Matched Analogue Analysis of Structural Responses Before and After Covalent Bond Formation in Wild-Type and A775_G776insYVMA HER2

Endpoint structure–activity relationships (SARs) for irreversible inhibitors can integrate reversible binding, covalent chemistry, conformational behavior, and cellular factors, making it difficult to assign a potency change to a single molecular event. We reanalyzed a previously reported series of irreversible inhibitors of wild-type (WT) HER2 and HER2 A775_G776insYVMA (HER2YVMA) as six matched analogue pairs. Three complementary analyses were kept physically distinct. Pre-reaction molecular dynamics (MD) trajectories were used to quantify the fraction of frames satisfying a predefined near-attack-conformation (NAC)-like distance–angle criterion; continuous structural descriptors calculated from OpenComplex2 (OC2)-generated covalently linked HER2–inhibitor structures were used to characterize structural differences after covalent linkage; and covalent-adduct MD trajectories were used to characterize post-bond-formation dynamics. The Michael addition step itself was neither simulated nor measured. Cyclization of compound 3 to compound 9c increased the NAC-like frame fraction in HER2WT from 0.0217% to 0.358% but decreased it in HER2YVMA from 0.745% to 0.333%, with the overall direction preserved across all six sensitivity definitions. In HER2YVMA, the modification distinguishing compounds 4a and 5a increased the NAC-like fraction from 2.620% to 6.244% despite a loss of cellular potency. The analogous tail modification increased this fraction in the 8c/10e series, whereas the HER2YVMA 9c/11f response was starting conformation sensitive. The distribution of OC2-generated covalently linked structures across the predefined structural coordinate and the final-adduct MD readouts likewise varied with HER2 context and scaffold, although some differential effects were sensitive to the coordinate reference. These analyses complement experimental SARs but do not provide a measure of reaction kinetics or thermodynamic populations.