An interdisciplinary approach provides insights into the pronounced selectivity of compound 42 for DPP9.

Dipeptidyl peptidase 8 (DPP8) and 9 (DPP9) are proteases gaining significant attention for their role in health and disease. Distinctive studies of these proteases are hampered by their close homology. Furthermore, designing selective compounds is a major challenge due to the highly conserved catalytic site. Here, we provide mechanistic insights underlying the DPP9-over-DPP8 selectivity of the semi-selective inhibitor "Compound 42". We performed enhanced sampling molecular dynamics simulations to investigate the binding pose of "Compound 42", which enabled the design of various DPP9 mutants that were characterized through a combination of biochemical (Ki determinations) and in silico approaches. Our findings show that DPP9 residue F253 is an important selectivity-determining factor. This work marks the discovery and validation of a structural feature that can be exploited for the design of DPP8 or DPP9 selective inhibitors.