Remodeling tumor-associated macrophage for anti-cancer effects by rational design of irreversible inhibition of mitogen-activated protein kinase-activated protein kinase 2

Abstract

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti-cancer therapies. The limitations of ATP-competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC₅₀ = 2.3 nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor-promoting macrophage M2 phenotype in vitro and in vivo. In macrophage-rich tumor model, compound 11 notably slowed growth in a dose-dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions.

Keywords

anti-tumor / irreversible inhibitor / kinase / macrophage / mitogen-activated protein kinase-activated protein kinase 2 (MK2)

References

1	D Hanahan, RA Weinberg. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674.
2	H Sandler, G Stoecklin. Control of mRNA decay by phosphorylation of tristetraprolin. Biochem Soc Trans. 2008;36(3):491-496.
3	EF Wagner, áR Nebreda. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer. 2009;9(8):537-549.
4	LR Coulthard, DE White, DL Jones, MF McDermott, SA Burchill. p38MAPK: stress responses from molecular mechanisms to therapeutics. Trends Mol Med. 2009;15(8):369-379.
5	MC Genovese. Inhibition of p38: Has the fat lady sung? Arthritis Rheum. 2009;60(2):317-320.
6	SB Cohen, TT Cheng, V Chindalore, et al. Evaluation of the efficacy and safety of pamapimod, a p38 MAP kinase inhibitor, in a double-blind, methotrexate-controlled study of patients with active rheumatoid arthritis. Arthritis Rheum. 2009;60(2):335-344.
7	N Damjanov, RS Kauffman, GT Spencer-Green. Efficacy, pharmacodynamics, and safety of VX-702, a novel p38 MAPK inhibitor, in rheumatoid arthritis: results of two randomized, double-blind, placebo-controlled clinical studies. Arthritis Rheum. 2009;60(5):1232-1241.
8	MC Genovese, SB Cohen, D Wofsy, et al. A 24-week, randomized, double-blind, placebo-controlled, parallel group study of the efficacy of oral SCIO-469, a p38 mitogen-activated protein kinase inhibitor, in patients with active rheumatoid arthritis. J Rheumatol. 2011;38(5):846-854.
9	RK Singh, AK Najmi, SG Dastidar. Biological functions and role of mitogen-activated protein kinase activated protein kinase 2 (MK2) in inflammatory diseases. Pharmacol Rep. 2017;69(4):746-756.
10	S Schreiber, B Feagan, G D'Haens, et al. Oral p38 mitogen-activated protein kinase inhibition with BIRB 796 for active Crohn's disease: a randomized, double-blind, placebo-controlled trial. Clin Gastroenterol Hepatol. 2006;4(3):325-334.
11	P Cohen. Targeting protein kinases for the development of anti-inflammatory drugs. Curr Opin Cell Biol. 2009;21(2):317-324.
12	PCF Cheung, DG Campbell, AR Nebreda, P Cohen. Feedback control of the protein kinase TAK1 by SAPK2a/p38α. EMBO J. 2003;22(21):5793-5805.
13	M Fiore, S Forli, F Manetti. Targeting mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2, MK2): medicinal chemistry efforts to lead small molecule inhibitors to clinical trials. J Med Chem. 2016;59(8):3609-3634.
14	D Gordon, ET Hellriegel, HR Hope, D Burt, JB Monahan. Safety, tolerability, pharmacokinetics, and pharmacodynamics of the MK2 inhibitor ATI-450 in healthy subjects: a placebo-controlled, randomized phase 1 study. Clin Pharmacol Adv Appl. 2021;13:123-134.
15	D Gordon, A Kivitz, A Singhal, et al. Selective inhibition of the MK2 pathway: data from a phase IIa randomized clinical trial in rheumatoid arthritis. ACR Open Rheumatol. 2023;5(2):63-70.
16	S Watanabe, M Alexander, AV Misharin, GRS Budinger. The role of macrophages in the resolution of inflammation. J Clin Invest. 2019;129(7):2619-2628.
17	L Cassetta, JW Pollard. A timeline of tumour-associated macrophage biology. Nat Rev Cancer. 2023;23(4):238-257.
18	MJ Pittet, O Michielin, D Migliorini. Clinical relevance of tumour-associated macrophages. Nat Rev Clin Oncol. 2022;19(6):402-421.
19	DG DeNardo, B Ruffell. Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol. 2019;19(6):369-382.
20	S Chen, AFUH Saeed, Q Liu, et al. Macrophages in immunoregulation and therapeutics. Signal Transduct Target Ther. 2023;8(1):207.
21	L Suarez-Lopez, G Sriram, YW Kong, et al. MK2 contributes to tumor progression by promoting M2 macrophage polarization and tumor angiogenesis. Proc Natl Acad Sci. 2018;115(18):E4236-E4244.
22	K Soukup, A Halfmann, B Dillinger, et al. Loss of MAPK-activated protein kinase 2 enables potent dendritic cell-driven anti-tumour T cell response. Sci Rep. 2017;7(1):11746.
23	KL Berggren, S Restrepo Cruz, MD Hixon, et al. MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma. Oncogene. 2019;38(48):7329-7341.
24	G Jomrich, F Maroske, J Stieger, et al. MK2 and ETV1 are prognostic factors in esophageal adenocarcinomas. J Cancer. 2018;9(3):460-468.
25	F Dietlein, B Kalb, M Jokic, et al. A synergistic interaction between Chk1- and MK2 inhibitors in KRAS-mutant cancer. Cell. 2015;162(1):146-159.
26	FMS Gurgis, W Ziaziaris, L Munoz. Mitogen-activated protein kinase–activated protein kinase 2 in neuroinflammation, heat shock protein 27 phosphorylation, and cell cycle: role and targeting. Mol Pharmacol. 2014;85(2):345-356.
27	PM Grierson, PB Dodhiawala, Y Cheng, et al. The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress. Sci Transl Med. 2021;13(622):eabb5445.
28	C Wang, S Hockerman, EJ Jacobsen, et al. Selective inhibition of the p38α MAPK–MK2 axis inhibits inflammatory cues including inflammasome priming signals. J Exp Med. 2018;215(5):1315-1325.
29	TW Traut. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem. 1994;140(1):1-22.
30	FM Gribble, G Loussouarn, SJ Tucker, C Zhao, CG Nichols, FM Ashcroft. A novel method for measurement of submembrane ATP concentration. J Biol Chem. 2000;275(39):30046-30049.
31	JF Schindler, A Godbey, WF Hood, et al. Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2. Biochim Biophys Acta. 2002;1598(1):88-97.
32	J Malona, C Chuaqui, BM Seletsky, et al. Discovery of CC-99677, a selective targeted covalent MAPKAPK2 (MK2) inhibitor for autoimmune disorders. Transl Res. 2022;249:49-73.
33	DR Anderson, MJ Meyers, WF Vernier, et al. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2). J Med Chem. 2007;50(11):2647-2654.
34	Y Wu, BP Zhou. TNF-α/NF-κB/Snail pathway in cancer cell migration and invasion. Br J Cancer. 2010;102(4):639-644.
35	M Rokavec, MG ?ner, H Li, et al. IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. J Clin Invest. 2014;124(4):1853-1867.
36	E Voronov, RN Apte. IL-1 in colon inflammation, colon carcinogenesis and invasiveness of colon cancer. Cancer Microenviron. 2015;8(3):187-200.
37	K Hamidzadeh, SM Christensen, E Dalby, P Chandrasekaran, DM Mosser. Macrophages and the recovery from acute and chronic inflammation. Annu Rev Physiol. 2017;79(1):567-592.
38	BB Phinney, AL Ray, AS Peretti, et al. MK2 regulates macrophage chemokine activity and recruitment to promote colon tumor growth. Front Immunol. 2018;9:1857.