Uncovering the potential of the anchor site for enhancing Golgi alpha-mannosidase II selectivity using molecular dynamics simulations and free energy calculations.

Designing small molecule inhibitors that are highly selective for Golgi alpha mannosidase II (GMII) over lysosomal alpha mannosidase (LM) remains crucial for the development of novel anticancer drugs targeting the N-glycosylation pathway. Studies have previously identified an unconserved 'anchor site' in GMII that represents an attractive target for achieving selectivity. In this study we conduct molecular dynamics simulations and free energy calculations of GMII and LM with their natural oligosaccharide substrates to investigate the potential of the anchor site. Our findings reveal that the corresponding N-acetylglucosamine residue remains tightly bound to the anchor site in GMII, helping stabilize overall substrate binding to GMII, while the lack of a similar conserved site in LM allows the substrate to fluctuate more freely. Our simulations also suggest that besides stabilizing the catalytic site mannose for cleavage, the anchor site residue may play a role in facilitating the release of the holding site mannose and its transition to the catalytic site in GMII. Subsequently, free energy calculations reveal that the anchor site N-acetylglucosamine contributes 4.107 kcal/mol to the free energy of binding in GMII, with a significantly smaller contribution of 1.035 kcal/mol in LM. This difference of 3.072 kcal/mol in the free energy of binding represents potential gains in selectivity that could be achieved by targeting the anchor site in GMII. Taken together, these findings provide further evidence on the potential of targeting the anchor site in the design of highly selective GMII inhibitors.