Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

Abstract

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

“…Other atypical PKCs (ζ and λ and ι) do not bind DAG and require only PS for activation. Mimics of DAG such as the natural products phorbol 12, 13-dibutyrate (PDBu), and bryostatin 1 (Figure ) activate conventional and novel PKCs by binding their C1 domains with nanomolar affinities. , Small molecules that engage the ATP binding site of PKCs such as bisindolylmaleimide I (BIM1, GF109203X, Figure ) block the catalytic activity of PKCs and enhance interactions with allosteric activators by promoting the accessibility of C1 domains . Early studies of activation of PKCs by phorbol esters suggested that they are oncogenes, − but some PKC activators such as bryostatin 1 are potent anticancer agents. ,− Because mutations in PKCs in cancer are generally loss of function, recent studies classify most PKCs as tumor suppressors. − Using FPCBA, we show here that PDBu and bryostatin 1 exhibit altered selectivity for specific PKC isozymes in cells compared with biochemical assays, offering a novel approach for optimization of related anticancer agents.…”

“…54,55 Small molecules that engage the ATP binding site of PKCs such as bisindolylmaleimide I (BIM1, GF109203X, Figure 2) 56 block the catalytic activity of PKCs and enhance interactions with allosteric activators by promoting the accessibility of C1 domains. 57 Early studies of activation of PKCs by phorbol esters suggested that they are oncogenes, 58−60 but some PKC activators such as bryostatin 1 61 are potent anticancer agents. 60,62−64 Because mutations in PKCs in cancer are generally loss of function, recent studies classify most PKCs as tumor suppressors.…”