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.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.

Mechanisms of Action of Novel Influenza A/M2 Viroporin Inhibitors Derived from Hexamethylene Amiloride.

The increasing prevalence of influenza viruses with resistance to approved antivirals highlights the need for new anti-influenza therapeutics. Here we describe the functional properties of hexamethylene amiloride (HMA)-derived compounds that inhibit the wild-type and adamantane-resistant forms of the influenza A M2 ion channel. For example, 6-(azepan-1-yl)-N-carbamimidoylnicotinamide ( 9: ) inhibits amantadine-sensitive M2 currents with 3- to 6-fold greater potency than amantadine or HMA (IC50 = 0.2 vs. 0.6 and 1.3 µM, respectively). Compound 9: competes with amantadine for M2 inhibition, and molecular docking simulations suggest that 9: binds at site(s) that overlap with amantadine binding. In addition, tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 27: ) acts both on adamantane-sensitive and a resistant M2 variant encoding a serine to asparagine 31 mutation (S31N) with improved efficacy over amantadine and HMA (IC50 = 0.6 µM and 4.4 µM, respectively). Whereas 9: inhibited in vitro replication of influenza virus encoding wild-type M2 (EC50 = 2.3 µM), both 27: and tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 26: ) preferentially inhibited viruses encoding M2(S31N) (respective EC50 = 18.0 and 1.5 µM). This finding indicates that HMA derivatives can be designed to inhibit viruses with resistance to amantadine. Our study highlights the potential of HMA derivatives as inhibitors of drug-resistant influenza M2 ion channels.

5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site.

Controlling epithelial sodium channels with light using photoswitchable amilorides.

Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane proteins, assembled from ß, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx into non-epithelial cells. The functional role of δßγENaC in various organs, including the human brain, is still poorly understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical control of ENaC channels, in particular the δßγ isoform, by switching between blue and green light, or by turning on and off blue light. PA1 was used to modify functionally δßγENaC in amphibian and mammalian cells. We also show that PA1 can be used to differentiate between δßγENaC and αßγENaC in a model for the human lung epithelium.

2-Amidino analogs of glycine-amiloride conjugates: inhibitors of urokinase-type plasminogen activator.

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 µM and were cytotoxic to human U87 malignant glioma cells.

Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA).

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 µM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.

Novel inhibitors of the sodium-calcium exchanger: benzene ring analogues of N-guanidino substituted amiloride derivatives.

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on 45Ca(2+) uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N'-(1-naphtylmethyl)guanidine (IC(50)=3.4 microM) was found more active than amiloride (IC(50)=690 microM) and 3,4-dichlorobenzamil (IC(50)=15.2 microM), the reference inhibitor.

Species specificity of amidine-based urokinase inhibitors.

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.

Preparation and diuretic properties of novel amiloride analogues.

Fifteen novel amiloride analogues were synthesized and their diuretic properties compared to amiloride and triamterene in white wistar rats. Whereas none of the 6-substituted derivatives exhibited significant natriuretic and antikaliuretic effects, five of the compounds modified in the 2-position were found equal or better than standards. The results are discussed with respect to chemical structure and physiochemical properties.

Synthesis and characterization of methylbromoamiloride, a potential biochemical probe of epithelial Na+ channels.

We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel.

Synthesis, properties and biological activity of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide -- a new ligand for epithelial sodium channels.

A method is described for the synthesis and purification of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide (benzamil). The tritium was inserted at the meta position of the benzyl ring, from which it apparently does not exchange with solvent hydrogen. When stored in ethanol at -4 degrees C the radioligand remains stable for at least 15 months. The pharmacology of benzamil is very similar to that of amiloride in terms of its effects on sodium transporting epithelia except that it has a higher affinity. The affinity of benzamil for sodium channels in amphibian epithelia in the absence of sodium is approximately 10(9) M-1. The new ligand can be used to label sodium channels in epithelia, and may be useful in channel isolation procedures.