of other inhibitors.
  • the compounds thus identified may serve as conventional "lead compounds" or may themselves be used as potential or actual therapeutics.
  • an agent which is capable of modulating activity of an enzyme of the "conventional" pathway in inflammatory/immune response cells together with a further agent which is capable of modulating activity of an enzyme of the "alternative" pathway in inflammatory/immune response cells, for the manufacture of a medicament for treating or ameliorating a disease or condition associated with an undesirable inflammatory/immune response.
  • inhibitors of MK2 and/or MK3 would facilitate to block the "alternative" pathway.
  • the invention also extends to appropriate methods of treating inflammatory/immune conditions by administering to a subject compounds as described herein or cells treated with such compounds.
  • Figure 1 shows MK2/3 but not MSK1/2 or MNK1/2 are involved in TLR- induced macropinocytosis.
  • DCs left untreated or pre-treated with 2 ⁇ M PD184352, 5 ⁇ M SB203580 or O.l ⁇ M BIRB796 were stimulated with 50 ng/ml LPS for 30 min followed by 8 min incubation with lmg/ml FITC-dextran. Results are expressed as 'fold induction' (median intensity values) relative to FITC-dextran uptake in unstimulated cells. Data are the mean ⁇ s.d. of triplicate stimulations and are representative of 3 to 4 experiments, (b) Signalling cascades leading to the activation of MKs.
  • Figure 2 shows RSK inhibitors BI-D 1870 and SLOlOl block TLR-induced macropinocytosis.
  • NTKD C-terminal kinase domain
  • Results are expressed as 'fold induction' (median intensity values) relative to FITC-dextran uptake in unstimulated cells, (c) FITC-dextran uptake in cells left untreated or stimulated with LPS in the absence or in presence of BI-D 1870 (bar 20 ⁇ m) as determined by confocal microscopy.
  • Data are representative of 2 independent experiments performed in duplicate, (d) FITC-dextran uptake in DCs left untreated or stimulated with LPS for 30 min in the presence of increasing concentrations of BI-Dl 870 (left) or SLOlOl (right), (e) Phosphorylation of Erkl/2, p38 and GSK3 ⁇ / ⁇ in splenic DCs stimulated with 50 ng/ml LPS for 30 min in the presence of 3 ⁇ M BI-D 1870 or lOO ⁇ M SLOlOl.
  • Data are representative of at least 3 independent experiments.
  • Data for panels (b) and (d) are the mean ⁇ s.d. of triplicate stimulations and are representative of 3 to 4 independent experiments.
  • FIG. 3 shows RSK activity in DCs is controlled by both Erkl/2 and p38 pathways
  • Figure 4 Shows the p38 MAP kinase activation of RSK in DCs but not other cell types. Phosphorylation of RSK(S386), Erkl/2 and p38 was assessed in lysates from (a) NIH3T3 cells left untreated or treated with the indicated inhibitors, then stimulated with 100 ng/ml EGF (left) or 100 ng/ml anisomycin (right) for 10 min at 37°C, (b) T cell blasts left untreated or treated with the indicated inhibitors, then stimulated with 30 ng/ml PMA for 15 min at 37°C, (c) MEF left untreated or treated with the indicated inhibitors, then stimulated at 37°C with TNF (10ng/ml) for 15min or LPS (100ng/ml) for 30min, or (d) splenic DCs left untreated or treated with the indicated inhibitors, then stimulated with 100 ng/ml Pam3CSK for 30 min at 37 0 C, 30 ng/ml PM
  • Figure 5 shows MK2 and MK3 in p38-induced RSK(S386) phosphorylation in DCs.
  • RSK2 In vitro phosphorylation of RSK2(S386).
  • RSK2 was immunoprecipitated from lysates of splenic DCs (left) or 3T3 cells (right) and was incubated with p42 MAPK (Erk2) or MK2 as indicated for 45 min at 30°C. PDKl was also included. Phosphorylation of T577, S386 and S227 of RSK2 was assessed and blots were reprobed to measure total Rsk2 content as a loading control.
  • Data are representative of at least 2 or 3 independent experiments.
  • Figure 6 shows RSK phosphorylation in MSKl, 2-DKO 5 MNKl ,2-DKO, MK2-K0 or MK3-K0 DCs.
  • Splenic DCs from indicated mice were left untreated or treated with the indicated inhibitors, and were stimulated with 50 ng/ml LPS for 30 min at 37°C. Phosphorylation of Rsk on S386 was assessed and blots were re-probed to measure total Rsk2 content as a loading control.
  • Data are representative of 2 experiments performed on spleen DCs derived from 2 wild-type or MK KO mice,
  • Wild-type (WT), MK2-K0 or MK3-K0 BM DCs were left untreated or treated with the indicated inhibitors, and were stimulated with 50ng/ml LPS for 30 min at 37 0 C.
  • Phosphorylation of S386 of Rsk was assessed and blots were reprobed to measure total Rsk2 as a loading control
  • c Phosphorylation of MNK and MSK in WT or MK2,3-DKO BM (left) and splenic (right) DCs treated as in Figure 5. Blots were reprobed to measure total MNKl and MSKl content as loading controls.
  • Data are representative of at least 2 independent experiments.
  • Figure 7 shows an ERK pathway inhibitor is sufficient to block completely LPS-induced macropinocytosis in MK2,3-DKO DCs.
  • Wild-type (WT) or MK2,3- DKO DCs were left untreated or were pre-treated with 2 ⁇ M PD 184352, 5 ⁇ M SB203580 or O.l ⁇ M BIRB0796 and were stimulated with 50 ng/ml LPS for 30 min followed by 8 min incubation with lmg/ml FITC-dextran. Results are expressed as 'fold induction' (median intensity values) relative to FITC-dextran uptake in unstimulated cells. Data are the mean ⁇ s.d. of triplicate stimulations and are representative of at least 3 to 4 independent experiments performed in either BM or spleen DCs.
  • FIG. 8 shows Rsk activation is regulated by both erkl/2 and p38 pathways in DCs.
  • Rsk acts at two different levels in the MAP kinase pathway.
  • the potential negative feedback of Erkl/2 activation mediated by Rsk activated through the p38 pathway is also shown (dotted line) and discussed in the text.
  • Figure 9 shows schematically enzymes which are found within the "conventional” and “alternative” pathways, leading to RSK and identifies compounds which serve to inhibit particular enzymes.
  • Figure 9b shows inflammatory cytokine production following addition of inhibitors of enzymes of the "conventional” and “alternative” pathways and shows that RSK controls inflammatory cytokine production.
  • the Rsk inhibitor Dl 870 blocks LPS-stimulated (TLR4) inflammatory cytokine production by bone marrow derived DCs (BMDCs).
  • TLR4 LPS-stimulated inflammatory cytokine production by bone marrow derived DCs
  • Panels show that production of IL-6 and IL12 following LPS stimulation of BMDCs is completely suppressed by 5 ⁇ M D 1870 (panels a) and b)). Suppression is maintained at least up to 22h following stimulation. TNFa production is also partially suppressed (panel c).
  • the Rsk inhibitor Dl 870 blocks inflammatory cytokine production induced by a TLR2 (Pam3CSK) and a TLR7 (R848) ligand in bone marrow derived DCs (BMDCs).
  • Panels show that production of IL-6 and IL 12 following TLR2 (a) and b)) or TLR7 (c) and d)) stimulation of BMDCs is completely suppressed by 5 ⁇ M D 1870. Suppression is maintained at last up to 22h following stimulation.
  • IL-6 panel a
  • IL- 12 panel b
  • TNFa panel c
  • IL-6 panel a
  • IL- 12 panel (b)
  • TNFa panel (c)
  • TLR-stimulated DC induce the development of Foxp3+ T cells.
  • DC were treated with 5 ⁇ M D 1870 Rsk inhibitor or vehicle and activated with different TLR ligands as shown in Figure 14(a).
  • the DC were additionally loaded with the ovalbumin peptide SIINFEKL. After 24h the cells were washed and co- cultured with OTII T cells which recognise the SIINFEKL peptide. After 10 days during which the differentiating T cells were expanded with IL-2 they were restimulated with PMA and ionomycin and their phenotype analysed by FACS. Increased levels of T cells expressing the Foxp3 protein are induced when DC are treated with D 1870 (see Figure 14(b)).
  • DCs were generated from the spleen or the bone marrow of wild-type C57BL/6 or C3H/HeJ mice or from mice lacking MSKl and MSK2 55 , MKNl and MNK2 (kindly provided by C. Proud (University of British Columbia) and R. Fukunaga (Osaka University 30 )) or MK2 26 .
  • a targeting vector for was designed whereby exon 3, which encodes for subdomains III and IV forming the major part of the ATP-binding site of MK3, was replaced with a neomycin resistance cassette.
  • Flanking upstream (1.2kb) and downstream (4.5kb) sequences were present to permit homologous recombination.
  • ES cells that had integrated the vector were selected with G418 and those in which homologous recombination occurred were identified by PCR with the forward primer 5'-CAGAATAAAACGCACGGGTGTTGGGTCG-S' and the reverse primer 5'- GGTAGGGCCACCACAGCTTCATCCCAGAG-3'.
  • Blastocyst injections were carried out by the Transgenics service at the University of Dundee and were used to generate chimeric mice by standard methods.
  • mice lacking both MK2 and MK3 were generated by mating and were identified by PCR using the following primers: MK2 forward (5'-CAT GCC ATG ATG AGG TGC CTC TGC-3') and MK2 reverse (wild- type allele 5'-CCC TCT CTA CCT CTT TCT GTG AAT GCC-3' and KO allele 5'- CTG TTG TGC CCA GTC ATA GCC G-3') MK3 forward (5'-GCCAATGTCCCGC ATTATCTCTGC-3') and MK3 reverse (wild-type allele 5'-CAGGGAGCACTC ACAGAGCAGTGGGC-3' and KO allele (5'-CTG TTG TGC CCA GTC ATA GCC G-3').
  • MK2 forward 5'-CAT GCC ATG ATG AGG TGC CTC TGC-3'
  • MK2 reverse wild- type allele 5'-CCC TCT CTA CCT CTT TCT GTG AAT GCC-3' and KO all
  • DCs from mouse spleen or bone marrow DCs were cultured in complete RPMI supplemented with 10 ng/ml recombinant GM-CSF (Peprotech) and 1 ng/ml TGF- ⁇ (R&D Systems) or 10 ng/ml recombinant GM-CSF, respectively, as described previously 10 .
  • NIH3T3 cells (ECCC) were cultured in DMEM supplemented with sodium pyruvate, penicillin, streptomycin and 10% calf serum (Invitrogen).
  • Murine embryonic fibroblasts (MEF) were generated and maintained as described 55 .
  • T cells blasts were generated from C57BL/6 splenocytes as previously described 56 .
  • Dextran uptake was measured as described previously 10 . Briefly, 2 x 10 5 spleen or BM DCs where left untreated or were treated with PDl 84352 (made and analized as described in 5S ), SB203580 (Calbiochem), BIRB796 (made and analized as described in 31 ), BI-D 1870 (made and analized as described in 38 ) and SLOlOl (Toronto Research Chemicals Inc.) at the indicated concentrations for 30 min or Ih (SLOlOl) at 37 0 C.
  • FITC-dextran (Invitrogen) at a final concentration of lmg/ml for 8 min at 37°C.
  • Cells were washed four times at 4°C with PBS containing 0.2% FCS, stained with APC-labelled anti-CD 1 Ic (BD biosciences) at 4°C and FITC- dextran uptake was measured on a FACS Calibur (BD biosciences).
  • FITC-dextran uptake was also analyzed on a LSM510 confocal microscope (Zeiss) as described previously I0 .
  • 2 x 10 4 3T3 cells were plated in 6-well plates. After 48 h at 37°C, cells were starved for 8 h at 37°C in DMEM supplemented with 2mg/ml BSA then pre-treated with the inhibitors indicated above and then stimulated with either 100 ng/ml anisomycin or 100 ng/ml EGF (Peprotech) for 10 min at 37°C. T cell blasts were cultured overnight at 37°C in the absence of IL-2, re-suspended at a concentration of 10 6 cells in 400 ⁇ l of RPMI containing 0.5% FCS, and incubated for 30 min at 37°C.
  • Cells were pre-treated with the inhibitors mentioned above for 30 min at 37°C and then stimulated with 30 ng/ml PMA for 15 min at 37°C.
  • MEF were starved overnight at 37 0 C, pre-treated with inhibitors as above and then stimulated either with 10ng/ml TNF for 15 min or with 100 ng/ml LPS for 30 min at 37°C.
  • cells were washed once in cold PBS and either frozen or immediately lysed in SDS sample buffer. Equal amounts of proteins were loaded on 4-12% NuPage gels (Invitrogen) and then transferred onto nitrocellulose membranes (Amersham).
  • p-RSK (S227) was from R&D Systems (clone AF892), p-RSK (S386) (clone 9341), p-RSK (T573) (clone 9346), p-RSK (S356/S360) (clone 9348), p-Erkl/2 (clone 9101), p-p38 (clone 9211), p-GSK3 ⁇ / ⁇ (S21/S9) (clone9331), P-MK2 (T334) (clone 3041), p-MNKl/2 (T197/202) (clone 2111), p-MSKl (S376) (clone 2111), p-CREB (clone 9198) and MK2 (clone 3042) were from Cell Signalling and MNKl (clone G-19), RSK2 (clone E-I), ERK2 (clone C- 14), p38
  • Splenic DCs were stimulated as described above and then lysed in lysis buffer (1% TritonX-100 containing 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, and 0.1% 2- ⁇ -mercaptoethanol and 1 tablet of protease inhibitors (Roche)) for 10 min on ice. Lysates were centrifuged at 14,000 r.p.m. at 4°C for 15 min and the supernatant was frozen in liquid nitrogen and stored at -8O 0 C.
  • lysis buffer 1% TritonX-100 containing 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose
  • RSK2 was immunoprecipitated from 25 ⁇ g of lysates with 5 ⁇ g of anti- RSK2 (from D. Alessi, University of Dundee, UK) coupled to G-protein Sepharose (Amersham) for 30 min at 4°C.
  • the beads were resuspended in 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% ⁇ -mercaptoethanol, 2.5 ⁇ M PKI (TTYADFIASGRTGRRNAIHD, peptide inhibitor of cyclic- AMP-dependent protein kinase), 1OmM magnesium acetate, 0.1 mM [ ⁇ 32 p]ATP and Crosstide (GRPRTSSFAEG, 30 ⁇ M) .
  • buffer A 5OmM Tris-HCl pH7.5, 0.1 mM EGTA and 0.1% ⁇ -mercaptoethanol
  • the kinase assay was carried out for 30 min at 3O 0 C and then terminated and analysed as described previously 57 .
  • 3T3 cells or 10 7 splenic DCs in a 10-cm dish were starved as above and then lysed in lysis buffer.
  • RSK2 was immunoprecipitated from 300 ⁇ g of cell lysate with 5 ⁇ g of anti-RSK2 (Santa Cruz Biotechnology) coupled to G-proteiii Sepharose (Amersham) as described above.
  • the beads were resuspended in 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% ⁇ -mercaptoethanol, 1OmM magnesium acetate, ImM ATP, and 5U active p42 MAPK or 1, 5 or 1OU of active MK2 (kindly provided by the DSTT unit, Dundee). 2U/ml PDKl was added to all samples which were incubated at 30°C for 45 min. The reaction was immersed in SDS lysis buffer and then loaded on a 4-12% NuPage gel. Nitrocellulose membranes were probed with antibodies specific for RSK phosphorylated at Ser386, Thr577 or Ser227.
  • Mouse spleen dendritic cells were expanded in GMCSF and TGFbeta in accordance with West et al (2004). 70,000 cells were stimulated with 50ng/ml LPS and after 3 hours the supernatents were tested for cytokine production using standard ELISA methods (kits from Peprotec). The concentrations were: 2 ⁇ M PD 184352, 5 ⁇ M SB203580, O.l ⁇ M BIRBb796 3 ⁇ M BH-Dl 870 or 100 ⁇ M SOlOl (see Figure 2 legand) and there was a preincubation period of 30 minutes. PDl 84352 is originally described in Sebalt-Leopold et al Nature Med.
  • BIRB796 is first described in Pargellis et al Nat. Struct. Bio 9: 268- (20002) and is an inhibitor of p38 MAP kinase.
  • 70,000 SDC or BMDC were pre-incubated with different concentrations of BI-D 1870 (1, 3, 5 and 10 ⁇ M) for 30 min at 37C and then stimulated with either 50 ng/ml LPS, 1 ⁇ g/ml R848 or 100 ng/ml Pam3CSK for 1, 3, 6, 8 or 24h at 37C.
  • the experiments were performed in a 96-well plate in a final volume of 200 ⁇ l 5% FCS/RPMI.
  • the supernatant was collected and cytokine production was measured by ELISA using the BD OptEIA ELISA kit (BD biosciences) to detect IL l ⁇ and IL-10 and the ELISA kit from Peprotech to detect IL- 6, IL-12 and TNF ⁇ .
  • Spleen DC were activated with one of the following TLR ligands, LPS (TLR4), R848 (TLR7) or Pam3CSK (TLR2) in the presence or absence of D 1870. All cells were additionally incubated with 0.1 or 10.0 ⁇ M peptide SIINFEKL (from Ovalbumin). After 24b. the cells were washed and co-cultured with na ⁇ ve OTII T cells isolated from the spleens of OTII transgenic mice. IL-2 was added on days 3,5 & 8. On day 10 the T cells were activated with PMA and ionomycin and the fraction of cells expressing the Foxp3 protein was measured by FACS. RESULTS
  • RSK activation requires both Erkl/2 and PDKl which act sequentially along with the RSK C-terminal kinase domain (CTKD; Figure 2a) to activate the RSK N- terminal kinase domain (NTKD) 18>33>34 .
  • CTKD C-terminal kinase domain
  • NNKD N- terminal kinase domain
  • RSK activation is thought to be exclusively controlled by the activation of Erkl/2.
  • Analysis of RSK-dependent signalling has been hampered by the existence of four potentially redundant isoforms and the fact that, to date, only a RSK2-deficient mouse has been described 36 .
  • the recent isolation and synthesis of RSK chemical inhibitors is an important development that has begun to allow dissection of its roles 37 ' 38 .
  • BI-Dl 870 is a highly specific inhibitor of all RSK isoforms in vitro and in cellular assays of RSK function. Importantly, this compound did not block other kinases in a large (>50 kinases) test panel even at concentrations >100 fold higher than those needed to block RSK 38 .
  • BI-D 1870 potently inhibited endocytosis as measured by flow cytometry ( Figure 2b) or by microscopy ( Figure 2c).
  • BI-Dl 870 was as effective as the combination of SB203580 plus PDl 84352 and blocked both TLR4- and TLR2- induced endocytosis (Figure 2b). Endocytosis was half-maximally blocked in the presence of ⁇ 1 ⁇ M and almost fully blocked at 5 ⁇ M ( Figure 2d) BI-D 1870; these concentrations are similar to those required to block phosphorylation of the RSK substrate GSK3 in the human cell line HEK293 38 . To confirm the involvement of RSK we also tested SLOlOl, a second recently reported and structurally different RSK inhibitor purified from the tropical plant Forsteronia refi-acta 39 .
  • a key step in the activation of RSK is the phosphorylation of Ser386 (using mouse RSK2 amino acid numbering) in the linker domain catalysed by the Erkl/2 activated CTKD of RSK 33>4 °.
  • Ser386 using mouse RSK2 amino acid numbering
  • CTKD the linker domain catalysed by the Erkl/2 activated CTKD of RSK 33>4 °.
  • TLR-activated DC displaying cognate peptides to CD4 T cells are able to drive the differentation and expansion of those T cells along distinct pathways (THl, TH2, THl 7, Foxp3+ Treg).
  • IL-6 suppresses the generation of anti-inflammatory Treg cells in favour of frequently pro-inflammatory TH 17 cells (Bettelli et al Nature 441: 235- (2006)) and that IL- 12 promote THl differentiation we tested the possibility that suppression of Rsk activity and consequently IL-6 and IL- 12 production would promote the differentiation of Treg cells.
  • BMDC were stimulated with various TLR ligands in the presence or absence of the Rsk inhibitor Dl 870 and in the presence of either 0.1 ⁇ M or lO ⁇ M antigenic peptide SIINFEKL. After 24h the cells were washed and co-cultured with na ⁇ ve OTII T cells which recognise the SIINFEKL peptide. The cultures were supplemented with IL-2 at intervals and on day 10 the proportion of Treg cells in the culture measured by the expression of the transcription factor Foxp3. Cultures containing BMDC treated with Dl 870 while under TLR stimulation showed a strikingly increased proportion of T cells expressing the Foxp3 protein (Figure 14). This result demonstrates that the suppression of Rsk activity in TLR stimulated, antigen-presenting DC, confers on DC enhanced capacity to drive the differentiation of Foxp3+ T cells.
  • mice lacking MK2 and/or MK3 and two different RSK-specific inhibitors we identified RSK 1-3 and MK2/3 as the MKs that control TLR-induced macropinocytosis downstream of both p38 and Erkl/2 in DCs.
  • DCs lacking both MK2 and MK3 showed a partial attenuation of the response, inhibition of RSK resulted in a potent and complete blockade of TLR-induced endocytosis.
  • the use of two structurally distinct RSK inhibitors made unlikely the possibility that the observed altered TLR-induced endocytosis was due to non-specific effects of the inhibitors.
  • RSK was strongly implicated by our systematic analysis of DCs lacking all other known MKs.
  • MK2 was able to phosphorylate Ser386 on RSK in vitro. It is also worth stressing that MK2 and MK3 are phylo genetically very closely related to the CTKD of RSK 43 . Earlier data support the idea that MK2 can phosphorylate the hydrophobic motifs of AGC family kinases in vitro but that this only occurs in certain cells in vivo. For example, MK2 was able to phosphorylate a homologous site on the serine- threonine kinase Akt (Ser473) and to contribute to its activation in vitro 4 ⁇ .
  • RSK has been implicated in the regulation of a variety of important cellular processes in other cell types including cell cycle control, gene transcription and cell survival. Several of these are likely to be relevant in DCs activated by microbial products.
  • stimuli that are weak Erkl/2 activators but strong p38 activators could engage signalling programs downstream of RsSKin DCs.
  • p38 activators can access all the known downstream MK pathways. This feature is likely to be important in eliciting the full range of possible cellular responses in stimulated DCs.
  • RSK substrate potentially relevant to the LPS-induced macropinocytosis is the Na + /H + exchange protein NHEl.
  • NHEl inhibitor amiloride 52 More recently, amiloride and its analogues have been shown to block macropinocytosis in human DCs 53 and in LPS-treated murine DCs (data not shown).
  • NHEl function is involved in stimulated macropinocytosis, it is possible that NHEl may be a relevant target of RSK in this context.
  • a further potentially relevant substrate of RSK is the actin cross-linking protein filamin A, which is phosphorylated by RSK on Ser2152 in human melanoma cells 54 .
  • DCs utilize a novel and unexpected configuration of the MAP kinase pathway, in which RSK is activated not only by the canonical Erk 1/2 pathway but also by p38 via either MK2 or MK3.
  • RSK is activated not only by the canonical Erk 1/2 pathway but also by p38 via either MK2 or MK3.
  • MAPK-activated protein kinases a family of protein kinases with diverse biological functions. Microbiol MoI Biol Rev 68, 320-44 (2004).
  • Neininger, A. et al. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post- transcriptional levels. J Biol Chem. 277, 3065-8. Epub 2001 Dec 6. (2002).
  • Sapkota, G.P. et al. BI-D 1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo. Biochem J. 401, 29-38. (2007).
  • Mnks are novel components in the control of TNF alpha biosynthesis and phosphorylate and regulate hnRNP Al. Immunity 23, 177-89 (2005).
  • Takahashi, E. et al. p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. J Biol Chem 274, 20206-14 (1999).
  • Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products [see comments]. J-Exp-Med 182, 389-400 (1995).

Abstract

La présente invention concerne des composés spécifiques pouvant servir à la régulation de la fonction de cellules inflammatoires, ainsi que leur utilisation potentielle pour moduler une réponse immune ou une inflammation. La présente invention concerne également l'identification d'une autre voie RSK, notamment dans les cellules dendritiques, et le développement d'essais destinés à l'identification de composés capables de moduler l'autre voie RSK associée dans des cellules dendritiques/ cellules macrophages, ainsi que leur utilisation potentielle pour augmenter ou réguler une réponse immune ou pour traiter une inflammation.
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US11944625B2 (en) 2016-07-13 2024-04-02 The Children's Medical Center Corporation Calmodulin inhibitors, Chk2 inhibitors and RSK inhibitors for the treatment of ribosomal disorders and ribosomapathies
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JP2021523115A (ja) * 2018-05-04 2021-09-02 ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッドＴｈｅ Ｂｒｉｇｈａｍ ａｎｄ Ｗｏｍｅｎ’ｓ Ｈｏｓｐｉｔａｌ， Ｉｎｃ． 炎症性疾患または障害を治療するための方法および組成物
KR102272653B1 (ko) 2019-10-29 2021-07-05 순천대학교 산학협력단 항염증 활성을 나타내는 켐페롤-3-오-글루코실-6¨-오-펜타디오닉산 화합물 및 이를 유효성분으로 포함하는 항염증용 조성물
KR20210050718A (ko) * 2019-10-29 2021-05-10 순천대학교 산학협력단 항염증 활성을 나타내는 켐페롤-3-오-글루코실-6¨-오-펜타디오닉산 화합물 및 이를 유효성분으로 포함하는 항염증용 조성물

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