A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

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

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 nonspecific 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 potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Antifungal activity of 6-substituted amiloride and hexamethylene amiloride (HMA) analogs.

Fungal infections have become an increasing threat as a result of growing numbers of susceptible hosts and diminishing effectiveness of antifungal drugs due to multi-drug resistance. This reality underscores the need to develop novel drugs with unique mechanisms of action. We recently identified 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of human Na+/H+ exchanger isoform 1, as a promising scaffold for antifungal drug development. In this work, we carried out susceptibility testing of 45 6-substituted HMA and amiloride analogs against a panel of pathogenic fungi. A series of 6-(2-benzofuran)amiloride and HMA analogs that showed up to a 16-fold increase in activity against Cryptococcus neoformans were identified. Hits from these series showed broad-spectrum activity against both basidiomycete and ascomycete fungal pathogens, including multidrug-resistant clinical isolates.

6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist.

P2X7 is an extracellular adenosine 5'-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1ß release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

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

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.

Systematic evaluation of structure-property relationships and pharmacokinetics in 6-(hetero)aryl-substituted matched pair analogs of amiloride and 5-(N,N-hexamethylene)amiloride.

The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.