Abstract Introduction We developed a novel strategy for treating ARDS by targeting small molecules to the substrate binding site on p38α that binds the proinflammatory substrate MK2. One compound developed using this strategy, GEn-1124, exerts anti-inflammatory and endothelial-barrier-stabilizing activity, reduced mortality in mouse models of acute lung injury (ALI) and is currently in a Phase II clinical trial in ARDS (NCT05795465). GEn-1124 has a unique mechanism of action. By disrupting p38α:MK2 binding it reduces the MK2-mediated nuclear export of p38α, thereby prolonging intranuclear signaling (including anti-inflammatory pathways). We have developed additional compounds using the same strategy to develop a pipeline of potential drugs to treat ARDS and other inflammatory diseases. This study focuses on four second-generation compounds with distinct biological effect profiles. Methods Using computer-aided drug design, we identified new lead compounds and over 200 analogs. The biological effects of four of these second-generation analogs, GEn-1915, GEn-1917, MP-135, and SP-2-117, were analyzed in detail. Endothelial barrier stabilizing activity was analyzed by pretreating human pulmonary artery endothelial cells (hPAECs) with compounds at 1.5-25µM for 1h, then with thrombin, and monitoring permeability using Electric Cell-substrate Impedance Sensing (ECIS). Effects on gene expression were assessed by pretreating hPAECs with each compound at 25µM for 1h, then with 10 ng/ml TNFα for 3h. RNA was analyzed by RNASeq and Ingenuity Pathway Analysis with z-score cut-off -1.5 and 1.5. Lung protective effects were assessed in a mouse model of ALI induced by i.t. instillation of 50µg LPS and hyperthermia to 39 °C. Mice were treated with 1 mg of each compound dissolved in 4% DMSO/30% SBECD administered i.p. 5h post-LPS instillation. Mice were euthanized 24h post-LPS and lung lavage analyzed for protein concentration (Bradford assay) and cell counts. Results The compounds exhibited variable endothelial barrier stabilizing activity (Table). MP135 was most active and SP-2-117 least active in this assay. The gene expression pathway analysis focused on direct p38α substrates. GEn-1915 was the least anti-inflammatory compound in this assay. MP135 upregulated three and GEn-1917 and SP2-117 each upregulated two anti-inflammatory pathways. GEn-1917 was the only compound that inhibited MK2 signaling. MP135 and GEn-1917 exerted the greatest lung protective effect in vivo but had greater effect on protein leak than neutrophil infiltration. Conclusions Our drug development strategy targeting p38α substrate binding sites can produce a pipeline of compounds with distinct biological effect profiles with therapeutic potential in ARDS and other disorders caused by endothelial leak and inflammation. This abstract is funded by: Merit Review BX004270
Tulapurkar et al. (Fri,) studied this question.