First-in-Class Substrate- and Function-Selective p38alpha Inhibitors with Anti-inflammatory and Endothelial-stabilizing Activities

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of MAPK14/p38a near the binding site of the proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. We developed an analog of UM101, GEn-1124, with improved aqueous stability and p38a binding affinity. Compared with UM101, GEn-1124 has 60-fold greater p38a binding affinity as measured by Surface Plasmon Resonance (SPR), 66-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in a mouse models of acute lung injury (ALI). GEn-1124 reduced lung injury and improved survival in a mouse model of ALI induced by intratracheal LPS instillation and exposure to febrile-range hyperthermia (FRH) from 10% to 40% and in a mouse influenza pneumonia model from 0% to 50%. RNASeq and pathway analysis of gene expression in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38a and MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the phosphorylated p38a:MK2 complex, dissociates nuclear export of phosphorylated p38a and MK2, thereby promoting intranuclear phosphorylated p38a retention and intranuclear signaling, and accelerating inactivation of p38-free cytosolic MK2 by unopposed phosphatases.