Measuring and modeling dynamics of regulators of G protein signaling to facilitate discovery of allosteric inhibitors of protein‐protein interactions (843.13)

Ischemic stroke morbidity and disability stem from neuronal death largely driven by glutamatergic excitotoxicity following hypoxia in brain tissue deprived of blood flow. Pharmacological inhibition of RGS4 with chemical probes significantly potentiates endogenous neuroprotection against excitotoxicity in vivo. The only known RGS4 inhibitors are unsuitable as clinical leads because they are relatively nonspecific covalent modifiers that inhibit through an uncharacterized, though putatively allosteric, mechanism. We seek to determine the structural mechanism by which current small‐molecule chemical probes allosterically inhibit RGS4. We hypothesize that inhibitors, binding to Cys148, trap a stochastically minor open conformation of RGS4 and disrupt a structurally key helical bundle to both prevent a unique conformational shift in RGS4 required to bind G‐alpha and to shift the population dynamics toward increasingly extended and unstable conformations. Structural changes in RGS proteins upon binding to inhibitory compounds measured with structural biological techniques including nuclear magnetic resonance, circular dichroism, light scattering, small‐angle X‐ray scattering, and atomistic molecular dynamics simulations support our hypothesis. Our investigations further show that RGS8, a homolog of RGS4, differs significantly in biophysical properties and we conclude that that these differences underly specificity of inhibitors for RGS4. Grant Funding Source : Supported by 5T32GM067795‐08 and pre‐doctoral fellowship from AFPE.