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

10 August 2021, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

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.

Keywords

urokinase plasminogen activator
species selectivity
molecular dynamics simulations
free energy perturbation
water network

Supplementary materials

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Supporting Information
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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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