Discovery and Characterization of a Substrate Selective p38α Inhibitor

Abstract

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

Syk‐dependent ERK activation regulates IL‐2 and IL‐10 production by DC stimulated with zymosan

“…The MAP kinase-activated protein kinase MK2 is a strong candidate for ERK-dependent post-transcriptional regulation of mRNA levels and has been previously implicated in the regulation of TNF-a and IL-6 mRNA levels in macrophages [42][43][44][45]. Consistent with this hypothesis, the specific MK2a inhibitor, CMPD1 [46], significantly decreased IL-2 but not IL-10 mRNA levels (Supporting Information Fig. 1).…”

Characterization of an ERK-binding Domain in Microphthalmia-associated Transcription Factor and Differential Inhibition of ERK2-mediated Substrate Phosphorylation

“…A peptide inhibitor of cyclin-dependent kinase-mediated Rb phosphorylation has been developed that does not affect cyclin-dependent kinase-mediated histone phosphorylation (78). CMPD1 is a small molecule inhibitor of p38␣ MAPK that exhibits substrate-selective inhibition of MK2 versus ATF-2 (79). In addition, the finding that the FXFP-site binds to a different part of the MAPK surface than the D-site (35) suggests that it should be possible to specifically inhibit FXFP interactions.…”

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

“…showed that targeting the non-ATP binding site of a MAP kinase could produce a substrate selective inhibitor [99]. They discovered a non-ATP p38 MAPKα inhibitor (16) that inhibits the phosphorylation of MK2a with a K i of ~330 nM.…”

“…Isothermal titration calorimetry analysis confirmed the binding of 16 to p38 MAPKα, but showed that this compound does not prevent the binding of MK2a. While a deuterium exchange mass spectrometry (DXMS) study suggested that 16 binds in the vicinity of the p38 MAPKα active site and disrupts the ATP binding site [99], the basis for its substrate selectivity is unknown.…”