Gaussian accelerated molecular dynamics uncovers binding mechanism differences between small-molecule inhibitors and...

Glucose-regulated protein 94 (Grp94), an endoplasmic reticulum-resident Hsp90 (heat shock protein 90) homolog, plays indispensable roles in the development and physiology of multicellular organisms by mediating the folding of client proteins. However, aberrant overexpression of Grp94 disrupts client protein homeostasis by inducing misfolding, thereby contributing to the pathogenesis of a range of diseases. Small-molecule inhibitors targeting the ATP binding site in Grp94 can inhibit its biological function, but their clinical utility is hampered by off-target binding to homologous Hsp90α, which causes undesirable side effects. Thus, the development of Grp94-selective small-molecule inhibitors remains an unmet need in drug discovery. In this study, three compounds (NPCA, PU-H36, and BnIm1) were selected to generate initial structures, followed by Gaussian accelerated molecular dynamics (GaMD) simulations to evaluate their performances. A multidisciplinary analytical approach, including principal component analysis (PCA), correlation network analysis (CNA), free energy landscape (FEL) analysis, molecular mechanics generalized Born surface area (MM-GBSA) calculations, Hirshfeld Surface analysis (HSA) and residue free energy decomposition, was employed to compare the binding mechanisms of these compounds to Grp94/Hsp90α and elucidate the molecular basis underlying Grp94 selectivity. Key findings showed that ligand binding causes significant perturbations in the conformational dynamics and energy state distribution of Grp94/Hsp90α, confirming the pivotal role of α1, α4, and α5 subdomains in orchestrating the conformational regulation of these chaperones. Conformational superimposition analyses revealed that residue displacements in Grp94 optimize the binding pose of inhibitors and facilitate their access to selective binding sites. Moreover, N107 (N51 in Hsp90α), M154 (M98 in Hsp90α), N162 (N106 in Hsp90α), F195 (F138 in Hsp90α), and T245 (T184 in Hsp90α) were identified as core residues that mediate ligand binding. Notably, H36 has strong interactions with the non-conservative residues F199 and I247 in Grp94, which contribute to the selectivity of H36 for Grp94. Overall, the results of the present study identify ligand-induced dynamic differences between Grp94 and Hsp90α and reveal the molecular mechanism governing Grp94 selectivity. This work provides novel mechanistic insights and a robust theoretical framework to guide the rational design of Grp94-selective therapeutic agents.