Search

Chemistry has been developed to specifically functionalize two structurally similar classes of indole-based MK2 inhibitors at positions prompted by a combination of X-ray crystallographic and computer assisted drug design. A gain in molecular potency was obtained by introducing aminomethyl groups to the lactam rings of 6-arylcarbamoyl-tetrahydro-beta-carbolinone and 6-arylcarbamoyl-dihydropyrazino[1,2-a]indolone MK2 inhibitors. In addition, improvements in molecular potency were achieved by expansion of the lactam from a 6- to 7-membered ring leading to 7-arylcarbamoyl-tetrahydro-[1,4]diazepino[1,2-a]indolones.

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.

The Pim family of Ser/Thr kinases has been implicated in the process of lymphomagenesis and cell survival. Known substrates of Pim kinases are few and poorly characterized. In this study we set out to identify novel Pim-2 substrates using the Kinase Substrate Tracking and Elucidation (KESTREL) approach. Two potential substrates, eukaryotic initiation factor 4B (eIF4B) and apoptosis inhibitor 5 (API-5), were identified from rat thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation sites of eIF4B and mouse BAD, the only other known Pim-2 substrate, revealed conserved amino acids preceding the phosphorylated serine residue. Stepwise replacement of the conserved residues produced a consensus sequence for Pim kinase recognition: RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this recognition sequence 20-fold more efficiently than the original (K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1 phosphorylation site. The identification of the novel Pim kinase consensus sequence provides a more sensitive and versatile peptide based assay for screening modulators of Pim kinase activity. [ABSTRACT FROM PUBLISHER]