Disruption of water networks is the cause of human/mouse species selectivity in urokinase plasminogen activator (uPA) inhibitors derived from hexamethylene amiloride (HMA)

Urokinase plasminogen activator (uPA) plays a critical role in tumour cell invasion and migration and is a promising anti-metastasis drug target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent uPA inhibitors while lacking the diuretic and anti-kaliuretic properties of the parent drug amiloride. However, the compounds as a class display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenografted mouse models of cancer. We applied molecular dynamics simulations, free energy perturbation, X-ray crystallography and biochemical assays to understand the molecular basis of this selectivity. Our findings revealed that residue 99 is a key contributor to human/mouse selectivity, whereby enthalpically unfavourable steric expulsion of a water molecule by the 5-N,N-hexamethylene ring of HMA and analogues occurs when residue 99 is Tyr (as in mouse uPA). The study will serve to guide further optimisation of dual-potent human/mouse uPA inhibitors from the amiloride class as anti-metastasis drugs.