Abstract
The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising anti-metastasis target. 6-Substituted analogs of 5-N,N-(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and anti-kaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenografted mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5-N,N-hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analog 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as anti-metastasis drugs.
Supplementary materials
Title
Supporting Information
Description
Binding affinities and poses from molecular docking, snapshots, RMSD, RMSF, analysis of hydrogen bond and non-bonded interactions and water density maps from MD simulations, thermodynamic cycles used and relative binding free energies calculated from FEP and X-ray crystallography refinement data and statistics (PDF).
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