First-in-class mitogen-activated protein kinase (MAPK) p38α: MAPK-activated protein kinase 2 dual signal modulator...

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38α mitogen-activated protein kinase (MAPK) near the binding site of its proinflammatory substrate, mitogen-activated protein kinase-activated protein kinase (MK)2. UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38α binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38α-binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38α-binding affinity as measured by surface plasmon resonance, 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells in vitro, and greater lung protection in vivo. GEn-1124 improved survival from 10%-40% in murine acute lung injury induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide instillation and febrile-range hyperthermia and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in tumor necrosis factor α-treated human pulmonary artery endothelial cells showed that the gene-modifying effects of GEn-1124 were much more restricted to tumor necrosis factor α-inducible genes than those of the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38α and MK2 subcellular trafficking, and surface plasmon resonance analysis of phosphorylated p38α:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38α:MK2 complex and dissociates nuclear export of MK2 and p38α, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38α and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. SIGNIFICANCE STATEMENT: We describe a novel analog of our first-in-class small molecule modulator of p38α/MK2 signaling targeted to a pocket near the glutamate-aspartate-containing substrate binding domain of p38α, which destabilizes the p38α:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream proinflammatory and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in acute respiratory distress syndrome.