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

Abstract Most of the available p38α 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-p38α scFv antibodies among which one fully inhibited p38α 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 p38α and induced a significant decrease in lipopolysaccharide-induced tumor necrosis factor α production. To gain insight into the mechanisms by which the scFv induced p38α inhibition, the scFv epitope was mapped by deep mutational scanning of p38α displayed on yeast surface and high-throughput selection by flow cytometry of p38α 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 p38α in an inactive form.