Dataset Information

Comparative chemical array screening for p38?/? MAPK inhibitors using a single gatekeeper residue difference between p38?/? and p38?/?.

ABSTRACT: Mammalian p38 mitogen activated protein kinases (MAPKs) are responsive to a variety of cellular stresses. The development of specific pyridinyl imidazole inhibitors has permitted the characterization of the p38 MAPK isoform p38?, which is expressed in most cell types, whereas the physiological roles of p38? and p38? are poorly understood. In this study, we report an approach for identifying selective inhibitors against p38? and p38? by focusing on the difference in gatekeeper residues between p38?/? and p38?/?. Using GST-fused p38? wild type and T106M mutant constructs, wherein the p38? gatekeeper residue (Thr-106) was substituted by the p38?/?-type (Met), we performed comparative chemical array screening to identify specific binders of the mutant and identified SU-002 bound to p38?T106M specifically. SU-002 was found to inhibit p38?T106M but not p38? kinase activity in in vitro kinase assays. SU-005, the analog of SU-002, had inhibitory effects against the kinase activity of p38? and p38? in vitro but not p38?. In addition, SU-005 inhibited both p38? and p38? auto-phosphorylation in HeLa and HEK293T cells. These results demonstrate that the comparative chemical array screening approach is a powerful technique to explore specific inhibitors for mutant proteins with even single amino-acid substitutions in a high-throughput manner.

SUBMITTER: Kondoh Y

PROVIDER: S-EPMC4949465 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Publications

Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/β and p38γ/δ.

Kondoh Yasumitsu Y Honda Kaori K Hiranuma Sayoko S Hayashi Teruo T Shimizu Takeshi T Watanabe Nobumoto N Osada Hiroyuki H

Scientific reports 20160719

Mammalian p38 mitogen activated protein kinases (MAPKs) are responsive to a variety of cellular stresses. The development of specific pyridinyl imidazole inhibitors has permitted the characterization of the p38 MAPK isoform p38α, which is expressed in most cell types, whereas the physiological roles of p38γ and p38δ are poorly understood. In this study, we report an approach for identifying selective inhibitors against p38γ and p38δ by focusing on the difference in gatekeeper residues between p3 ...[more]

PMID: 27431267

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Project description:p38 Mitogen-activated protein kinase (p38 MAPK) is a protein kinase critically involved in the progress of inflammation/stress-associated diseases. Our data suggested that javamide analogues may contain strong anti-inflammation activities, but there is little information about their effects on p38 MAPK. Therefore, in this paper, the effects of thirty javamide analogues on p38 MAPK were investigated using in silico screening and in vitro p38 MAPK assay methods. The javamide analogues were synthesized and their chemical structures were confirmed using nuclear magnetic resonance (NMR) spectroscopic methods. Then, the javamide analogues were screened using an in silico modeling program. The screened analogues demonstrated a wide range of binding energy (ΔE; -20 to -39) and several analogues with ΔE; -34 to -39 showed strong binding affinity to p38 MAPK. In vitro p38 MAPK assay, the kinase was significantly inhibited by the analogues with great binding energy (ΔE; -34 to -39) and in silico scores (Avg. score; -27.5 to -29.3). Furthermore, the comparative analysis of both assays showed a positive correlation between the in silico scores and p38 MAPK inhibition. In fact, the javamide analogues with top five in silico scores (Avg. score; -27.5 to -29.3) were found to inhibit p38 MAPK by 27-31% (p < 0.05) better than those with less scores (ΔE < -27.0). Especially, javamide-II-O-ethyl ester with relatively high in silico score (Avg. score; -29.2) inhibited p38 MAPK (IC50 = 9.9 μM) a little better than its methyl ester with best in silico score (Avg. score; -29.3). To support the ability to inhibit p38 MAPK, the treatment of javamide-II-ethyl and -methyl esters could suppress the production of IL-8 and MCP-1 protein significantly by 22-73% (p < 0.05) in the differentiated THP-1 cells, and the inhibition was slightly stronger by the ethyl ester than the methyl ester. Altogether, this study suggests that javamide-II-O-ethyl ester may be a most potent p38 MAPK inhibitor among the tested compounds and the combining in silico and in vitro assay approach may be a useful and efficient solution as a functional screening approach in searching new lead compounds for targeted molecules.

Dataset Information

Comparative chemical array screening for p38?/? MAPK inhibitors using a single gatekeeper residue difference between p38?/? and p38?/?.

Publications

Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/β and p38γ/δ.

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