Synthesis and Characterization of Novel Mono- and Bis-Guanyl Hydrazones as Potent and Selective ASIC1 Inhibitors Able to Reduce Brain Ischemic Insult

Abstract

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca 2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazeneinspired diaryl mono-and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo-and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

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

“…Furthermore, proton-activated currents elicited by exposure to pH 5.5 were inhibited by the ASIC1-selective peptide toxin Mambalgin 1 with a K D of 27.7 ± 2.8 nM ( n = 6) (Figure D), and were absent upon siRNA knockdown of ASIC1 transcripts encoding for ASIC1 (GFP CTR = −36.7 ± 3.2 pA/pF, n = 6; GFP – ASIC1 = −5.2 ± 3.4 pA/pF, n = 6; unpaired t test, p = < 0.0001) (Figure E). Positive immunostaining for the canonical isoform ASIC1a (Figure S1A–D) that correlated with the pattern observed using the pan-ASIC1MaB cocktail (Figure S1E–H) and the lack of ASIC3 signal, are consistent with previous reports , supporting ASIC1a channels as the primary ASIC isoform mediating acid-sensitive currents in HEK-293 cells. Thus, the functional characterization of ASIC-mediated currents in tsA-201 cells supports their use as a readily available and affordable option for library screening at physiologically relevant levels of ASIC expression.…”

“…Acid-sensitive currents mediated by ASIC1 channels have been described in human embryonic kidney (HEK-293) cells. , We initially assessed pH-sensitive (ASIC) currents in tsA-201 (an SV40 T antigen-transformed HEK-293 clone) for automated patch clamp (APC) screening of large compound libraries as a low-cost alternative to proprietary ASIC-overexpressing cell lines. From a conditioning pH (pH C ) 7.4, tsA-201 cells exposed to acidic stimuli (pH S = 5.5) displayed robust whole-cell inward currents (35.2 ± 3.1 p A /p F , n = 20) that activated monoexponentially with a time constant of 81.2 ± 4.9 ms ( n = 20) under APC (holding potential, V h = −60 mV).…”

“…The aminoindazole lead 4-fluoro-6-((1-(5-fluoro-2-methylpyridin-4-yl)­azetidin-3-yl)­oxy)-1 H -indazol-3-amine ( 13 ) showed potent activity against wild-type ASIC1 at pH 6.0 (WT IC 50 = 25 nM), with target engagement demonstrated by significant decreases in inhibition against heterologously expressed ASIC1 bearing single alanine substitutions at the putative binding site (F98A IC 50 = 746 nM, Y340A IC 50 = 261 nM). Continuing from the diminazine precedent, Gornati and co-workers recently reported an extensive optimization campaign aimed at developing blood-brain barrier (BBB) permeable leads for treating ischemic brain injury . These efforts produced a series of 3,5-pyrazolyl­bisguanylhydrazone and bisimidazolyl­hydrazone leads with activity against human ASIC1 in the 0.4–2.6 μM.…”

Hemorrhagic Stroke Induces a Time-Dependent Upregulation of miR-150-5p and miR-181b-5p in the Bloodstream

“…All these membrane proteins are implied in cellular ionic homeostasis, and their activity has been strongly linked to stroke pathophysiology (36)(37)(38). Similarly, among miR-180 targets linked to stroke pathophysiology, the membrane channel acid sensing ionic channel, ASIC1, and the Na + /H + exchanger should be mentioned (52,53).…”