WO2009040512A2 - Modulation of rsk - Google Patents

Abstract

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.

Description

MODULATION QF RSK Field of the Invention

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.

Background to the Invention

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. Some responses, such as cytokine secretion, influence events in the external environment while others influence the capacity of the DC itself to capture, process and present antigen. For example, 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 ^π. DC vacuolar and cytoskeletal systems are also affected as well as protein synthesis and turnover. Whereas 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.

Besides the activation of transcription factors and some other substrates Erkl/2 and p38 phosphorylate and activate a group of kinases named MAP kinase activated protein kinases (MKs) which include MK2 and 3 (also known as MAPKAP- K2 and 3), the mitogen and stress activated kinases MSKl and 2, the MAP kinase interacting kinases MNKl and 2, the p90 ribosomal S6 kinases RSK 1, 2,3 and 4 (reviewed in ¹³-^14>15). Detailed studies in various cells have shown that while 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 and 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. In the case of MSK, the C-terminal domain activates the N-terminal¹⁶'²¹ 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.

The MKs have been shown to control a broad range of cellular functions. MK2 controls the stability and translation of TNFα 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. Thus Rsk emerges as a key mediator of TLR signalling and dendritic cell activation.

Summary of the Invention

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.

In accordance with a first aspect of the present invention there is provided use of a compound represented by the general structure (I):

wherein Ri is H or OH, and R₂, R₃, R₄, R₅ are independently selected at each position from the group consisting of H, OH, C₁-C₄ alkyl -OCOR₅, -COR₆, Ci-C₄ alkoxy, -O-glucoside and -O-rhamnoside, and R₆ is H or -CH₃ 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.

Preferably R₁ is OH and R₂, R₃ and R₄ is independently selected from the group consisting of hydroxy and -OCOCH₃ and R₅ is CH₃.

More particularly, in a preferment, the compound is as represented by formula

(HI)

In accordance with the invention a further suitable class of compounds is based on the compounds of formula (II).

wherein X is O or S; R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected, at each position, from C₁-C₆ alkyl, OH, H, NH₂, NO₂, H, halo; and R₇ is a substituted or unsubstituted aromatic ring, when substituted, the ring may be independently substituted at one or more positions by a Ci-C₆ alkyl, OH, NH₂, NO₂ or halo.

Preferably X is O.

Preferably R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected, at each position, from H, or Ci-C₆ alkyl. More preferably Ri, R₄ and R₆ are H and R₂, R₃ and R₅ are C₁-C₆ alkyl. More preferably Ri, R₄ and R₆ are H and R₂ and R₃ are methyl and R₅ is isopentyl.

Preferably R₇ is phenyl substituted by OH and/or halo. More preferably R₇ is phenyl substituted by F at positions 3 and 5 of the ring and by OH at position 4.

A most preferred compound in accordance with the above is the compound represented by formula (IV).

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. As is demonstrated in Examples section that follows, two representative compounds of the present invention (see formulae III and IV) 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.

Hence, according to another aspect of the present invention, there is provided uses and methods of treating 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 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. Takeda, Nature Reviews Immunology 4: 499 - 511 (2004); TLR signalling and the function of dendritic cells H. Hemi & S. Akira Chem. Immunol. Allergy 86: 120-135 (2005); and NOD-like proteins in immunity, inflammation and disease, Fritz J.H. et al Nature Immunology 7: 1250-1257 (2006)). 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. 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.

The term "administering" as used herein 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. As used herein, 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.

Herein, the term "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.

Herein, the term "preventing" refers to a method for barring an organism from acquiring a disorder or disease in the first place.

The term "therapeutically 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.

For any compound used in this invention, a therapeutically effective amount, also referred to herein as a therapeutically effective dose, can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ 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.

Moreover, 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 LD₅₀ and the ED₅₀. The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD₅₀ and ED₅₀. 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 ED₅₀ 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. Preferably, therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake, 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.

As used herein, 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.

As DCs are also involved in antigen presentation, 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. For example 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.

For use according to the present invention, 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.

Pharmaceutical formulations 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.

Pharmaceutical formulations 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.

Pharmaceutical formulations 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. Alternatively, 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.

As one possibility 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, and 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.

As a further possibility 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.

It should be understood that in addition to the aforementioned carrier ingredients 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.

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.

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. Alternatively, 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. In a further aspect there is provided 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.

In a further aspect there is provided 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.

In a further aspect 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.

Thus, in a further aspect there is provided 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, the method 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.

Typically, the cells may be dendritic or macrophage cells.

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that possess the ability to stimulate native T 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 naϊve 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. Indeed, 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).

Thus, 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.

It may be desirable for any compound identified using an assay of the present invention to be generally specific for the alternative pathway described herein. Thus, 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.

Contacting of the test compound with said DCs may be by any appropriate means. 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). In one preferred embodiment, 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. When screening for modulators, a positive assay result need not indicate that particular test agent is a good pharmaceutical. Rather, a positive test result can simply indicate that the 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.

In view of the identification in inflammatory response/immune response cells, such as dendritic cells and macrophages, of an alternative pathway comprising RSK, it will be understood that known compounds which may inhibit or activate enzymes of the conventional pathway (see Figure 6) may not be optimal in terms of affecting an inflammatory response/immune response, in view of the newly identified pathway.

Thus, in a further aspect there is provided use of 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.

For example, 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.

Detailed Description

The present invention will now be further described by way of example and with reference to the figures which show:

Figure 1 shows MK2/3 but not MSK1/2 or MNK1/2 are involved in TLR- induced macropinocytosis. (a) DCs left untreated or pre-treated with 2μM PD184352, 5μM SB203580 or O.lμM 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. (c,d) Left, phosphorylation of MSK1/2, MNKl/2 or MK2 in splenic DCs stimulated with 50 ng/ml LPS for 30 min in the presence of 2μM PDl 84352 or/and O.lμM BIRB796, as assessed by immunoblot with indicated antibodies. Data are representative of at least 3 independent experiments. Right, LPS-induced FITC-dextran uptake in DCs generated from wild-type (WT) mice or from mice lacking MSKl and MSK2 (MSKl, 2-DKO)₅ MNKl and MNK2 (MNKl, 2- DKO) ,MK2 (MK2-K0), MK3 (MK3-K0) or MK2 and MK3 (MK2,3-DKO). Data are the mean ± s.d. of triplicate stimulations. They are representative of 3 to 4 experiments performed with both BM or spleen DCs derived from 2 to 3 mice.

Figure 2 shows RSK inhibitors BI-D 1870 and SLOlOl block TLR-induced macropinocytosis. (a) Model of RSK activation by Erkl/2 and PDKl. Numbering corresponds to the mouse RSK2 sequence. CTKD: C-terminal kinase domain, NTKD: C-terminal kinase domain, (b) DCs left untreated or pre-treated with 2μM PD 184352, 5μM SB203580, 3μM BI-D1870 or 100 μM SLOlOl were stimulated with 50 ng/ml LPS or 100 ng/ml Pam3CSK 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, (c) FITC-dextran uptake in cells left untreated or stimulated with LPS in the absence or in presence of BI-D 1870 (bar 20 μm) 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 GSK3α/β in splenic DCs stimulated with 50 ng/ml LPS for 30 min in the presence of 3μM BI-D 1870 or lOOμM 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.

Figure 3 shows RSK activity in DCs is controlled by both Erkl/2 and p38 pathways, (a) RSK2 activity was measured in splenic DCs left untreated or stimulated with LPS for 30 min in the absence or presence of 2μM PD184352 or/and 5μM SB203580. Data are the mean ± s.d. of triplicates and are representative of at least 2 independent experiments, (b) LPS-induced phosphorylation of the four RSK phosphorylation sites (S356/T369, T573, S386 and S227) in splenic DCs left untreated or stimulated with 50 ng/ml LPS for 30 min in the absence or presence of 2μM PDl 84352 5μM SB203580 or 0.1 μM BIRB796. As a loading control membranes were re-probed with anti-RSK2. Data are representative of at least 4 independent experiments, (c) LPS-induced phosphorylation of S386 on RSKl, RSK2 and RSK3 immunoprecipitated from DCs treated as in b. Data in each panel are representative of at least 2 independent experiments.

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 37⁰C, 30 ng/ml PMA or 100 ng/ml anisomycin for 30 min at 37°C. Data in each panel are representative of at least 3 independent experiments.

Figure 5 shows MK2 and MK3 in p38-induced RSK(S386) phosphorylation in DCs. (a) Sequence alignment of the S386 site and the Erkl/2 -binding site of the four RSK isoforms. The MK2 recognition sequence is shown in the grey box. (b) Phosphorylation of RSK on T573 and S386, and of Erkl/2, p38 and CREB in wild- type (WT) or MK2,3-DKO BM (left) or splenic (right) DCs treated as in (a). Blots were reprobed to measure total RSK2, Erkl/2 and p38 as loading controls. Data are representative of at least 2 independent experiments, (c) 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-DKO₅ MNKl ,2-DKO, MK2-K0 or MK3-K0 DCs. (a) 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, (b) 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 37⁰C. 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 2μM PD 184352, 5μM SB203580 or O.lμM 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.

Figure 8 shows Rsk activation is regulated by both erkl/2 and p38 pathways in DCs. (a) Conventional pathway of Rsk activation induced by Erkl/2 in fibroblasts and other cell types (e.g. 3T3). (B) In DCs, Rsk activation is mediated not only by Erkl/2 but also by the p38 pathway through direct phosphorylation of Rsk on S386 by MK2 and Mk3. Thus 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.

Figure 10

The Rsk inhibitor Dl 870 blocks LPS-stimulated (TLR4) inflammatory cytokine production by bone marrow derived DCs (BMDCs). Panels show that production of IL-6 and IL12 following LPS stimulation of BMDCs is completely suppressed by 5μM D 1870 (panels a) and b)). Suppression is maintained at least up to 22h following stimulation. TNFa production is also partially suppressed (panel c).

Figure 11

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 5μM D 1870. Suppression is maintained at last up to 22h following stimulation.

Figure 12

The suppression of cytokine production in spleen DC is dependent on the concentration Rsk inhibitor used. IL-6 (panel a)), IL- 12 (panel b)) and TNFa (panel c)) production were measured following LPS stimulation of spleen DC in the presence of varying concentrations of D 1970.

Figure 13

The suppression of cytokine production in bone marrow DCs is dependent on the concentration Rsk inhibitor used. IL-6 (panel a)), IL- 12 (panel (b)) and TNFa (panel (c)) production were measured following LPS stimulation of bone marrow derived DC in the presence of varying concentrations of D 1970.

Figure 14

Rsk inhibitor treated, TLR-stimulated DC induce the development of Foxp3+ T cells. DC were treated with 5μM 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)).

Methods

Mice and cell culture. 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 MSK2⁵⁵, MKNl and MNK2 (kindly provided by C. Proud (University of British Columbia) and R. Fukunaga (Osaka University ³⁰)) or MK2²⁶. To generate MK3- deficient mice, 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'). Animal work was overseen by a local Ethical Review Committee and was conducted in accordance with UK Home Office Project Licences. 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-β (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. FITC-dextran uptake was measured as described previously ¹⁰. Briefly, 2 x 10⁵ 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 37⁰C. Cells were then stimulated with either 50 ng/ml LPS (Alexis) or 100 ng/ml Pam3CSK (EMC Microcollections) for 30 min at 37°C followed by the addition of 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.

Cell stimulation and cell lysate preparation. 10⁶ spleen DCs were incubated for 5 h at 37°C in RPMI containing 0.3% FCS in 6-well plates. Cells left untreated or treated with either DMSO or 2μM PDl 84352, 5μM SB203580, 0.1 μM BIRB796 or 3μM BI-Dl 870 for 30 min at 37°C were stimulated for 30 min with either 50 ng/ml LPS or lOOng/ml Pam3CSK for 30 min at 37°C or with 30 ng/ml PMA (Sigma) or 100 ng/ml anisomycin (Sigma) for 15 min at 37⁰C. 2 x 10⁴ 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 10⁶ cells in 400 μl 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 37⁰C, 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. In each experiment, 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). Membranes were probed with the following antibodies: 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-GSK3α/β (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 (clone C-20), MSKl (clone H- 19) were from Santa Cruz Biotechnology).

RSK kinase assay. 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-β-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 -8O⁰C. RSK2 was immunoprecipitated from 25 μg of lysates with 5 μg of anti- RSK2 (from D. Alessi, University of Dundee, UK) coupled to G-protein Sepharose (Amersham) for 30 min at 4°C. After 4 washes with buffer A (5OmM Tris-HCl pH7.5, 0.1 mM EGTA and 0.1% β-mercaptoethanol), the beads were resuspended in 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 2.5μM PKI (TTYADFIASGRTGRRNAIHD, peptide inhibitor of cyclic- AMP-dependent protein kinase), 1OmM magnesium acetate, 0.1 mM [_γ32p]ATP and Crosstide (GRPRTSSFAEG, 30μM) . The kinase assay was carried out for 30 min at 3O⁰C and then terminated and analysed as described previously ⁵⁷. To analyze phosphorylation of RSK Ser386 in vitro, 3T3 cells or 10⁷ splenic DCs in a 10-cm dish were starved as above and then lysed in lysis buffer. RSK2 was immunoprecipitated from 300 μg of cell lysate with 5 μg 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% β-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.

Analysis of SKl -3 Ser386 phosphorylation. Cells (2x10⁶) were stimulated and lysed as described above. RSKl, RSK2 or RSK3 were immunoprecipitated with antibodies specific for RSKl (clone C-21), RSK2 (clone E-I) or RSK3 (clone C-20) (Santa Cruz Biotechnology) coupled to G-protein Sepharose for 60 min at 4⁰C. After 4 washes, the beads were resuspended in SDS lysis buffer and the loaded on 4-12% NuPage gels as described above. Nitrocellulose membranes were probed with antibodies specific for p-RSK(S386).

Inflammatory cytokine production.

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: 2μM PD 184352, 5μM SB203580, O.lμM BIRBb796 3μM BH-Dl 870 or 100 μM 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. 5: 810 (1999) and is an inhibitor of MEK the kinase upstream of Erk. BIRB796 is first described in Pargellis et al Nat. Struct. Bio 9: 268- (20002) and is an inhibitor of p38 MAP kinase.

Alternatively, 70,000 SDC or BMDC were pre-incubated with different concentrations of BI-D 1870 (1, 3, 5 and 10 μM) for 30 min at 37C and then stimulated with either 50 ng/ml LPS, 1 μg/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 μl 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 lβ and IL-10 and the ELISA kit from Peprotech to detect IL- 6, IL-12 and TNFα.

T cell polarisation

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 μM peptide SIINFEKL (from Ovalbumin). After 24b. the cells were washed and co-cultured with naϊve 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

Kinase requirements of LPS-induced macropinocytosis

Stimulation of murine DCs with TLR ligands leads to a number of striking cell biological changes including transiently enhanced actin-dependent macropinocytosis measurable by uptake of FITC-dextran or enhanced antigen presentation ¹⁰. Preincubation with the p38 inhibitor SB203580, or PDl 84352, which targets the Erkl/2 activators MEK1/2, resulted in a 20% reduction in TLR-induced endocytosis; however, treatment with both inhibitors virtually eliminated the response (Figure Ia). Similar results were obtained when PD 184352 was used with the structurally unrelated p38 inhibitor BIRB0796 ³¹ (Figure Ia). These results suggested that this acute response to TLR signalling might be mediated by a downstream target common to both p38 and Erkl/2.

Many direct substrates of p38 and Erkl/2 are transcription factors but because the acute endocytic response occurs independently of de novo transcription¹⁰ we focused on MKs, the other major group of p38 and Erkl/2 substrates (Figure 2b). Only MSK1/2 and MNKl are known to be activated by both p38 and Erkl/2. Both MNK and MSKl were phosphorylated in cultured spleen DCs following LPS stimulation and this phosphorylation was reduced to baseline in the presence of both p38 and MEK1/2 inhibitors (Figure Ic). However there was no decrease, relative to wild-type DCs, in LPS-induced endocytosis of FITC-dextran by DCs established from the spleen and/or bone marrow of mice lacking MSKl and MSK2 or MNKl and MNK2 (Figure Ic). Thus, MSK1/2 and MNK1/2 were not required for acute TLR- induced endocytosis. Another possibility was that two different MKs, each downstream of either Erkl/2 or p38, could be involved. We first assessed whether MK2 and MK3, which are only activated by p38 (Figure Id), were required for LPS- stimulated FITC-dextran uptake. Although the response was largely intact in DCs lacking either MK2 or MK3, DCs lacking both MK2 and MK3 exhibited a blunted response (Figure Id). However, p38 amounts have been shown to be significantly lower in MK2/3 -deficient cells, and that may result in reduced TLR-induced endocytosis ³². Together these results rule out an obligatory role for the MSKs and MNKs and leave open the possibility that MK2 or MK3 might be involved in TLR- induced macropinocytosis. Next we examined the effect of ablating the activity of RSK kinases, the remaining class of MKs downstream of p38 and Erkl/2.

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. As PDKl is constitutively active ³⁵, 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. However the recent isolation and synthesis of RSK chemical inhibitors is an important development that has begun to allow dissection of its roles ³⁷'³⁸. We decided to test a particularly well characterized dihydropteridineone inhibitor of the N-terminal kinase domain of RSK . 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. When added to LPS- or Pam3CSK-stimulated spleen- or bone marrow-derived DC cultures BI-D 1870 potently inhibited endocytosis as measured by flow cytometry (Figure 2b) or by microscopy (Figure 2c). Notably, 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 μM and almost fully blocked at 5μM (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. SLOlOl also blocked LPS- stimulated endocytosis completely, although at higher concentrations than BI-D 1870 (Figure 2d). Neither compound interfered with activation of p38 or Erkl/2 (Figure 2e). In fact Erkl/2 activation by LPS was stronger in the presence of BI-D 1870 consistent with previous reports ' (Figure 2e and data not shown). As expected, phosphorylation of the known RSK substrate GSK3α/β was reduced to basal amounts in the presence of BI-D 1870 (Figure 2e). DCs utilize a novel pathway of RSK activation

The above results were initially puzzling as the TLR-driven response was virtually eliminated by two different inhibitors of RSK but not by inhibition of its upstream activator Erkl/2. To directly examine RSK activity, we immunoprecipitated RSK from DCs stimulated with LPS in the presence or absence of MAP kinase inhibitors. We focused on RSK2 because immunoprecipitating antibodies were readily available. RSK2 was potently activated by LPS in murine DCs as shown by the ability of immunoprecipitated RSK2 from stimulated DCs to phosphorylate a peptide substrate (Figure 3a). Notably, although the Erkl/2 inhibitor PDl 84352 substantially reduced the activity of RSK in this assay, significant activity remained that, surprisingly, was quenched when DCs were also exposed to the p38 inhibitor SB203580. In fact the p38 inhibitor suppressed RSK2 activity to some extent (Figure 3a). This result contrasts with those observed in other cell types where RSK activation is completely blocked by PDl 84352 but not at all by SB203580 ^I6'³⁸.

As noted above, 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°. We examined the phosphorylation status of Ser386 and of other sites on RSK in TLR-stimulated DCs and under conditions where the upstream kinases Erkl/2 and p38 were blocked. Phosphorylation of Ser386 was not detected in unstimulated DCs but was rapidly induced in the presence of LPS, indicating activation of the CTKD by Erkl/2 (Figure 3b). Consistent with this observation, LPS stimulated phosphorylation of the Erkl/2 target sites Ser369, Thr365, and Thr577 on RSK. However, whereas treatment with PDl 84352 blocked the phosphorylation of Ser369, Thr365 and Thr577, phosphorylation of Ser386 on RSK was surprisingly resistant to ERK1/2 inhibition (Figure 3b). However, treatment with PDl 84352 together with either SB203580 or BIRB0796 completely suppressed TLR-induced Ser386 phosphorylation. Enhanced phosphorylation of the PDKl target site Ser227 was similarly affected by the inhibitors, consistent with PDKl binding only to phospho-Ser386 (Figure 3b). Note that basal Ser227 phosphorylation does not lead to substantial RSK activity in the absence of Ser386 phosphorylation.

Next we asked whether all three of the inducible RSK isoforms were subject to this unusual mode of activation. DCs were activated by LPS and each isoform was immunoprecipitated and analysed separately for evidence of Erkl/2-independent modification of Ser386. Phosphorylation of Ser386 in all three RSK isoforms was inhibited only to a limited extent by PD 184352 alone but was completely blocked in the additional presence of a p38 inhibitor (Figure 3c). Analysis of Erkl/2- independent RSK Ser386 phosphorylation in DCs lacking MSK1/2 ruled out the possibility that our antibody was cross-reacting with phosphorylated MSK 1/2 which are closely related to RSK and are known to be activated by p38 (Figure 5a online)

These results indicate that LPS signalling in DCs can trigger a second mode of RSK activation whereby the normal requirement for Erkl/2 activation of the C- terminal kinase domain is bypassed. Instead a second input from the p38 MAP kinase pathway leads to Ser386 phosphorylation and RSK activation. This explains the SB203580-sensitive Erkl/2-independent RSK activity noted here and, presumably, why both PDl 84352 and SB203580 were required to quench RSK-dependent LPS- stimulated macropinocytosis (Figure Ia). Cell type-specific p38-dependent RSK activation

To determine whether the distinct mode of p38-mediated RSK activation is cell type- or stimulus-specific we compared RSK activation induced by different stimuli in DCs and in three other cell types. In 3T3 cells, EGF and anisomycin activated Erkl/2 and p38, respectively (Figure 4a). However, RSK was activated only by EGF and not by anisomycin. Moreover in 3T3 cells, RSK activation, as measured by Ser386 phosphorylation, was completely blocked by PD 184352 and unaffected by the p38 inhibitors SB203580 and BIRB0796. Similarly, in PMA- treated T cell blasts, phosphorylation of RSK on Ser386 was completely blocked by PD 184352 (Figure 4b). In mouse embryonic fibroblasts (MEF), tumor necrosis factor (TNF) and LPS activate both EEK1/2 and p38. However, RSK activation induced by either stimulus was fully blocked by PD 184352, demonstrating that p38 activation in fibroblasts, even when TLR-ligand driven, does not result in RSK activation (Figure 4c). hi DCs, consistent with the results already presented, the TLR2 ligand Pam3CSK activated RSK via both Erkl/2 and p38 pathways (Figure 4d). Moreover, in contrast to the situation in fibroblasts, the p38 activator anisomycin also activated RSK in DCs, albeit to a lesser extent than seen with PMA, a very potent Erkl/2 activator (Figure 4d). Taken together, these results show that the non- canonical p38-dependent pathway of RSK activation is cell type-specific rather than stimulus-specific.

We wanted to define the p38-mediated pathway of RSK activation in DCs more precisely. Unlike the related kinase MSK which is known to be activated by p38, the C-terminal region of RSK does not contain a p38 docking site. Nor is Ser386 followed by proline, the residue found adjacent in most p38 target sites ⁴¹. It therefore seemed unlikely that p38 was directly responsible for RSK activation and that, more likely, an enzyme(s) downstream of p38 (e.g. MSK1/2, MKNl/2 or MK2/3) activated RSK. These MKs have kinase domains homologous to the CTKD of RSK and their activities can be regulated by p38. However the fact that LPS-induced endocytosis was normal in DCs lacking MSKl and MSK2 or MNKl and MNK2 (Figure lc,d) made it unlikely that these kinases contributed to RSK activation. Indeed, phosphorylation of RSK Ser386 occurred in DCs generated from mice lacking MSKl and MSK2 or MNKl and MNK2 under conditions where Erkl/2 activity was blocked (Figure 5b). Notably, closer inspection of the RSK sequence around Ser386 revealed a perfect MK2/3 consensus phosphorylation site, 0XRXXS/T0, where 0 is a hydrophobic residue (Figure 5a). Moreover, as shown above (Figure Id), the LPS- stimulated endocytic response was blunted in MK2/3 -deficient DCs raising the possibility that the involvement of these MKs in RSK activation might be direct and not simply in stabilizing p38 MAP kinase. We therefore tested the possibility that MK2 and/or MK3, acting downstream of p38, were responsible for Erkl/2- independent RSK activation. We stimulated DCs lacking both MK2 and MK3 with LPS in the presence of PD 184352. Whereas in wild-type cells and in cells lacking either MK2 or MK3, phosphorylation of Ser386 on RSK persisted, DCs lacking both MK2 and MK3 failed to sustain this key phosphorylation event (Fig. 5b and Figure 6a). Consistent with data from other cell types ⁴², p38 quantities were lower in DCs lacking MK2 and MK3 (Figure 5b). However reduced p38 amounts were unlikely to account for the impaired RSK activation because p38-dependent activation of MNK and CREB (which occurs via MSK) was largely unaffected in the absence of MK2 and MK3 (Fig. 5b and Figure 6c). To show directly that MK2 can phosphorylate RSK Ser386 we incubated RSK2, immunoprecipitated from either spleen DCs or NIH3T3 cells, with purified active Erk or graded doses of active MK2. Purified PDKl was also added to allow analysis of the phosphorylation of Ser227. As expected the inclusion of active Erk led to phosphorylation of Thr577 in the CTKD of RSK and as a consequence of CTKD activation, to phosphorylation of Ser386 in the hydrophobic motif of NTKD (Figure 5c). Notably, addition of MK2 also led to Ser386 phosphorylation in a dose-dependent manner and in the absence of any CTKD Thr577 phosphorylation (Figure 5c). Because PDKl was also present, the addition of MK2 also led to increased Ser277 phosphorylation. Similar results were seen in DCs and NIH3T3 cells. Thus, as predicted by the foregoing data Ser386 was a direct target of MK2 and MK3. Taken together these results show that in DCs RSK is activated not only by the canonical Erk 1/2 pathway but also by p38 via either MK2 or MK3 (Figure 6).

Finally, if MK2/3 were responsible for Erkl/2-independent RSK activation, LPS-stimulated macropinocytosis should be completely sensitive to PD 184253 in DCs lacking MK2 and MK3. This was indeed the case (Figure 7). In contrast to the situation in wild-type cells and in DCs lacking only MK2, DCs lacking both MK2 and MK3 exhibited a complete block in LPS-induced uptake of FITC-dextran uptake in the presence of PDl 84352. As expected, p38 inhibitors were without effect in DCs lacking both MK2 and MK3. Evidence that RSK controls inflammatory cytokine production

To test the hypothesis that the "alternative" pathway, as well as the "conventional" pathway are directed through RSK and that RKS controls production of inflammatory cytokines, such as IL-6 and IL- 12, the inventors have tested inhibitors (see Figure 8a) of the two pathways and an inhibitor of RSK and observed their effect on inflammatory cytokine production. Figure 9b shows that inhibitors of each pathway alone are not as effective as when added together to dendritic cells, or as an inhibitor of RSK, in reducing production of the proinflammatory cytokines IL-6 and IL- 12.

In more detailed experiments over a longer time courses and testing different concentrations of the Rsk inhibitor Dl 870, profound suppression of production the inflammatory cytokines IL-6 and IL- 12 was observed. TNFa production was also partially blocked. This was true when LPS was used to stimulated BMDCs (Figure 10) and when other TLR ligands such as the TLR2 ligand Pam3CSK and the TLR7 ligand (R848) were used (Figure 11). Moreover, Dl 870 blocked production of these cytokines by spleen derived DC and in a concentration dependent manner (Figure 12). Almost complete inhibition of IL-6 and IL- 12 production was achieved at 3μM D 1870. In the case of BMDCs, slightly higher levels of Rsk inhibitor were required (~5μM) to achieve complete inhibition (Figure 13).

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). As it is known that 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 μM or lOμM antigenic peptide SIINFEKL. After 24h the cells were washed and co-cultured with naϊve 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.

DISCUSSION

Many MAP kinase-dependent TLR-driven responses in DCs are likely to be executed by MKs. However, to date, the role of MKs in DC responses to microbial stimuli has not been studied. Here, we investigated how a specific MAP kinase- dependent response is propagated in murine DCs by systematically testing the MKs downstream of p38 and Erkl/2 for their involvement in the rapid actin-dependent endocytic response triggered by TLR signalling.

Using 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. Whereas 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. Moreover, RSK was strongly implicated by our systematic analysis of DCs lacking all other known MKs.

Notably, RSK inhibition was equivalent to the combined effect achieved by SB203580 and PD184253. This result was unexpected because blockade of Erkl/2, the upstream activator of RSK produced only a modest inhibitory effect. We resolved this apparent contradiction by identifying a new configuration of the MAP kinase pathway in murine DCs. In DCs, phosphorylation of Ser386, the docking site for PDKl and allosteric activator of the NTKD of RSK²³, was incompletely suppressed by PDl 84352 suggesting that the Erkl/2-activated CTKD of RSK could be by-passed in response to TLR signalling. Ser386 phosphorylation was completely quenched, however, in the presence of Erkl/2 and p38 inhibitors. Several lines of evidence support the idea that p38-dependent RSK activation proceeds through MK2/3 in DCs. First, we observed some reduction in LPS-stimulated endocytosis in DCs lacking both MK2 and MK3. Second, blockade of the Erkl/2 pathway eliminated the LPS response in MK2/3 -deficient but not wild-type DCs. Third, the key PDKl recognition site in RSK, Ser386, is a perfect MK2/3 consensus phosphorylation site. Fourth, phosphorylation of this site was no longer observed when the Erkl/2 pathway was blocked in MK2/3 -deficient DCs. Fifth, purified 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 ^4Λ. However, to date, of the many cell types studied, only neutrophils appear to use MK2 to activate Akt ⁴⁵; this occurs via Hsp27, which mediates Akt-MK2 interactions ⁴⁶. Therefore an important question is why MK2 can phosphorylate the hydrophobic motif of RSK in DCs but not in other cell types examined here. Although RSK isolated from 3T3 cells and DCs could be phosphorylated by MK2 in vitro, RSK only became an MK2 substrate in DCs. These data suggest that DCs may express one or more 'scaffolding' proteins that permit MK2/3 to engage RSK and phosphorylate Ser386. RSK was not as strongly activated via the p38 pathway compared with canonical ERK-driven activation. Nonetheless, approximately 80% of the biological response, LPS- stimulated endocytosis, was produced when the Erk pathway was blocked, demonstrating that p38-driven RSK activation is physiologically important.

Whereas BI-Dl 870 and SLOlOl target the NTKD of RSK, other inhibitors, which target the CTKD of some RSK isoforms, have recently been described ³⁷. Notably, in LPS-stimulated macrophages, phosphorylation of Ser386 persisted in the presence of a CTKD inhibitor (fmk-pa), consistent with the proposal that a second pathway of RSK activation may exist ⁷. However, the pathway was not defined and nor was it clear what biological response in macrophages might utilize such a pathway. Although RSK3, whose CTKD is resistant to this inhibitor, might have accounted for some of the Ser386 phosphorylation seen in that study, our data suggest that the p38-MK2/3 pathway of RSK activation may also be active in macrophages and may account for the alternative pathway of RSK activation observed by Cohen et al⁴⁷.

What could be the significance of this dual mode of RSK activation in DCs? 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. By relaxing the normally exclusive role of Erkl/2 in RSK activation, stimuli that are weak Erkl/2 activators but strong p38 activators, could engage signalling programs downstream of RsSKin DCs. In other words, in 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. Other evidence suggests that the p38 and Erkl/2 pathways cross-regulate each other (e.g. in uninfected and Leishmania infected macrophages)⁴⁸. Indeed, consistent with data from RSK2-deficient cells ³⁶ and other cells treated with BI-D 1870 ³⁸ DCs treated with BI-Dl 870 showed enhanced Erkl/2 activation indicating that RSK can 'feed back' and suppress Erkl/2 activity ⁹. In addition, other cell types treated with p38 inhibitors exhibited an increase in Erkl/2 activation ⁵⁰ and our data suggest a potential alternative mechanism for this cross-inhibitory effect. Thus in DCs challenged with stimuli that strongly activate p38 but not Erkl/2, RSK activated by p38 might quench residual Erkl/2-driven pathways but leave intact the RSK pathway. Further dissection of the role of MKs in DCs is likely to refine our understanding of how DCs translate innate immune stimuli into specific responses that inform and regulate the adaptive immune system. How does RSK regulate TLR-mediated endocytosis? RSK is capable of phosphorylating many substrates but in the absence of specific inhibitors or a knockout of all four RSK isoforms it has been difficult to validate proteins as physiological RSK targets. Such validation should now be easier with the development of compounds like BI-D 1870. One previously documented RSK substrate potentially relevant to the LPS-induced macropinocytosis is the Na⁺/H⁺ exchange protein NHEl. In fibroblasts, growth factor-induced Na⁴VH⁺ exchange activity required RSK phosphorylation of Ser703 on NHEl ⁵¹, and macropinocytosis stimulated by EGF can be selectively blocked by the 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). Although it is not clear how 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 cells⁵⁴.

We also show that Rsk activation is required for the production of the inflammatory cytokines IL-6 and IL-12. Further studies are required to establish if this is due to a block in cytokine transcription, translation or secretion. The implications of this result are potentially very important. TLR-ligand activated DC bearing specific antigen are able to polarise T cell responses along different pathways. In essence, the production by DC of IL-6 and IL-12 promotes the differentation of T cells that are likely to have a pro-inflammatory phenotype ie. THl and THl 7 cells. Since inhibitors of Rsk selectively suppress the production of these cytokines, we tested the notion that regulatory T cells might more readily be induced. This indeed was the case. Other recent studies have used inhibitors of upstream kinases (p38 and Erk) to manipulate DC prior to their contact with T cells. For example, the treatment of DC with a p38 inhibitor suppressed DC production of IL-IO and promoted IL- 12 production (Jarnicki et al J.Immunol. 180: 3797 (2008). Consequently, the emergence of Tregs was suppressed and in vivo responses against a tumour challenge was boosted. Our study demonstrates that the opposite outcome may be achieved when Rsk is inhibited. We propose that TLR-activated DC additionally treated with Rsk inhibitors will promote anti-inflammatory outcomes in vivo, for example in inflammatory disease of the intestine. Targeting the kinases downstream of p38 and Erk (the MAP kinase activated kinases) of which Rsk is one, is likely to have more specific and targeted effects and will generally be more desirable than targeting p38 and Erk. The studies shown here demonstrate the potential of this appoach.

In summary here we linked specific acute and longer term TLR responses in DCs with a specific downstream MK. In addition, we showed that 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. Thus, in effect the cis-acting CTKD that is activated by Erkl/2 is by-passed and substituted by MK2 and/or MK3 which are related to the CTKD but activate RSK in trans rather than in cis and are themselves activated by p38. Perhaps CTKD shares a common ancestor with MK2 and MK3, and became fused to the NTKD of RSK to serve as its dedicated activator but in DCs, MK2 and MK3 still retain the ability to activate the NTKD. The NTKD of RSK is in effect a kinase activated by a MAP kinase activated kinase (KMK), which can be either its own CTKD or MK2/3. References

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Claims

1. Use of a compound represented by the general structure (I):

wherein R₁ is H or OH, and R₂, R₃, R₄, R₅ are independently selected at each position from the group consisting of H, OH, Ci-C₄ alkyl -OCOR₅, -COR₆, C₁-C₄ alkoxy, -O-glucoside and -O-rhamnoside, and R₆ is H or -CH₃ 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.

2. Use according to claim 1 wherein Rj is OH and R₂, R₃ and R₄ is independently selected from the group consisting of hydroxy and -OCOCH₃ and R₅ is CH₃.

3. Use according to claim 1 wherein the compound is as represented by formula (III)

4. Use of a compound represented by the general structure (II):

wherein X is O or S; R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected, at each position, from Ci-C₆ alkyl, OH, H, NH₂, NO₂, H, halo; and R₇ is a substituted or unsubstituted aromatic ring, when substituted, the ring may be independently substituted at one or more positions by a Ci-C₆ alkyl, OH, NH₂, NO₂ or halo, 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.

5. Use according to claim 4 wherein X is O.

6. Use according to claims 4 or 5 wherein Ri, R₂, R₃, R₄, R₅ and R₆ are independently selected, at each position, from H, or C₁-C₆ alkyl.

7. Use according to claims 4 or 5 wherein R₁, R₄ and R₆ are H and R₂, R₃ and R₅ are C₁-C₆ alkyl.

8. Use according to claims 4 or 5 wherein R₁, R₄ and R₆ are H and R₂, R₃ are methyl and R₅ is isopentyl.

9. Use according to claims 4 - 8 wherein R₇ is phenyl substituted by OH and/or halo.

10. Use according to claim 9 wherein R₇ is phenyl substituted by F at positions 3 and 5 of the ring and by OH at position 4.

11. Use of a compound according to claim 4 wherein the compound is represented by formula (IV)

12. Use according to any preceding claim for use in treating diseases associated with an undesirable inflammatory response and/or associated with an undesirable immune response, such as Rheumatoid arthritis, multiple sclerosis, osteoarthritis, chronic obstructive pulmonary (lung) disease, inflammatory bowel disease, psoriasis, arthroscelerosis, pelvic inflammatory disease, allergy, graft vs host disease, autoimmune disease.

13. Use of an RSK inhibitor or cell treated with an RSK inhibitor for treating 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.

14. Use according to claim 13 wherein the cell treated with an RSK inhibitor is additionally activated through a Toll-like receptor or other pathway such as a NOD-like receptor pathway or other pathway of pathogen sensing.

15. Use according to claim 13 or 14 wherein the inhibitor is selected from a compound as defined in any of claims 1 — 12.

16. Use according to claims 13 - 15 wherein the cell treated with an RSK inhibitor with or without additional stimulus is a dendritic or macrophage cell.

17. A cell which has been treated with an RSK inhibitor for use in treating 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.

18. The cell according to claim 17, which is a dendritic or macrophage cell.

19. Use of a compound capable of activating or enhancing RSK activity in the manufacture of a medicament for enhancing immune function.

20. A method of screening for modulators of RSK of an alternative RSK associated pathway in cells which produce an inflammatory response or are associated with an immune response, the method comprising the steps of: d) providing an inflammatory/immune response cell in which an Erkl/2 associated RSK activation pathway has been inhibited; e) contacting a test compound with said cell; and f) detecting if a RSK of said alternative pathway displays a modulation in activity in response to the addition of the test compound.

21. The method according to claim 20 further comprising contacting the test agent with a cell having a functional Erkl/2 associated RSK activation pathway, in order to ascertain the specificity or otherwise of the agent for the alternative RSK activation pathway.

22. Use of 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.