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International Journal of Molecular... Mar 2021The 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 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 H 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 uPA IC = 175 nM, NHE1 IC = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine uPA IC = 86 nM, NHE1 IC = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino ( NHE1 IC = 129 nM, uPA IC = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) ( NHE1 IC = 85 nM, uPA IC = 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.
Topics: Amiloride; Breast Neoplasms; Cell Line, Tumor; Diuretics; Female; Humans; Models, Molecular; Neoplasm Invasiveness; Sodium-Hydrogen Exchanger 1; Structure-Activity Relationship; Urokinase-Type Plasminogen Activator
PubMed: 33804289
DOI: 10.3390/ijms22062999 -
Scientific Reports Aug 2022Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways...
Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways of how amiloride may reduce interstitial fibrosis. Amiloride was administered to a rat model of lithium induced interstitial fibrosis over a long term (6 months), as well as for short terms of 14 and 28 days. Kidney cortical tissue was subjected to RNA sequencing and microRNA expression analysis. Gene expression changes of interest were confirmed using immunohistochemistry on kidney tissue. Pathways identified by RNA sequencing of kidney tissue were related to 'promoting inflammation' for lithium and 'reducing inflammation' for amiloride. Validation of candidate genes found amiloride reduced inflammatory components induced by lithium including NF-κB/p65 and activated pAKT, and increased p53 mediated regulatory function through increased p21 in damaged tubular epithelial cells. Amiloride also reduced the amount of Notch1 positive PDGFrβ pericytes and infiltrating CD3 cells in the interstitium. Thus, amiloride attenuates a multitude of pro-inflammatory components induced by lithium. This suggests amiloride could be repurposed as a possible anti-inflammatory, anti-fibrotic agent to prevent or reduce the development of chronic interstitial fibrosis.
Topics: Amiloride; Animals; Fibrosis; Inflammation; Kidney; Lithium; Lung Diseases, Interstitial; Rats
PubMed: 36028651
DOI: 10.1038/s41598-022-18825-1 -
Molecules (Basel, Switzerland) Nov 2023This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of...
This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA-AML complex. Our findings revealed that AML specifically binds to Sudlow's site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA-AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible.
Topics: Humans; Serum Albumin, Human; Molecular Docking Simulation; Amiloride; Protein Binding; Binding Sites; Circular Dichroism; Thermodynamics; Leukemia, Myeloid, Acute; Spectrometry, Fluorescence
PubMed: 38067419
DOI: 10.3390/molecules28237688 -
Journal of Thrombosis and Haemostasis :... Feb 2014Blood-induced joint damage is characterized by synovitis and cartilage damage. Recently, we demonstrated that joint bleeding in hemophilic mice results in elevated...
BACKGROUND
Blood-induced joint damage is characterized by synovitis and cartilage damage. Recently, we demonstrated that joint bleeding in hemophilic mice results in elevated synovial levels of urokinase plasminogen activator (u-PA) and plasmin, and in plasmin-mediated cartilage damage.
OBJECTIVE
To evaluate whether treatment with amiloride (an inhibitor of u-PA) or antiplasmin attenuates synovitis and cartilage damage following joint bleeding in hemophilic mice.
METHODS
Following the induction of joint bleeding, hemophilic mice were randomized between daily oral treatment with amiloride (1 mg kg⁻¹) or control, or weekly intra-articular treatment with amiloride (2.5 mg mL⁻¹), antiplasmin (2.5 mg mL⁻¹), or control. After 5 weeks of treatment, synovitis and cartilage damage were determined on hematoxylin and eosin-stained (Valentino score) and Safranin O-stained sections, respectively.
RESULTS
No effects of oral and intra-articular treatment with amiloride were found. In contrast, intra-articular treatment with antiplasmin resulted in significant (P < 0.01) reductions in both synovitis (score 1, 11.1% vs. 0%; score 2, 11.1% vs. 4.2%; score 3, 61.1% vs. 16.7%; score 4, 5.6% vs. 29.2%; score 5, 11.1% vs. 20.8%; score 6, 7.7% vs. 8.3%; score 7, 0% vs. 8.3%; and score 8, 0% vs. 12.5%) and cartilage damage (score 2, 10% vs. 8.3%; score 3, 50% vs. 12.5%; score 4, 30% vs. 33.3%; score 5, 10% vs. 33.3%; and score 6, 0% vs. 16.7%) as compared with controls.
CONCLUSIONS
Intra-articular treatment with antiplasmin (but not amiloride) following joint bleeding prevented synovitis and cartilage damage in hemophilic mice. These data offer promise for the use of antiplasmin as a new therapeutic intervention for patients who suffer from joint bleeds despite administration of clotting factor.
Topics: Amiloride; Animals; Antifibrinolytic Agents; Cartilage; Hemarthrosis; Hemophilia A; Mice; Synovitis
PubMed: 24283895
DOI: 10.1111/jth.12467 -
The Journal of Biological Chemistry Sep 1987Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for...
Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for DNA synthesis to occur in situ. In experiments to determine the mechanism of inhibition of DNA synthesis by amiloride, we observed that amiloride inhibited both the catalytic activity of purified DNA topoisomerase II in vitro and DNA topoisomerase II-dependent cell functions in vivo. Many compounds capable of inhibiting DNA topoisomerase II are DNA intercalators. Thus, we performed studies to determine if and how amiloride bound to DNA. Results indicated that amiloride 1) shifted the thermal denaturation profile of DNA, 2) increased the viscosity of linear DNA, and 3) unwound circular DNA, all behavior consistent with a DNA intercalation mechanism. Furthermore, quantitative and qualitative measurements of amiloride fluorescence indicated that amiloride (a) bound reversibly to purified DNA under conditions of physiologic ionic strength, and (b) bound to purified nuclei in a highly cooperative manner. Lastly, amiloride did not promote the cleavage of DNA in the presence of DNA topoisomerase II, indicating that the mechanism by which amiloride inhibited DNA topoisomerase II was not through the stabilization of a "cleavable complex" formed between DNA topoisomerase II, DNA, and amiloride. The ability of amiloride to intercalate with DNA and inhibit topoisomerase II is consistent with the proposed planar, hydrogen-bonded, tricyclic nature of amiloride's most stable conformation. Thus, DNA and DNA topoisomerase II must be considered as new cellular targets of amiloride action.
Topics: Amiloride; Carcinogens; Cell Adhesion; Cell Line; DNA; Humans; Intercalating Agents; Novobiocin; Nucleic Acid Denaturation; Phorbol 12,13-Dibutyrate; Phorbol Esters; Plasmids; Topoisomerase II Inhibitors; Viscosity
PubMed: 2820967
DOI: No ID Found -
American Journal of Physiology.... Dec 2022Amiloride has been shown to inhibit acid-sensing ion channels (ASICs), which contribute to ischemia-related muscle pain during exercise. The purpose of this study was to... (Randomized Controlled Trial)
Randomized Controlled Trial
Amiloride has been shown to inhibit acid-sensing ion channels (ASICs), which contribute to ischemia-related muscle pain during exercise. The purpose of this study was to determine if a single oral dose of amiloride would improve exercise tolerance and attenuate blood pressure during blood-flow-restricted (BFR) exercise in healthy adults. Ten subjects (4 females) performed isometric plantar flexion exercise with BFR (30% maximal voluntary contraction) after ingesting either a 10-mg dose of amiloride or a volume-matched placebo (random order). Time to failure, time-tension index (TTI), and perceived pain (visual analog scale) were compared between the amiloride and placebo trials. Mean blood pressure, heart rate, blood pressure index (BPI), and BPI normalized to TTI (BPI) were also compared between trials using both time-matched (TM and TM) and effort-matched (T and T) comparisons. Time to failure (+69.4 ± 63.2 s, < 0.01) and TTI (+1,441 ± 633 kg·s, = 0.02) were both significantly increased in the amiloride trial compared with placebo, despite no increase in pain (+0.4 ± 1.7 cm, = 0.46). In contrast, amiloride had no significant influence on the mean blood pressure or heart rate responses, nor were there any significant differences in BPI or BPI between trials when matched for time (all ≥ 0.13). When matched for effort, BPI was significantly greater in the amiloride trial (+5,300 ± 1,798 mmHg·s, = 0.01), likely owing to an increase in total exercise duration. In conclusion, a 10-mg oral dose of amiloride appears to significantly improve the tolerance to BFR exercise in healthy adults without influencing blood pressure responses.
Topics: Adult; Female; Humans; Male; Amiloride; Blood Pressure; Heart Rate; Hemodynamics; Regional Blood Flow; Resistance Training
PubMed: 36222880
DOI: 10.1152/ajpregu.00190.2022 -
JCI Insight Nov 2023Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that...
Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.
Topics: Mice; Male; Animals; Amiloride; Lipoylation; Epithelial Sodium Channels; Sodium
PubMed: 37707951
DOI: 10.1172/jci.insight.172051 -
PLoS Pathogens May 2021SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope...
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.
Topics: Amiloride; Animals; Antiviral Agents; Binding Sites; COVID-19; Chlorocebus aethiops; Coronavirus Envelope Proteins; Humans; Ion Channels; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Protein Domains; SARS-CoV-2; Vero Cells; Virus Assembly; COVID-19 Drug Treatment
PubMed: 34003853
DOI: 10.1371/journal.ppat.1009519 -
The Journal of Biological Chemistry Jul 2005The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several...
The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are alphaSer-583, betaGly-525, and gammaGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the beta and gamma glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the alpha subunit resulted in moderate (<5-fold) weakening of amiloride K(i). We investigated the role of alphaSer-583 in amiloride binding by systematically mutating alphaSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a beta-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at alpha583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some alphaSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that alphaSer-583 may have a role in ion permeation through ENaC.
Topics: Amiloride; Animals; Binding Sites; Diuretics; Dose-Response Relationship, Drug; Epithelial Sodium Channels; Glycine; Histidine; Hydrogen-Ion Concentration; Ions; Kinetics; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Oocytes; Patch-Clamp Techniques; Protein Binding; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; RNA, Complementary; Serine; Sodium; Sodium Channels; Xenopus laevis
PubMed: 15908426
DOI: 10.1074/jbc.M503500200 -
Journal of Korean Medical Science Aug 2013Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in...
Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%±12% and 76%±14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.
Topics: Acid Sensing Ion Channels; Amiloride; Analgesics; Animals; Disease Models, Animal; Male; Neuralgia; RNA, Messenger; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Transcription, Genetic
PubMed: 23960454
DOI: 10.3346/jkms.2013.28.8.1238