Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2 (MAPKAPK2) Inhibitors

Abstract

ABSTRACT:A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through highthroughput screening using the affinity selectionÀmass spectrometry-based Automated Ligand Identification System platform. Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective inhibitors of MK2 should be feasible by targeting the outside ATP pocket. KEYWORDS:Mitogen-activated protein kinase-activated protein kinase 2, non-ATP-competitive inhibitors, Automated Ligand Identification System (ALIS), saturation-transfer-difference (STD) NMR, 1 H/ 15 N-HSQC (heteronuclear single quantum coherence) U nregulated protein kinase activity is often associated with inflammatory diseases, such as rheumatoid arthritis, which is characterized by autoimmune-mediated cartilage and bone destruction that leads to intolerable joint pain and crippling and afflicts about 1% of the world's population. 1 Activation of the p38/mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2) pathway has been implicated in promoting pro-inflammatory cytokine production and related inflammatory diseases. 2À4 However, despite numerous assessments of potent inhibitors in early stage clinical trials, no p38 MAPK inhibitors have progressed beyond phase II trials largely because of dose-dependent toxicities and unimpressive efficacies. 4,5 The broad involvement of p38R MAPK and its downstream substrates in diverse cellular processes may be a major cause of related clinical trial failures. 4,6,7 To minimize the apparent side effects caused by directly targeting p38R MAPK, several discovery teams have attempted inhibiting targets downstream of p38R MAPK that are responsible for its pro-inflammatory cytokine-regulating properties in macrophages. 4,7 Of these MK2, a serine/threonine protein kinase that directly associates with p38R MAPK to form a heterodimer and is phosphorylated and activated by p38R MAPK has emerged as the most attractive downstream target. 7À10 For example, in studies employing genetically engineered mouse models wherein MK2, MK3, and MK5 genes were knocked out, it was shown that MK2 is the primary MAPKAPK family member required for lipopolysaccharide (LPS)-induced tumor necrosis factor R (TNFR) and interleukin 6 (IL6) production. 11À13 Furthermore, MK2-null mice were shown to have reduced joint damage in a collagen-induced arthritis model and no induction of asthma in a lung ovalbumin sensitization model. 14,15 In contrast to the embryonic lethality associated with knocking out the geneencoding p38R MAPK, MK2-null mice were fertile and healthy, suggesting that inhibition of MK2 in inflammatory disease states may provide efficacy comparable to direct p38R MAPK in...

IL-33 promotes type 1 cytokine expression via p38 MAPK in human NK cells

“…Previous studies showed that MAPK-MAPK (MK2) is down-stream of p38 MAPK, and that MK2 mediates inflammatory responses. 24,25 Addition of a specific MK2 inhibitor (MK2 IV) to NK-cell cultures diminished the enhancing effect of IL-33 on IL-12-stimulated IFNproduction ( Fig. 8).…”

Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials

“…94 Different NMR studies and biochemical (enzymatic analysis of MK2 inhibition) experiments were set up to demonstrate that 103 bound MK2 without competing with ATP (non-ATP-competitive inhibition) and that its binding was site-specific. This compound showed a single-digit micromolar activity in a DELFIA immunoassay (5500 nM) and toward SW1353 chondrosarcoma cells (about 2000 nM) without appreciable cytotoxicity, as well as a significant kinase selectivity in an in house profiling panel assay.…”

Interleukin-33 promotes type 1 cytokine expression via p38 MAPK in human natural killer cells

“…Moreover, IL-33 amplification of IFN- is dependent on p38 MAPK activity and associated with increased stability of IFNG mRNA. The p38 MAPK regulates inflammatory responses via phosphorylation of downstream mediators, including MK2 27,28 . Yet, MK2 inhibitors had only a partial inhibitory effect in contrast to the abrogation of IL-12/IL-33 synergistic interactions following p38 MAPK inhibition.…”