Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective
Benjamin J Buckley1,2,3,4, Ashna Kumar1,2,3, Ashraf Aboelela1,2
1Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.
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.
Insights
Amiloride analogs were screened for anti-cancer activity, revealing selective inhibitors for urokinase-type plasminogen activator (uPA) and sodium-hydrogen exchanger isoform-1 (NHE1). These compounds offer new tools for cancer research.
Area of Science:
- Oncology
- Pharmacology
- Medicinal Chemistry
Background:
- Amiloride, a K+-sparing diuretic, exhibits anti-cancer properties by inhibiting urokinase-type plasminogen activator (uPA) and sodium-hydrogen exchanger isoform-1 (NHE1).
- uPA is implicated in matrix degradation and tumor invasiveness, while NHE1 regulates pH and supports cancer progression.
Purpose of the Study:
- To co-screen a library of 5- and 6-substituted amilorides against uPA and NHE1.
- To identify selective and dual-targeting inhibitors as pharmacological probes for cancer research.
Main Methods:
- A library of amiloride analogs was synthesized and screened against purified uPA and NHE1.
- Inhibitory concentrations (IC50) and selectivity ratios were determined for key compounds.
Main Results:
- Pyrimidine analog 24 demonstrated dual uPA/NHE1 inhibition (IC50s 175 nM and 266 nM, respectively).
- Methoxypyrimidine analog 26 showed high uPA selectivity (IC50 86 nM, selectivity ratio 143).
- Morpholino (29) and oxazepine (30) analogs exhibited potent and selective NHE1 inhibition (IC50s 129 nM and 85 nM, respectively).
Conclusions:
- Novel amiloride derivatives provide selective and dual-targeting inhibitors for uPA and NHE1.
- These chemotype-matched, non-cytotoxic probes facilitate the study of selective versus dual inhibition of uPA and NHE1 in cancer.
- This research expands the pharmacological toolkit for investigating cancer mechanisms involving uPA and NHE1.
