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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262079 | GEO | 2024/04/01

REPOSITORIES: GEO

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Project description:Background: Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2 h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.