Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors

Abstract

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co‑screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5,715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.

Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in Vibrio cholerae

“…The mammalian Na + /H + exchanger isoform 1 (NHE1) is ubiquitously expressed integral membrane protein present in mammalian cells and dysregulation of NHE1 is often associated with heart disease and cancer (Fliegel, L. 2008). The NHE1 plays an important role as a central regulator of transmembrane pH supporting carcinogenic progression (Buckley et al 2021) After extensive exploration of ADMET and QSAR properties, we used in silico methods to determine the pharmacological consequences of AMLRD and its analogues (Supplementary file 1). Among them, six AMLRD analogs with modest pharmacokinetic features and the lowest toxicities were obtained for molecular docking simulations against Vc-NhaP2.…”

“…Amiloride was the first drug discovered to inhibit mammalian Na + /H + exchanger isoform 1 (NHE1) (Pedersen et al 2007). The NHE1 plays an important role as a central regulator of transmembrane pH supporting carcinogenic progression (Buckley et al 2021;Teiwes et al 2007). Amiloride, a potassium-sparing diuretic, is capable of inhibiting the Na + /H + exchangers (NHE) and prevents cancer progression (Buckley et al 2021;Teiwes et al 2007) In time, several amiloride analogs have been synthesized to have a marked increase in their specificity to inhibit the NHE (Buckley et al 2021;Teiwes et al 2007).…”

“…The NHE1 plays an important role as a central regulator of transmembrane pH supporting carcinogenic progression (Buckley et al 2021;Teiwes et al 2007). Amiloride, a potassium-sparing diuretic, is capable of inhibiting the Na + /H + exchangers (NHE) and prevents cancer progression (Buckley et al 2021;Teiwes et al 2007) In time, several amiloride analogs have been synthesized to have a marked increase in their specificity to inhibit the NHE (Buckley et al 2021;Teiwes et al 2007). There have been several studies showing amiloride and its analogs exerting robust anti-cancer effects in multiple rodent models at supra-clinical doses (Buckley et al 2021).…”

“…Amiloride, a potassium-sparing diuretic, is capable of inhibiting the Na + /H + exchangers (NHE) and prevents cancer progression (Buckley et al 2021;Teiwes et al 2007) In time, several amiloride analogs have been synthesized to have a marked increase in their specificity to inhibit the NHE (Buckley et al 2021;Teiwes et al 2007). There have been several studies showing amiloride and its analogs exerting robust anti-cancer effects in multiple rodent models at supra-clinical doses (Buckley et al 2021). In a recent study, 5-and 6substituted amilorides has been synthesised that acted in the inhibition of two different cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator, a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the NHE1 (Buckley et al 2021).…”

“…There have been several studies showing amiloride and its analogs exerting robust anti-cancer effects in multiple rodent models at supra-clinical doses (Buckley et al 2021). In a recent study, 5-and 6substituted amilorides has been synthesised that acted in the inhibition of two different cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator, a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the NHE1 (Buckley et al 2021).…”