Identification of a Novel Inhibitory Allosteric Site in p38α

Abstract

In the present study, we report the discovery of a novel allosteric inhibitory site for p38α, a subclass of the mitogen-activated protein kinases (MAPK) family. The putative site was discovered after inspection of the crystallographic structure of the p38α-MK2 complex. MK2 (MAPK-activated protein kinase 2) is an interesting protein playing a dual role as modulator and substrate of p38α. This intriguing behavior is due to the ability of the two proteins to form distinctive heterodimers when p38α is phosphorylated or not. We hypothesized that the regulatory action of MK2 is due to its capability to keep p38α in an inactive conformation and consequently, we investigated the atomic structure of the p38α-MK2 complex to understand such regulatory behavior at the molecular level. After inspection of the complex structure, two peptides designed from the MK2 regulatory loop in contact with p38α with sequences Tyr1-Ser2-Asn3-His4-Gly5-Leu6 (peptide-1) and [Phe0]-peptide-1 (peptide-2) in their zwitterionic form were investigated for their phosphorylation inhibitory capability in vitro. Since both peptides exhibited inhibitory capability of the p38α kinase mediated phosphorylation of MEF2A, in a subsequent step we pursued the discovery of small molecule peptidomimetics. For this purpose we characterized in detail the peptide-p38α interaction using molecular dynamics simulations, leading to the definition of a pharmacophore for the peptide-protein interaction. This hypothesis was used as query for a in silico screening, leading to the discovery of a fused ring compound with micromolar inhibitory activity. Site-directed mutagenesis studies support that the compound binds to the putative novel allosteric site in p38α.

New Insights into the Stereochemical Requirements of the Bombesin BB1 Receptor Antagonists Binding

“…The chemical structures of the six compounds were found to displace the radioligand used in the binding assays by more than 15%, as listed in Table 1 . These results yielded a success rate of about 50%, as found in other cases [ 39 , 40 ]. Table 1 also lists the results of radioligand displacement experiments for the BB1R and BB2R, suggesting that the ligands are BB1R selective.…”

Identification of Potential Small Molecule Binding Pockets in p38α MAP Kinase

“…Finally, the procedure also identifies an allosteric site recently reported by this laboratory, located between the ACE motif of the activation loop, the αEF helix, the N-terminal end of the αG helix, and part of the N-terminal segment of the activation. 57 Additionally, ten prospective binding sites, not previously described for the p38α protein or other MAP kinases named NP1 to NP10, are shown in Figure 6b. The NP1 site is located at the back of the beta sheets between the ends N-terminal and C-terminal, which is in an area with a high flexibility.…”

In silico Methods for Design of Kinase Inhibitors as Anticancer Drugs

“…MD simulations permitted definition of pharmacophoric features of small peptide inhibitors derived from sequence of MK2. Subsequent virtual screening study resulted in first small molecule allosteric inhibitor for identified binding site (Gomez-Gutierrez et al, 2016). Cournia et al verified existence of allosteric site on human PI3Kα previously described in murine PI3Kα using combination of FTMap, MD, and in vitro assays.…”