Drug design using unique conformations to preferentially target a specific site on collagen-bound MMP1

Abstract

Precise site-specific drug design remains a challenge in structure-based drug discovery. Most existing approaches screen for ligands to target binding pockets on a protein surface based on static structures obtained from techniques such as X-ray, NMR, cryo-EM, and AlphaFold. However, the structure-function paradigm is, in reality, a structure-dynamics-function relationship that determines a proteins binding and activity. As such, drug screening or design without evaluating binding competition across the protein surface or considering the receptors dynamic, substrate-dependent conformational states is incomplete. Substrate-specific unique protein conformations are underexplored and offer novel opportunities for selective therapeutic targeting, though systematic workflows for identifying and exploiting such sites remain limited. Previously, we showed that collagen alters matrix metalloprotease-1 (MMP1) dynamics and that R405 is an allosteric residue on the MMP1 surface that exhibits strong dynamic correlations with its active site. Here, we present a substrate-specific allosteric drug-design framework that targets specific sites on a protein, using collagen-bound MMP1 as a model system. We determined the conformational dynamics of free and collagen-bound MMP1 using all-atom molecular dynamics (MD) simulations and categorized conformations into clusters of similar conformations. We then compared and identified unique conformations that occur only in collagen-bound MMP1 to design drugs against them using a machine-learning approach. The top three unique clusters were used to generate approximately 150,000 candidate compounds that were then screened against both the R405-centered region and all detectable binding pockets across the MMP1 surface. We have found several compounds that bind preferentially around R405 by at least 0.3 kcal/mol relative to competing sites across the surface. This strategy establishes a generalizable framework for designing ligands that preferentially target substrate-specific allosteric sites, providing new opportunities for precision therapeutics that modulate proteins in their biologically relevant functional states. Simple SummaryIn this paper, we establish a substrate-specific allosteric drug-design strategy that integrates all-atom molecular dynamics simulations, conformational clustering, machine-learning-based ligand design, and surface-wide binding-selectivity screening, using collagen-bound MMP1 as a model system. We show that collagen binding reshapes the conformational ensemble of MMP1, creating unique conformational states that are absent or inaccessible in the free enzyme. By identifying these substrate-specific conformations, generating ligands based on the corresponding dynamic fingerprints around the collagen-specific allosteric residue R405, and screening compounds across all binding pockets on the MMP1 surface, we demonstrate preferential targeting of the collagen-specific site relative to competing pockets. These results establish a generalizable framework for designing ligands that selectively recognize biologically relevant substrate-bound conformations rather than static protein structures alone. Substrate-specific allosteric targeting may enable selective modulation of individual protein functions while minimizing off-target interactions, providing new opportunities for precision therapeutics against dynamic protein systems.