Disruption of Water Networks is the Cause of Human/Mouse Species Selectivity in Urokinase Plasminogen Activator (uPA) Inhibitors Derived from Hexamethylene Amiloride (HMA)

Abstract

The serine protease 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 that show anti-metastatic effects in vivo and high selectivity over closely related trypsin-like serine proteases, 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. To understand the molecular basis of this selectivity, we performed molecular dynamics simulations and alchemical free energy perturbation calculations using human and mouse uPA and their complexes with amiloride, HMA and 6-substituted HMA analogues and compared the results to enzyme inhibitory potencies. X-ray structures of selected compounds bound to partially murinised human uPA (H99Y) were also obtained. Collectively, the computational and experimental findings revealed that residue 99 is a key contributor to human/mouse uPA species 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). Analogue 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 highlights the crucial role water molecules can play in protein ligand binding and will serve to guide further optimisation of dual-potent human/mouse uPA inhibitors from the amiloride class as anti-metastasis drugs.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor

“…Relative to amiloride, 6-iodoamiloride 94 shows moderately decreased ENaC activity (the K + -sparing and diuretic target of the parent drug), and modestly improved potency at the sodium–hydrogen exchanger, isoform 1 (NHE1). ,, 6-iodoamiloride has been used as a probe of Na + -mediated processes in diverse settings, including in elucidation of the mechanisms of Na + -driven bacterial flagellar motion . In addition to differing selectivity for the classical Na + -driven targets of amiloride, 94 also shows improved inhibitory activity against the uPA, a TLSP known to play roles in cancer progression and inflammatory disorders. , The enhanced activity of 94 has been attributed to enhanced occupation of the S1β subsite of the uPA proteolytic domain, a tractable amiloride pharmacophore that has been extensively characterized by multiple groups. , Furthermore, we recently published an evaluation of 94 and related analogs for activity at the trimeric ligand-gated ion channel P2X7, a key regulator of peripheral and central inflammation and known off-target of amiloride, finding no significant difference in inhibitory activity when compared to amiloride in whole-cell functional assays …”

Identification of novel candidates for inhibition ofLasR, a quorum-sensing receptor of multidrug resistantPseudomonas aeruginosa, through a specialized multi-levelin silicoapproach

“…This is a typical cut-off value for studies with MD simulations followed by MM/GBSA calculations. 21,22,[69][70][71][72][73][74] SHAKE algorithm was used to constrain bonds with hydrogen atoms, enabling the application of an integration time-step of 2 fs.…”

Ion Transport and Inhibitor Binding by Human NHE1: Insights from Molecular Dynamics Simulations and Free Energy Calculations

“…Our previous work focused on the selective optimization of the side-activities of amiloride and its analogues as anticancer agents. ,− It has been reported that NHE1 is among the potential targets for the observed anticancer effects of amiloride and its analogues. ,,, To begin, we constructed a three-dimensional homology model of NHE1. With the publication of a long-awaited experimental structure and our interest in developing novel NHE1 inhibitors, we subsequently conducted molecular dynamics (MD) simulations of the apo- and ligand-bound states as well as relative binding free energy perturbations (FEP) MD simulations.…”