First-in-Class Mitogen-Activated Protein Kinase p38α: MAPK-Activated Protein Kinase-2 (MK2) Dual Signal Modulator with Anti-inflammatory and Endothelial-stabilizing Properties.

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38aMitogen-activated Protein Kinase (MAPK) near the binding site of its proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by Surface Plasmon Resonance (SPR), 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in vivo GEn-1124 improved survival from 10% to 40% in murine acute lung injury (ALI) induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide (LPS) instillation and febrile-range hyperthermia (FRH) and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38aand MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex, dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention, and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance Statement We describe an analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream pro- and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in ARDS.