Characterization of the p38α MAPK allosteric inhibition by a single chain Fv antibody

Abstract

Most of the available p38 kinase inhibitors target the highly conserved ATP-binding site, thus explaining their off-target effects and toxicity. Here, we report the use of single-chain fragment variable (scFv) antibodies to identify new highly specific inhibitors. Using phage display, we selected five anti-p38 scFv antibodies among which one fully inhibited p38 kinase activity in vitro. We showed that this scFv did not affect ATP binding, while still acting as a competitive inhibitor of ATP hydrolysis. When expressed as intrabody in the nucleus of the THP-1 human monocytic cell line, the scFv inhibited the function of endogenous p38 and induced a significant decrease in lipopolysaccharide-induced tumor necrosis factor production. To gain insight into the mechanisms by which the scFv induced p38 inhibition, the scFv epitope was mapped by deep mutational scanning of p38 displayed on yeast surface and high-throughput selection by flow cytometry of p38 mutants no longer bound by the scFv. By this approach, we showed that the inhibitory scFv interacts with two helices within the C-terminal lobe of the kinase distant from the ATP-binding pocket and the docking groove. Altogether, our data show that the scFv antibody behaves as an allosteric inhibitor, constraining and maintaining p38 in an inactive form.