Automated patch clamp screening of amiloride and 5-N,N-hexamethyleneamiloride (HMA) analogs identifies 6-iodoamiloride as a potent acid-sensing ion channel inhibitor

Abstract

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate non-specific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6 disubstituted amiloride analogs using a custom-developed automated patch-clamp protocol and identified 6-iodoamiloride as a more potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 97 (hASIC1 97 IC50 88 nM cf. amiloride 11 IC50 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat small diameter dorsal root ganglion neurons (rDRG single-concentration 97 IC50 230 nM cf. 11 IC50 2.7 μM). 6-iodoamiloride represents the amiloride analogue of choice for studying the effects of ASIC inhibition on cell physiology.

4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors

“…After this fundamental step, we performed a high-throughput virtual screening of the Enamine screening library. In contrast to the well-published efforts of Merck [49] and recently published Kuduk and Finol-Urdaneta works [51,74] directed to the evolution of the parent amiloride molecule, the current in silico investigation discloses a new scaffold (Figure 6). The quality of our in silico model was further confirmed by electrophysiological assay resulting in the identification of three hit compounds.…”

“…However, for now, there are no available analogs in the chemical databases. Recently some novel analogs were suggested, using the isometric replacement of the amidine group, which usually causes a decrease in activity [71][72][73][74]. As opposed to this, synthesis and validation are also demanding.…”

“…Figure 6. Finalized outcome of the study represented in the form of a chemical flow charts: initial ancestor and its evolution based on selected papers and our findings[51,74].Author Contributions: Conceptualization, M.P., O.M. and D.M.V.…”