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. Overall design: Human pulmonary artery endothelial cells (hPAEC) monolayers were pretreated for 60 min with DMSO or 10 µM SB203580, or one of the four novel inhibitors at 25 µM in an 0.1% final DMSO concentration. The cells were then stimulated with 10 ng/ml human TNFa for 3h. Untreated controls received neither pretreatment nor TNFa.  Poly(A)-enriched samples were reverse transcribed and sequenced using the Illumina HiSeq platform. All treatments were done in triplicate.