In Silico Structural Analysis of Human β‐Glucuronidase for Antibody–Drug Conjugates Optimization

Human β-glucuronidase (HGUSB), a key lysosomal glycosyl hydrolase for the degradation pathway of glycosaminoglycans (GAGs), plays a crucial role in cell proliferation and inflammation, making it a promising target for novel therapeutic strategies including antibody-drug conjugates (ADCs) with β-glucuronic linkers. In this study, molecular docking and molecular dynamics (MD) simulations were performed to investigate the conformational stability of HGUSB in complex with different ligands, including substrates, inhibitors, and β-glucuronic linkers. Our rationale approach includes the evaluation of commercial substrates and a known inhibitor with different binding stoichiometries to identify the most favorable configuration and the most stable conformation of the enzyme. Based on the binding mechanism of HGUSB to these well-known ligands, the interaction with commercial linkers was evaluated, providing a structural determination of the recognition mechanism between the enzyme and ADCs. MD simulations on HGUSB::Linker complexes revealed that the maleimide-containing hydrophilic β-glucuronide, exhibited the most stable binding making it the best fitting linker among those analyzed in this study. Overall, this study identifies the optimal binding configuration of the HGUSB enzyme for investigating small molecule interactions and, despite the endogenous homotetrameric assembly, justifies the use of a simplified monomeric model for the study of larger macromolecular complexes, like linker analysis, ensuring an efficient and accurate computational approach. These findings lay the groundwork for a rationale optimization of β-glucuronic linker-based ADCs, offering new perspectives for targeted cancer therapies.