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 = 5715 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.
Highlights
Regulation of transmembrane pH gradients is an essential requirement for cellular homeostasis and healthy function in virtually all cell types
Substitution at the 5-position of the benzofuran ring was not favoured in either series except for 5-fluoro HMA analog 9, which showed a modest improvement relative to the unsubstituted parent 5. 5-Furopyridine 19 and 4-furopyridine
We identified 6-substituted amiloride and HMA analogs showing dualand single-target selective activity against urokinase-type plasminogen activator (uPA) and NHE1
Summary
Regulation of transmembrane pH gradients is an essential requirement for cellular homeostasis and healthy function in virtually all cell types. Upregulation of acidic metabolism (i.e., the Warburg Effect) [5] promotes activation of NHE1, leading to acidification of the extracellular tumor microenvironment and alkalinisation of cytosolic pH [6] These transmembrane pH perturbations confer a survival advantage to transformed cells, promoting carcinogenic progression [7]. Amiloride (i.e., 5-NH2 ) [27] analogs targeting the uPA S1β subsite resulted in uPA potency gains exceeding 100-fold Lead compounds from these series showed low nM uPA potency, high selectivity for uPA across the serine hydrolase superfamily, no ENaC activity, and in vivo anti-metastatic effects in xenografted mouse models of lung and pancreatic cancer [26,27]. We co-screened our 5- and 6-substituted amiloride libraries for NHE1 and uPA activity and report the discovery of non-cytotoxic, dual-uPA/NHE1 active, and single target-selective amilorides
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
