Biomarker of MK2 activity

Abstract

The present invention relates generally to the identification of a new biomarker for MK2 activity, and in particular methods of screening for inhibitors of MK2 and methods of measuring MK2 activity in a biological sample. Further, the invention also relates to kits suitable for use in said methods.

Description

[0001] BIOMARKER OF MK2 ACTIVITY

[0002] FIELD THE INVENTION

[0003] The present invention relates generally to the identification of a new biomarker for MK2 activity, and in particular methods of screening for inhibitors of MK2 and methods of measuring MK2 activity in a biological sample. Further, the invention also relates to kits suitable for use in said methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Breast cancer is the world’s most prevalent and most frequently diagnosed type of cancer and the number one reason for a loss of disability -adjusted life years in women (Fahad Ullah 2019, who.int 2022). In the year 2020, 2.3 million women were diagnosed with breast cancer, while an additional 685.000 related deaths were registered globally. Fortunately, the uprising breast cancer research in both molecular and pre-clinical context has improved the prognosis upon diagnosis drastically. But despite the huge progression with screening methods, diagnostic markers and therapeutic options, there are still points of concern that require extensive fundamental and applied biological research. One of these is the poor prognosis related to the aggressive triple-negative breast cancer (TNBC) subtype, another one is the arising resistance to drugs (who.int 2022, Nagini 2017, Vagia 2020).

[0006] In breast cancer, the nuclear receptors for estrogen (ER) and progesterone (PR) are well recognized to play part in tumor development, and the expression status of these receptors are used for classification and evaluation of treatment options for individual patients (Yip 2014 Future Oncol). The glucocorticoid receptor (GR), also a member of the nuclear receptor family, has traditionally not been linked to cancer development. GR is ubiquitously expressed, and it mediates the effects of its ligands, the glucocorticoids (GCs), which are natural stress hormones contributing to homeostasis by regulating glucose and lipid metabolism, immune responses, and brain functions (Buckingham 2006 Br J Pharmacol). GR is a transcription factor and cooperates with other transcription factors and co-factors to regulate gene expression (Ratman 2013 Mol Cell Endocrin). Synthetic GCs are widely used in the clinic for treatment of inflammation, severe asthma, and several lymphoid malignancies (Couthino 2011 Mol Cell Endocrinol, Barnes 1995 Nengl J Med, Inaba 2010 Lancet Oncol). In breast cancer therapy, GCs are routinely used to help patients to manage pain and side effects of chemotherapy and to stimulate appetite and energy levels (Kalfeist 2022 Cells, and references therein). In the last years, however, a potential contribution of GR and its ligands to cancer development within several tissues has been reconsidered, as several reports describe a link between high GR expression or GC concentrations and tumor development, metastasis, and poor prognosis (Tangen 2017 Gynecol Oncol, Puhr 2018 Clin Cancer Res, Huang 2017 Cancer Letters, Obradovic 2019 Nature, Mao 2020 Investig Clin Urol).

[0007] GR can be modified by post-translational modifications, of which phosphorylations are the most studied ones (Weikum 2017 Nat Rev Mol Cell Biol). The N -terminus of GR contains several phosphorylation sites, and their modification status will influence GR functions such as ligand binding, interaction with co -factors, scaffolding proteins, nuclear translocation, and transcriptional regulation (Irusen 2002, Wang 2002, Chen 2008, Nader 2010, Galiher-Beckley 2011, Bouazza 2014, Khan 2017). The Serine 134 (S134) phosphorylation site stands out by being phosphorylated independent of ligand binding and are mediated by an active p38MAPK pathway. Phosphorylation of GR at S134 is ligand independent but has profound effects on GR target genes after ligand stimulation, indicating that the level of cellular stress mediated by activation of the p38MAPK pathway will ultimately determine the response of cells and tissues to GCs (Galliher-Beckley 2011).

[0008] The p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway is activated by exogenous and endogenous stimuli such as stress factors or cytokines, ultimately leading to appropriate cell and tissue specific biological responses (Canovas and Nebrada 2021 Nat Rev Mol Cell Biol). p38 MAPK, similarly to glucocorticoid receptor (GR), is expressed in most human tissues and has gained research interest in regards of therapeutic options for various diseases within the last decades (Kumar 2003 Nat Rev Drug Discov, Dominguez 2005 Curr Opin Drug Discov Develop). It is involved in cancer-related processes such as cell proliferation, differentiation, migration, and survival (Canovas and Nebrada 2021 Nat Rev Mol Cell Biol). In triple-negative breast cancer (TNBC), the p38 kinase and its downstream effector mitogen-activated protein kinase-activated protein kinase-2 (MK2) have been connected to tumorigenesis and tumor progression (Chen 2021 NPJ Breast Cancer). In other breast cancer subtypes, p38 / MK2 signaling is involved in cancer early dissemination, later metastasis, and chemotherapy -involved bone loss, revealing chemical p38 inhibitors as potential therapeutic targets in breast cancer therapy (Wang 2020 Oncogene, Murali 2018 Cancer Res).

[0009] The clinical efficiency of small molecule p38 MAPK inhibitors, however, has been disappointing and their use in the clinic has been limited due to toxicity. This has resulted in efforts to focus more on the inhibition of key downstream targets of p38 such as MK2 (Martinez-Limon 2020 Int J Mol Sci). Additionally, several chemotherapeutic agents are known to activate the p38 MAPK pathway (Sanchez- Prieto 2000 Cancer Res, de Oleano 2012 Mol Cancer Res), and MK2 activity is reported to be involved in chemotherapy resistance, although the mechanism for this is unknown (Heijink et al 2019 Cell Reports). Clinical trials with oral MK2 inhibitors show good tolerance and promising effects for the treatment of rheumatoid arthritis (RA) and other immune-mediated inflammatory diseases. Moreover, MK2 inhibitors are currently also being evaluated for potential uses in cancer therapy (Fiore 2016 J Med Chem, Ray 2016 Int J Cancer, Guo 2019 Front Oncol). Hence, there is an urgent need in the art for identifying further MK2 inhibitors and methodologies for measuring MK2 inhibitor treatment effectiveness.

[0010] US 7,667,036 discloses a number of compounds, in particular pyrazolo[l,5-a] pyrimidine derivatives, exhibiting MK2 inhibitory activity. Further, a general procedure for determining MK2 inhibitory activity of a compound is also being disclosed. It is suggested that the inhibitory activity may be determined either by radiolabelling the compound prior to binding and then isolating the inhibitor-MK2 complex and determining the amount of the radiolabel bound; or by running a competition experiment where new inhibitors are incubated with MK2 bound to known radioligands. However, binding of a compound to MK2 does not necessarily mean that the MK2 activity is inhibited and radiolabelling is not suitable for all areas of application. Hence, there is still a need in the art for improved methods of measuring MK2 activity and MK2 inhibitor treatment effectiveness.

[0011] Summary of the invention

[0012] The present inventors have solved the above needs by having identified the glucocorticoid receptor (GR) as a downstream target for mitogen-activated protein kinase-activated protein kinase-2 (MK2) activity, and in particular that GR is phosphorylated at residue Ser 134 in response to MK2 activity.

[0013] In-vitro cell assay

[0014] A first aspect relates to an in-vitro method of screening for an inhibitor of MK2, comprising: a) incubating cells in the presence or absence of a potential MK2 inhibitor; b) measuring level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells; c) comparing the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the presence of the potential MK2 inhibitor; to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the absence of the potential MK2 inhibitor; wherein a higher measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the absence of the potential MK2 inhibitor relative to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in cells incubated in the presence of the potential MK2 inhibitor indicates that the potential MK2 inhibitor has MK2 inhibitory activity.

[0015] According to certain embodiments, the cells are human cells.

[0016] According to certain embodiments, the cells express MK2, GR and optionally p38 MAPK.

[0017] According to certain embodiments, the cells express MK2, GR and p38 MAPK.

[0018] According to certain embodiments, the cells have higher level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 when incubated in the absence of a MK2 inhibitor relative to level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 when incubated in the presence of a MK2 inhibitor.

[0019] According to certain embodiments, the cells are of a cell line selected from the group consisting of MDA-MB-231 (American Type Culture Collection (ATCC) Virginia, USA, HTB-26TM); MDA-MB-468 (ATCC, HTB-132TM); SKBR3 (ATCC, HTB-30TM); HCC1569 (ATCC, CRL-2330TM); BT-549 (ATCC, HTB- 122TM); T-47D (ATCC, HTB-133TM); A549 (ATCC, CRM-CCL-185TM); HEK- 293 (ATCC, CRL-1573TM); and Jurkat, Clone E6-1 (ATCC, TIB-152TM).

[0020] According to certain embodiments, the cells in step a) are incubated with a MK2 activation agent in the presence or absence of a potential MK2 inhibitor. In some embodiments, the cells express p38 MAPK and the MK2 activation agent is an agent that causes activation of p38 MAPK, such as Anisomycin.

[0021] In certain embodiments the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1. In some embodiments, the binding agent is p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0022] According to certain embodiments, the level of GR phosphorylated at residue Ser subjecting the incubated cells to lysis to obtain a cell lysate;

[0023] - reacting the cell lysate with a binding agent; and

[0024] - measuring the level of binding of the binding agent to the GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1. In some embodiments, the binding agent is p- GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0025] A second aspect relates to a kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, wherein the kit comprises:

[0026] - a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 , in particular p-GR(Serl34) mAb referred to in the materials and methods section of the present application;

[0027] - a MK2 activation agent, such as an agent that causes activation of p38 MAPK, for example Anisomycin;

[0028] - instructions for use of the kit components; and

[0029] - optionally a cell lysis buffer.

[0030] An alternative second aspect relates to a kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, wherein the kit comprises:

[0031] - a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 , in particular p-GR(Serl34) mAb referred to in the materials and methods section of the present application;

[0032] - a cell lysis buffer; and

[0033] - instructions for use of the kit components.

[0034] According to certain embodiments, the agent that causes activation of p38 MAPK is Anisomycin. In-vitro kinase assay

[0035] A third aspect relates to an in-vitro method of screening for an inhibitor of MK2, comprising:

[0036] - incubating GR, ATP and active MK2 in the presence or absence of a potential MK2 inhibitor;

[0037] - measuring the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition;

[0038] - comparing

[0039] - the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor; to

[0040] - the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor; wherein a higher measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor relative to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor indicates that the potential MK2 inhibitor has MK2 inhibitory activity.

[0041] According to certain embodiments, the active MK2 is obtained by incubating MK2 in the presence of a MK2 activation agent.

[0042] According to certain embodiments, the active MK2 is obtained by incubating MK2 in the presence of p38 MAPK and an agent that causes activation of p38 MAPK, such as Anisomycin.

[0043] According to certain embodiments, the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; the binding agent being an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1. In certain embodiments the binding agent is p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0044] A fourth aspect relates to a kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, e.g. suitable for use in the method according to the third aspect, wherein the kit comprises: i) MK2; ii) GR; iii) ATP; iv) a binding agent, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; and v) instructions for use of the kit components; wherein the MK2 is active MK2; or the kit further comprises a MK2 activation agent.

[0045] In certain embodiments the binding agent is p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0046] In certain embodiments the kit further comprises p38 MAPK and the MK2 activation agent is an agent that causes activation of the p38 MAPK, such as Anisomycin.

[0047] In some embodiments the active MK2 and the GR are in separate compartments and / or the MK2 and the MK2 activation agent are in separate compartments.

[0048] Use of a binding agent

[0049] A fifth aspect relates to use of a binding agent in an in-vitro method of screening for a MK2 inhibitor, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1. In some embodiments the binding agent is p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0050] A sixth aspect relates to use of a binding agent in an in-vitro method of measuring and / or visualizing MK2 activity in a sample, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1. In some embodiments the binding agent is p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0051] In some embodiments the sample is a tissue sample.

[0052] In some embodiments the MK2 activity is measured and / or visualized by immunohistochemistry staining of the sample, in particular a tissue sample, with the binding agent. A seventh aspect relates to use of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 as a biomarker for MK2 activity.

[0053] An eight aspect relates to an in-vitro method of monitoring MK2 activity in a tissue, the method comprising the following steps:

[0054] - measuring the level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in at least a first- and a second sample of the tissue; wherein the first sample has been taken from the tissue at a first point in time, the second sample has been taken from the tissue at a second point in time and the first point in time is different from the second point in time;

[0055] - comparing the measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the first sample to the measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the second sample; wherein

[0056] - a higher measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the first sample relative to the measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the second sample indicates that the MK2 activity is higher in the first sample relative to the second sample;

[0057] - a lower measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the first sample relative to the measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the second sample indicates that the MK2 activity is lower in the first sample relative to the second sample; and

[0058] - substantially equal measured level of glucocorticoid receptor phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the first- and second samples indicates no change in MK2 activity from the first point in time to the second point in time.

[0059] An ninth aspect relates to an in-vitro method of measuring MK2 activity in cells, the in-vitro method comprising the following step(s):

[0060] - measuring level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells; wherein

[0061] - the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells is positively correlated with the MK2 activity in the cells. In one embodiment, the cells are human cells.

[0062] In one embodiment the cells express p38 MAPK.

[0063] In one embodiment, the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.

[0064] In one embodiment, the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured by a method comprising the following steps:

[0065] - subjecting the cells to lysis to obtain a cell lysate;

[0066] - reacting the cell lysate with a binding agent; the binding agent being an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; and

[0067] - measuring the level of binding of the binding agent to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.

[0068] In one embodiment the binding agent is attached to a label, such as a visually detectable label. A non-limiting list of detectable labels are:

[0069] - horseradish peroxidase (HRP) and alkaline phosphatase (AP) which are enzymes that catalyse colour-changing reactions with substrates; a fluorescent chemical compound that absorb light at one wavelength and then emit light at a longer wavelength; and radiolabel that allows detection by its emitted radiation.

[0070] BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure la is a western blot showing that phosphorylation of GR at SI 34 is dependent on p38-MAPK in MDA-MB-231 cells.

[0072] The intensity of each band reflects total protein levels of phosphorylated GR (S134), GR, phosphorylated p38-MAPK, actin and p38-MAPK; after 30 minutes of treatment with Anisomycin, SB202190 or a combination of Anisomycin and SB202190; in MDA-MB-231 cells. Dexamethasone stimulation is used for comparison and actin protein staining as loading control.

[0073] Figure lb illustrates that phosphorylation of GR at SI 34 is dependent on p38- MAPK. SB202190 is a potent and selective inhibitor of p38-MAP kinases

[0074] Figure 2 is a western blot showing that both GR (S226) and GR (S211), but not GR (SI 34), are substrates for ERK2-MAPK, p38-MAPK, and JNK1-MAPK in an in- vitro kinase reaction.

[0075] The intensity of each band reflects total protein levels of phosphorylated GR (S226), phosphorylated GR (S211), phosphorylated GR (S134) and GR; after treatment with active ERK2-MAPK, active p38-MAPK or active JNK1-MAPK; at 30 °C for 0, 10 or 40 minutes in an in-vitro kinase reaction.

[0076] Figure 3 is a western blot showing that GR (S134) is a substrate for MK2 and that GR (S226) and GR (S211) are substrates for p38-MAPK in an in-vitro kinase reaction.

[0077] The intensity of each band reflects total protein levels of phosphorylated GR (S226), phosphorylated GR (S211), phosphorylated GR (S134) and GR; after treatment with active p38-MAPK or active MK2; at 30 °C for 0, 10, 20, 30 or 40 minutes in an in-vitro kinase reaction.

[0078] Figure 4a is a western blot showing that phosphorylation of GR at SI 34 is dependent on MK2 in MDA-MB-231 cells.

[0079] The intensity of each band reflects total protein levels of phosphorylated GR (SI 34), GR, MK2, phosphorylated Hsp27 and actin; after 30 minutes of treatment with Anisomycin, PF3644022, a combination of Anisomycin and PF3644022, dexamethasone, and a combination of dexamethasone and PF3644022; in MDA- MB-231 cells.

[0080] Figure 4b illustrates that phosphorylation of GR at SI 34 is dependent on MK2. PF3644022 is a potent and selective inhibitor of MK2.

[0081] Figure 5a is a western blot showing that phosphorylation of GR at SI 34 is dependent on MK2 in A549 cells (human lung carcinoma cell line A549). The intensity of each band reflects total protein levels of phosphorylated GR

[0082] (SI 34), GR, MK2, phosphorylated Hsp27 and actin; after 30 minutes of treatment with Anisomycin, Zunsemetinib, a combination of Anisomycin and Zunsemetinib, and dexamethasone; in A549 cells.

[0083] Figure 5b is a western blot showing that phosphorylation of GR at SI 34 is dependent on MK2 in HEK-293 cells (human embryonic kidney cell line HEK-293).

[0084] The intensity of each band reflects total protein levels of phosphorylated GR

[0085] (SI 34), GR, MK2, phosphorylated Hsp27 and actin; after 30 minutes of treatment with Anisomycin, Zunsemetinib, a combination of Anisomycin and Zunsemetinib, and dexamethasone; in HEK-293 cells.

[0086] Figure 5c is a western blot showing that phosphorylation of GR at SI 34 is dependent on MK2 in Jurkat cells (immortalized line of humane T-lymphocyte cells).

[0087] The intensity of each band reflects total protein levels of phosphorylated GR

[0088] (SI 34), GR, MK2, phosphorylated Hsp27 and actin; after 30 minutes of treatment with Anisomycin, Zunsemetinib, a combination of Anisomycin and Zunsemetinib, and dexamethasone; in Jurkat cells (immortalized line of humane T-lymphocyte cells).

[0089] DEFINITIONS

[0090] “GR” is used herein as an abbreviation for glucocorticoid receptor.

[0091] “p-GR” is used herein as an abbreviation for phosphorylated glucocorticoid receptor.

[0092] “GC” is used herein as an abbreviation for glucocorticoid.

[0093] 'DEX” is used herein as an abbreviation for dexamethasone. “MK2” is used herein as an abbreviation for “mitogen-activated protein kinase- activated protein kinase-2”.

[0094] MAPK is used herein as an abbreviation for mitogen-activated protein kinase. The terms “MAPK” and “MAP kinase” are used interchangeably herein.

[0095] Amino acids and their one-letter codes:

[0096] Alanine A Leucine L

[0097] Arginine R Lysine K

[0098] Asparagine N Methionine M

[0099] Aspartic acid D Phenylalanine F

[0100] Cysteine C Proline P

[0101] Glutamic acid E Serine S

[0102] Glutamine Q Threonine T

[0103] Glycine G Tryptophan W

[0104] Histidine H Tyrosine Y

[0105] Isoleucine I Valine V

[0106] The terms “p-GR”, “phosphorylated GR” and “phosphorylated glucocorticoid receptor” are used interchangeably herein.

[0107] When reference is made to a phosphorylated protein (such as phosphorylated glucocorticoid receptor) with a specific amino acid in brackets, it is the specific amino acid in brackets that is phosphorylated. Hence, the term “phosphorylated GR (SI 34)” refers to glucocorticoid receptor phosphorylated at a serine residue in position 134 of the glucocorticoid receptor.

[0108] Anisomycin is a potent activator of p38-MAPK (CAS-number 22862-76-6).

[0109] SB202190 is a potent and selective inhibitor of p38-MAP kinases (CAS-number 152121-30-7).

[0110] PF3644022 is a potent and selective inhibitor of MK2 (CAS-number 1276121-88- 0).

[0111] Zunsemetinib is an orally active and selective inhibitor of MK2 pathway (CAS- number 1640282-42-3).

[0112] Dexamethasone is a potent glucocorticoid which binds to the glucocorticoid receptor. The term “active MK2” refers to the form of MK2 that will phosphorylate GR at residue Ser 134 of SEQ ID NO: 1 in the presence of ATP.

[0113] A MK2 activation agent is an agent that causes MK2 activation either directly or indirectly. Anisomycin activates p38 MAPK and the active p38 MAPK activates MK2. Anisomycin is therefore to be considered a MK2 activation agent in the presence of p38 MAPK.

[0114] To be positively correlated means that as one variable increases, the other variable also increase - and as one decreases, the other decreases.

[0115] Sequence listing

[0116] DETAILED DESCRIPTION OF THE INVENTION

[0117] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of genetics, biochemistry, molecular biology and medicine.

[0118] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail.

[0119] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0120] It is well known that phosphorylation of GR at residue Seri 34 is independent of ligand binding and is mediated by an active p38MAPK pathway. This fact has been verified in example 1, where Anisomycin (potent activator of p38MAPK) was shown to cause an increase in GR phosphorylation at residue Serl34 (figure la, band 2A and 2C) while no such effect (figure la, band 4A and 4C) was provided by a combination of Anisomycin and SB202190 (inhibitor of p38MAPK). p38MAPK has a number of downstream targets, including MK2, but it is not known if active p38MAPK per se is able to phosphorylate GR at residue Serl34 or if the phosphorylation is actually caused by one of the downstream targets.

[0121] In order to investigate whether active p38MAPK per se was able to phosphorylate GR at residue Seri 34, the inventors designed an in-vitro kinase assay where recombinant active p38MAPK was incubated in the presence of GST-GR fusion protein and ATP (example 2). As shown in figure 2, in particular lanes 4 and 5 which are positive for active p38MAPK, it was not detected any GR that was phosphorylated at residue Ser 134. Hence, active p38MAPK per se is not causing any phosphorylation of GR at residue Seri 34.

[0122] Based on the knowledge that phosphorylation of GR at residue Serl34 is mediated by an active p38MAPK pathway, and that active p38MAPK per se is not sufficient to cause phosphorylation of GR, it is plausible to assume that the responsible agent is one of the downstream targets of p38MAPK. Based on the hypothesis that MK2 may be the responsible agent, the inventors designed an in-vitro kinase assay where recombinant active MK2 was incubated in the presence of GST-GR fusion protein and ATP (example 3). As shown in figure 3, in particular lanes 2-5 which are positive for active MK2, it was a significant increase with time of GR that was phosphorylated at residue Ser 134. Hence, active MK2 per se is sufficient to cause phosphorylation of GR at residue Seri 34.

[0123] To verify that MK2 is in fact the downstream target of p38MAPK that is responsible for phosphorylation of GR at residue Seri 34, the inventors designed a cell study (example 4) where the cells were exposed to Anisomycin (potent activator of p38MAPK) or a combination of Anisomycin and PF3644022 (inhibitor of MK2). As shown in figure 4a, in particular band 2A and 4A, the increased levels of GR phosphorylated at residue Seri 34 observed in the presence of Anisomycin were totally abolished in the presence of a MK2 inhibitor. Hence, the hypothesis that MK2 is the downstream target of p38MAPK that is responsible for phosphorylation of GR at residue Seri 34 has been verified.

[0124] Examples 1 and 4 were conducted in MDA-MB-231 cells and examples 2 and 3 were in-vitro kinase assays. In order to verify that the above results are not cell specific, the inventors designed a cell assay that repeated the basic setup of example 3 except from the cell type being used (example 5). This cell assay was used to study the effect of a MK2 inhibitor (Zunsemetinib) on phosphorylation of GR at residue Seri 34 in human lung carcinoma cells A549 (5a), human embryonic kidney cells HEK-293 (5b) and human T-lymphocyte cells Jurkat (5c). As shown in figure 5a, 5b and 5c (in particular bands 2A and 4A), it was verified that MK2 is the downstream target of p38MAPK that is responsible for phosphorylation of GR at residue Seri 34.

[0125] I view of the above, it is clear that the present inventors have identified the glucocorticoid receptor (GR) as a downstream target for MK2 activity, in particular that active MK2 phosphorylates GR at residue Seri 34. This new discovery makes it possible to monitor MK2 activity in cells in a simple, precise, and cost -efficient manner. Monitoring changes in MK2 activity in response to various stimuli is of particular interest when it comes to screening for new MK2 inhibitors.

[0126] A first aspect relates to an in-vitro cell-based method of screening for an inhibitor of MK2. The cells are incubated in the absence or presence of a potential MK2 inhibitor. In the absence of a MK2 inhibitor, the intracellular MK2 will phosphorylate GR at residue Ser 134 of SEQ ID NO: 1. However, in the presence of a MK2 inhibitor, the intracellular MK2 will be inhibited from phosphorylating any of its downstream targets, including GR. Hence, there will be a reduced level of GR phosphorylated at residue Ser 134 in cells that have been exposed to a MK2 inhibitor.

[0127] If the in-vitro cell-based method is intended to be used for identifying compounds with MK2 inhibitory activity in humans, it is preferred that the cells used in the assay also are human cells. Hence, in one preferred embodiment the cells are human cells.

[0128] In order to be able to detect MK2 inhibition, the cells need to have a basal MK2 activity level that is reduced in the presence of a MK2 inhibitor. Said in other words, that there is a detectable reduction in the level of GR phosphorylated at Ser 134 when the cells are incubated in the presence of a MK2 inhibitor relative to cells incubated in the absence of the MK2 inhibitor.

[0129] A low basal MK2 activity level is believed to negatively affect the sensitivity of the assay. Hence, it is preferred to use cells with a high basal MK2 activity level. However, if cells with a low basal MK2 activity level is to be used, the sensitivity of the assay is believed to be increased by incubating the cells in the presence of a MK2 activating agent. A MK2 activation agent may of course also be used in assays where the cells have high basal level of MK2 activity to increase the sensitivity of the assay further.

[0130] Based on the data shown in Figure 3, in particular lanes 2 to 5 (+MK2 for 10, 20, 30 and 40 minutes), it is clear that the level of GR phosphorylated at Ser 134 increases with time of exposure to active MK2. Hence, the sensitivity of the assay may also be increased by increasing the time of exposure to active MK2. A second aspect therefore relates to a kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, wherein the kit comprises a binding agent; a MK2 activation agent, such as an agent that causes activation of p38 MAPK, for example Anisomycin; instructions for use of the kit components; and optionally a cell lysis buffer; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 .

[0131] The MK2 activating agent may be any agent that is able to directly or indirectly activate intracellular MK2 activity. It is well known that phosphorylation of GR at residue Seri 34 is mediated by an active p38MAPK pathway, and it has been shown that Anisomycin (potent activator of p38MAPK) causes phosphorylation of GR at residue Seri 34 (figure la, band 2A; figure 4a, band 2A; figure 5a-c, band 2A). Hence, in the context of the present application Anisomycin will be considered to represent a MK2 activating agent when present in combination with p38MAPK. In sum, a MK2 activating agent is an agent which causes activation of MK2 in the assay of interest.

[0132] For those embodiments where the cells have sufficiently high level of basal MK2 activity, and no MK2 activation agent is needed, the kit may comprise a binding agent; a cell lysis buffer; and instructions for use of the kit components; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, in particular p-GR(Serl34) mAb referred to in the materials and methods section of the present application.

[0133] Having identified MK2 as the downstream target of p38MAPK that is responsible for phosphorylation of GR at Seri 34 is an important finding. However, to translate this finding into a commercially interesting application requires methodology to specifically measure the levels of GR that has been phosphorylated in Seri 34. There are many different options for specifically measuring the levels of GR that has been phosphorylated in Serl34, of which the use of a specific monoclonal antibody represents one of these alternatives (e.g. Phospho-Glucocorticoid Receptor (Seri 34) (E9R9W) Rabbit mAb #85060 provided by Cell Signaling Technology). Such an antibody may be used in a number of different assays for measuring or visualizing the amount / localization of GR that has been phosphorylated in Serl34, including western blotting, ELISA and immunohistochemistry. The latter methodology may be useful not only for measuring the level of GR that has been phosphorylated in Serl34 (which indicates MK2 activity), but also for visualizing location of MK2 activity in e.g. a tissue sample. While the first aspect relates to an in-vitro cell-based method of screening for an inhibitor of MK2, there are also in-vitro non-cell based methods of screening for an inhibitor of MK2.

[0134] A third aspect relates to an in-vitro method of screening for an inhibitor of MK2, comprising incubating GR, ATP and active MK2 in the presence or absence of a potential MK2 inhibitor; measuring the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition; comparing the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor; to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor; wherein a higher measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor relative to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor indicates that the potential MK2 inhibitor has MK2 inhibitory activity.

[0135] The active MK2 may be obtained by incubating MK2 in the presence of a MK2 activation agent. When MK2 is in its active state in the presence of ATP, it will phosphorylate GR at residue Seri 34. In order to avoid unintentional phosphorylation, it is preferred that GR has been incubated with the potential MK2 inhibitor prior to exposing GR for ATP and MK2 in an active state. Optionally, the potential MK2 inhibitor, MK2 in active state and ATP are simultaneously brought in contact with the GR.

[0136] A fourth aspect relates to a kit suitable for use in the method according to the third aspect, wherein the kit comprises: i) MK2; ii) GR; iii) ATP; iv) a binding agent, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; and v) instructions for use of the kit components; wherein the MK2 is active MK2; or the kit further comprises a MK2 activation agent.

[0137] In order to avoid unintentional phosphorylation, it is preferred that GR is not in contact with MK2 in an active state in the presence of ATP. This may e.g. be achieved by having ATP in a compartment separate from the other ingredients, active MK2 in a compartment separate from the other ingredients and / or MK2 activation agent in a compartment separate from the other ingredients.

[0138] A sixth aspect relates to use of a binding agent in an in-vitro method of measuring, visualizing and / or monitoring MK2 activity in a sample, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.

[0139] In some embodiments the sample is a tissue sample.

[0140] In some embodiments the MK2 activity is measured and / or visualized by immunohistochemistry staining of the sample, in particular a tissue sample, with the binding agent.

[0141] Being able to measure, visualize and / or monitor MK2 activity in a sample, such as a biological sample (e.g. a tissue sample), enables a number of different industrial applications. Measuring MK2 activity in a cell sample that has been exposed to different stimuli, such as potential MK2 inhibitors, may be used as a research tool e.g. in a method of screening for MK2 inhibitors.

[0142] Having generally described this concept, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

[0143] EXAMPLES

[0144] Materials and Methods:

[0145] Cell lines and culture conditions:

[0146] MDA-MB-231 (American Type Culture Collection (ATCC) Virginia, USA, HTB- 26TM) was maintained in GibcoTM RPMI-1640 Medium (Thermo Fisher Scientific, MA, USA).

[0147] A549 (ATCC, CRM-CCL-185TM), HEK-293 (ATCC, CRL-1573TM) and Jurkat, Clone E6-1 (ATCC, TIB-152TM) were maintained in DMEM.

[0148] All cell line media were supplemented with 10 % fetal bovine serum (FBS;

[0149] Millipore, MA, USA, TMS-013-B) penicillin (100 units / ml) and streptomycin (100 mg / ml) and cells were kept in a humified 5 % CO2 atmosphere at 37 °C. Cell lines were routinely screened for mycoplasma and mycoplasma-free cells were used in all experiments. p-GR (Serl34) specific antibody:

[0150] Phospho-Glucocorticoid Receptor (Serl34) (E9R9W) Rabbit mAb #85060 is a commercially available antibody provided by Cell Signaling Technology, Danvers, MA, USA.

[0151] Product Number: 85060;

[0152] Product Name: Phospho-Glucocorticoid Receptor (Seri 34) (E9R9W) Rabbit mAb #85060; Source / Isotype: Rabbit IgG;

[0153] Molecular weight: 91,94 kDa

[0154] The antibody may be obtained by following the procedure described in Mol Cell Biol. 2011 Dec; 31(23): 4663-4675 and has been referred to in scientific literature (Mol Cell Endocrinol. 2020 Dec 1; 518: 110873).

[0155] The antibody is herein referred to as p-GR(Serl34) mAb.

[0156] Other reagents:

[0157] Antibodies specific for GR (Purified Mouse anti-GR (cat# 611227), BD Biosciences), p-p38 MAPK (phospho-Thrl80 / Tyrl82 p38 MAPK (cat#4631), Cell signaling technology), Actin (Anti -Actin antibody (cat#A2066), Sigma Merck), p- GR (S226)( phospho-Glucocorticoid Receptor (Ser226) (D9D3V) Rabbit mAb (cat# 972855), Cell signaling technology) and p-GR (S211) (Phospho-Glucocorticoid Receptor (Ser211) Antibody (cat#4161), Cell signaling Technology.

[0158] Recombinant active p38a, ERK2 and JNK1 were purchased from MRC PPU reagents and services, Dundee, UK.

[0159] GST-GR fusion protein (1-400) was prepared according to standard procedure and ATP was purchased from Sigma Merck

[0160] Western blot:

[0161] Total cellular extract was obtained by lysis of the cells in NuPAGETM LDS Sample Buffer and NuPAGETM Sample Reducing Agent (Thermo Fisher Scientific). The cellular lysate was then denaturated by heating for 10 min at 98 °C and further used or stored at -20 °C. For protein separation, lysates were applied to 4-12 % Invitrogen Bis-Tris Gels (Thermo Fisher Scientific # NW04122BOX), with a running time of 45 min at 200 V / 120 mA in MES SDS Running Buffer (Thermo Fisher Scientific #NP0002-02). Chameleon® Duo Pre-stained Protein Ladder (LI- COR Biosciences, NE, USA #NP928 -60000) was used as molecular weight marker in all experiments. Western blot transfer to Odyssey® Nitrocellulose Membranes (LI-COR Biosciences #NP926-31090) was performed at 30 V / 150 mA for 2 h in blotting buffer containing 48 mM Trisbase, 384 mM glycine and 20 % MeOH. Subsequently, the membranes were blocked for 1 h at room temperature while shaking in Odyssey® Blocking Buffer (LI-COR Biosciences #927-40000), followed by incubation in the indicated primary antibody overnight at 4 °C. The next day, membranes were washed 3x 15 min with IX TBS-Tween at room temperature, then incubated with IRDye secondary antibodies (LI-COR Biosciences) in for 1 h at room temperature, with a repetition of the washing steps and detection via the Odyssey Sa detection system (LI-COR Biosciences) afterwards. In vitro kinase assays:

[0162] Recombinant active p38a, ERK2, JNK1 and MK2 were purchased from MRC PPU Reagents and Services, Dundee, UK. For in vitro protein kinase assays, the active kinase was incubated with GST-GR fusion protein (1-400) and 60 pM ATP in 50 pl kinase buffer (50 mM Tris HC1 pH 7.5, 0.1 mM EGTA, 1 mM sodium vanadate, 1 mM DTT, 10 mM Mg (CH3COO)2 / MgC12). The reaction was carried out at 30 °C for 10-40 min, terminated with LDS Sample Buffer (Thermo Fisher Scientific #NP0008) and Sample Reducing Agent (Thermo Fisher Scientific #B0009) and finally analyzed by western blotting.

[0163] Example 1:

[0164] GR is phosphorylated at SI 34 in the presence of a p38 MAPK activator in MDA- MB-231 cells

[0165] Five samples of MDA-MB-231 cells were prepared. A first sample was not subjected to any treatment for 30 minutes, a second sample was subjected to treatment with Anisomycin (10 pM) for 30 minutes, a third sample was subjected to treatment with SB202190 (10 pM) for 30 minutes, a fourth sample was subjected to treatment with a combination of Anisomycin (10 pM) and SB202190 (10 pM) for 30 minutes and a fifth sample was subjected to treatment with synthetic GC dexamethasone (100 nM) for 30 minutes.

[0166] Each of the five samples were then subjected to western blotting according to the protocol disclosed in the above materials and methods section of the present application. Antibodies specific for p-GR (SI 34), GR, p-p38 MAPK, Actin and p38 MAPK were used as primary antibodies.

[0167] The results are presented in figure la.

[0168] Example 2:

[0169] GR is NOT phosphorylated at SI 34 in the absence of active MK2 in an in vitro kinase assay

[0170] Recombinant active p38a, ERK2 and JNK1 were separately incubated with GST- GR fusion protein (1-400) and 60 pM ATP in 50 pl kinase buffer according to the protocol above for in vitro kinase assays. The reaction was carried out at 30 °C for 0, 10 and 40 minutes and terminated with LDS Sample Buffer and Sample Reducing Agent.

[0171] Each of the samples were then analyzed by western blotting according to the protocol disclosed in the above materials and methods section of the present application. Antibodies specific for p-GR (S226), p-GR (S211), p-GR (S134) and GR were used as primary antibodies.

[0172] The results are presented in figure 2.

[0173] Example 3:

[0174] GR is phosphorylated at SI 34 in the presence of active MK2 in an in vitro kinase assay

[0175] Recombinant active p38a and MK2 were separately incubated with GST-GR fusion protein (1-400) and 60 pM ATP in 50 pl kinase buffer according to the protocol above for in vitro kinase assays. The reaction was carried out at 30 °C for 0, 10, 20, 30 and 40 minutes and terminated with LDS Sample Buffer and Sample Reducing Agent.

[0176] Each of the samples were then analyzed by western blotting according to the protocol disclosed in the above materials and methods section of the present application. Antibodies specific for p-GR (S226), p-GR (S211), p-GR (S134) and GR were used as primary antibodies.

[0177] The results are presented in figure 3.

[0178] Example 4:

[0179] GR is NOT phosphorylated at SI 34 in the presence of a MK2 inhibitor in MDA- MB-231 cells

[0180] Six samples of MDA-MB-231 cells were prepared. A first sample was not subjected to any treatment for 30 minutes, a second sample was subjected to treatment with Anisomycin (10 pM) for 30 minutes, a third sample was subjected to treatment with PF3644022 (10 pM) for 30 minutes, a fourth sample was subjected to treatment with a combination of Anisomycin (10 pM) and PF3644022 (10 pM) for 30 minutes, a fifth sample was subjected to treatment with synthetic GC dexamethasone (100 nM) for 30 minutes and a sixth sample was subjected to treatment with a combination of synthetic GC dexamethasone (100 nM) and PF3644022 (10 pM) for 30 minutes.

[0181] Each of the six samples were then subjected to western blotting according to the protocol disclosed in the above materials and methods section of the present application. Antibodies specific for p-GR (SI 34), GR, MK2, p-Hsp27 and Actin were used as primary antibodies.

[0182] The results are presented in figure 4. Example 5:

[0183] GR is NOT phosphorylated at SI 34 in the presence of a MK2 inhibitor in A549 (lung), HEK-293 (kidney) and Jurkat (blood) cells

[0184] Five samples of A549 cells, five samples of HEK-293 cells and five samples of Jurkat cells were prepared. A first sample of each cell line was not subjected to any treatment for 30 minutes, a second sample of each cell line was subjected to treatment with Anisomycin (10 pM) for 30 minutes, a third sample of each cell line was subjected to treatment with Zunsemetinib (10 pM) for 30 minutes, a fourth sample of each cell line was subjected to treatment with a combination of Anisomycin (10 pM) and Zunsemetinib (10 pM) for 30 minutes and a fifth sample of each cell line was subjected to treatment with synthetic GC dexamethasone (100 nM) for 30 minutes.

[0185] Each of the fifteen samples were then subjected to western blotting according to the protocol disclosed in the above materials and methods section of the present application. Antibodies specific for p-GR (SI 34), GR, MK2, p-Hsp27 and Actin were used as primary antibodies.

[0186] The results are presented in figure 5a (A549, lung), figure 5b (HEK-293, kidney) and figure 5c (Jurkat, blood) respectively.

Claims

CLAIMS1.An in-vitro method of screening for an inhibitor of MK2, comprising: a) incubating cells in the presence or absence of a potential MK2 inhibitor; b) measuring level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells; c) comparing- the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the presence of the potential MK2 inhibitor; to- the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the absence of the potential MK2 inhibitor; wherein a higher measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells incubated in the absence of the potential MK2 inhibitor relative to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in cells incubated in the presence of the potential MK2 inhibitor indicates that the potential MK2 inhibitor has MK2 inhibitory activity.2.The in-vitro method according to claim 1, wherein the cells are human cells.3.The in-vitro method according to any one of the preceding claims, wherein the cells have higher level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 when incubated in the absence of a MK2 inhibitor relative to level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 when incubated in the presence of a MK2 inhibitor.4.The in-vitro method according to any one of the preceding claims, wherein the cells in step a) are incubated with a MK2 activation agent in the presence or absence of a potential MK2 inhibitor.5.The in-vitro method according to claim 4, wherein the cells express p38 MAPK and the MK2 activation agent is an agent that causes activation of p38 MAPK, such as Anisomycin.6.The in-vitro method according to any one of the preceding claims, wherein the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.7.The in-vitro method according to any one of the preceding claims, wherein the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured by a method comprising the following steps:- subjecting the incubated cells to lysis to obtain a cell lysate;- reacting the cell lysate with a binding agent; the binding agent being an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 ; and- measuring the level of binding of the binding agent to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 .8.A kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, wherein the kit comprises- a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1;- a MK2 activation agent, such as an agent that causes activation of p38 MAPK, for example Anisomycin; instructions for use of the kit components; andoptionally a cell lysis buffer.9.An in-vitro method of screening for an inhibitor of MK2, comprising:- incubating GR, ATP and active MK2 in the presence or absence of a potential MK2 inhibitor;- measuring the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition;- comparing- the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor; to- the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor; wherein a higher measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the absence of the potential MK2 inhibitor relative to the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the composition incubated in the presence of the potential MK2 inhibitor indicates that the potential MK2 inhibitor has MK2 inhibitory activity.10.The in-vitro method according to claim 9, wherein the active MK2 is obtained by incubating MK2 in the presence of a MK2 activation agent.11.The in-vitro method according to any one of claims 9-10, wherein active MK2 is obtained by incubating MK2 in the presence of p38 MAPK and an agent that causes activation of p38 MAPK, such as Anisomycin.12.The in-vitro method according to any one of claims 9-11, wherein the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated moleculecomprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.13.A kit suitable for use in an in-vitro method of screening for an inhibitor of MK2, wherein the kit comprises i) MK2; ii) GR; iii) ATP; iv) a binding agent, wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; and v) instructions for use of the kit components; wherein the MK2 is active MK2; or the kit further comprises a MK2 activation agent.14.The kit according to claim 13, wherein the kit further comprises p38 MAPK and the MK2 activation agent is an agent that causes activation of the p38 MAPK, such as Anisomycin.15.The kit according to any one of claims 13-14, wherein the active MK2 and the GR are in separate compartments and / or the MK2 and the MK2 activation agent are in separate compartments.16.An in-vitro method of measuring MK2 activity in cells, the in-vitro method comprising the following step(s):- measuring level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells; wherein- the measured level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 in the cells is positively correlated with the MK2 activity in the cells.17.The in-vitro method according to claim 16, wherein the cells are human cells.18.The in-vitro method according to any one of claims 16-17, wherein the cells express p38 MAPK.19.The in-vitro method according to any one of claims 16-18, wherein the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured using a binding agent; wherein the binding agent is an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.20.The in-vitro method according to any one of claims 16-19, wherein the level of GR phosphorylated at residue Ser 134 of SEQ ID NO: 1 is measured by a method comprising the following steps:- subjecting the cells to lysis to obtain a cell lysate;- reacting the cell lysate with a binding agent; the binding agent being an agent which specifically binds to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1, such as an isolated molecule comprising an antigen binding portion of an antibody specific for GR phosphorylated at residue Ser 134 of SEQ ID NO: 1; and- measuring the level of binding of the binding agent to GR phosphorylated at residue Ser 134 of SEQ ID NO: 1.21.The in-vitro method according to any one of claims 19-20, wherein the binding agent is attached to a label, such as a visually detectable label.

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