Modifying inhibitor specificity for homologous enzymes by machine learning

Selective inhibitors are essential for targeted therapeutics and for probing enzyme functions in various biological systems. The two main challenges in identifying such protein-based inhibitors lie in the extensive experimental effort required, including the generation of large libraries, and in tailoring the selectivity of inhibitors to enzymes with homologous structures. To address these challenges, machine learning (ML) is being used to improve protein design by training on targeted libraries and identifying key interface mutations that enhance affinity and specificity. However, such ML-based methods are limited by inaccurate energy calculations and difficulties in predicting the structural impacts of multiple mutations. Here, we present an ML-based method that leverages HTS data to streamline the design of selective protease inhibitors. To demonstrate its utility, we applied our new method to find inhibitors of matrix metalloproteinases (MMPs), a family of homologous proteases involved in both physiological and pathological processes. By training ML models on binding data for three MMPs (MMP-1, MMP-3, and MMP-9), we successfully designed a novel N-TIMP2 variant with a differential specificity profile, namely, high affinity for MMP-9, moderate affinity for MMP-3, and low affinity for MMP-1. Our experimental validation showed that this novel variant exhibited a significant specificity shift and enhanced selectivity compared with wild-type N-TIMP2. Through molecular modeling and energy minimization, we obtained structural insights into the variant's enhanced selectivity. Our findings highlight the power of ML-based methods to reduce experimental workloads, facilitate the rational design of selective inhibitors, and advance the understanding of specific inhibitor–enzyme interactions in homologous enzyme systems.