Repurposing of triamterene as a histone deacetylase inhibitor to overcome cisplatin resistance in lung cancer treatment.

PURPOSE: Cisplatin is the core chemotherapeutic drug used for first-line treatment of advanced non-small cell lung cancer (NSCLC). However, drug resistance is severely hindering its clinical efficacy. This study investigated the circumvention of cisplatin resistance by repurposing non-oncology drugs with putative histone deacetylase (HDAC) inhibitory effect. METHODS: A few clinically approved drugs were identified by a computational drug repurposing tool called "DRUGSURV" and evaluated for HDAC inhibition. Triamterene, originally indicated as a diuretic, was chosen for further investigation in pairs of parental and cisplatin-resistant NSCLC cell lines. Sulforhodamine B assay was used to evaluate cell proliferation. Western blot analysis was performed to examine histone acetylation. Flow cytometry was used to examine apoptosis and cell cycle effects. Chromatin immunoprecipitation was conducted to investigate the interaction of transcription factors to the promoter of genes regulating cisplatin uptake and cell cycle progression. The circumvention of cisplatin resistance by triamterene was further verified in a patient-derived tumor xenograft (PDX) from a cisplatin-refractory NSCLC patient. RESULTS: Triamterene was found to inhibit HDACs. It was shown to enhance cellular cisplatin accumulation and potentiate cisplatin-induced cell cycle arrest, DNA damage, and apoptosis. Mechanistically, triamterene was found to induce histone acetylation in chromatin, thereby reducing the association of HDAC1 but promoting the interaction of Sp1 with the gene promoter of hCTR1 and p21. Triamterene was further shown to potentiate the anti-cancer effect of cisplatin in cisplatin-resistant PDX in vivo. CONCLUSION: The findings advocate further clinical evaluation of the repurposing use of triamterene to overcome cisplatin resistance.

Drug repositioning of Clopidogrel or Triamterene to inhibit influenza virus replication in vitro.

Influenza viruses cause respiratory tract infections and substantial health concerns. Infection may result in mild to severe respiratory disease associated with morbidity and some mortality. Several anti-influenza drugs are available, but these agents target viral components and are susceptible to drug resistance. There is a need for new antiviral drug strategies that include repurposing of clinically approved drugs. Drugs that target cellular machinery necessary for influenza virus replication can provide a means for inhibiting influenza virus replication. We used RNA interference screening to identify key host cell genes required for influenza replication, and then FDA-approved drugs that could be repurposed for targeting host genes. We examined the effects of Clopidogrel and Triamterene to inhibit A/WSN/33 (EC50 5.84 uM and 31.48 uM, respectively), A/CA/04/09 (EC50 6.432 uM and 3.32 uM, respectively), and B/Yamagata/16/1988 (EC50 0.28 uM and 0.11 uM, respectively) replication. Clopidogrel and Triamterene provide a druggable approach to influenza treatment across multiple strains and subtypes.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney.

Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.

The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Mesenchymal subtype neuroblastomas are addicted to TGF-ßR2/HMGCR-driven protein geranylgeranylation.

The identification of targeted agents with high therapeutic index is a major challenge for cancer drug discovery. We found that screening chemical libraries across neuroblastoma (NBL) tumor subtypes for selectively-lethal compounds revealed metabolic dependencies that defined each subtype. Bioactive compounds were screened across cell models of mesenchymal (MESN) and MYCN-amplified (MYCNA) NBL subtypes, which revealed the mevalonate and folate biosynthetic pathways as MESN-selective dependencies. Treatment with lovastatin, a mevalonate biosynthesis inhibitor, selectively inhibited protein prenylation and induced apoptosis in MESN cells, while having little effect in MYCNA lines. Statin sensitivity was driven by HMGCR expression, the rate-limiting enzyme for cholesterol synthesis, which correlated with statin sensitivity across NBL cell lines, thus providing a drug sensitivity biomarker. Comparing expression profiles from sensitive and resistant cell lines revealed a TGFBR2 signaling axis that regulates HMGCR, defining an actionable addiction in that leads to MESN-subtype-dependent apoptotic cell death.

Systematic analysis and identification of unexpected interactions from the neuroprotein drug interactome in hydrocephalus pharmacological intervention.

Hydrocephalus is a neurological condition caused by an abnormal accumulation of cerebrospinal fluid; pharmacological intervention of the disease has been found to elicit a variety of adverse drug reactions (ADRs) in central nervous system (CNS) by unexpectedly targeting certain functional neuroproteins. Here, a systematic neuroprotein drug interactome (SNDI) is created for 11 hydrocephalus drugs/metabolites plus 20 control drugs across 518 druggable pockets on the surface of 472 CNS neuroproteins via a large-scale molecular docking approach. Heuristic clustering analysis of the SNDI profile divides the 31 investigated drug ligands into a distinct panel and a background panel; the former consists of two hydrocephalus drugs (Furosemide and Triamterene) and their respective metabolites (Furosemide glucuronide and Hydroxytriamterene) that are inferred to have generally high affinity towards the whole array of neuroprotein pockets. A total of 13 neuroproteins are enriched in gene ontology semantic mining as putative unexpected targets of the distinct panel, and their intermolecular interactions with hydrocephalus drugs/metabolites are investigated in detail using dynamics simulation and energetics analysis. We also perform kinase assay and viability test to substantiate the interactome analysis. It is found that the Furosemide and Triamterene have significant cytotoxic effects on normal human astrocytes, in which the Triamterene can inhibit the neurokinase ROCK2, a representative of putative unexpected targets, with a high activity, which is comparable with the sophisticated ROCK2 inhibitor Fasudil.

In vitro cytotoxicity and DNA/HSA interaction study of triamterene using molecular modelling and multi-spectroscopic methods.

The anticancer activity of triamterene on HCT116 and CT26 colon cancer cells lines was investigated. Furthermore, the mechanism of interaction between triamterene and calf thymus DNA (ct-DNA) and also human serum albumin (HSA) was conducted using spectroscopic and molecular docking techniques. In vitro cytotoxicity of triamterene against HCT116 and CT26 cells showed promising anticancer effects with IC50 values of 31.30 and 24.45 µM, respectively. Competitive studies of the triamterene with NR (neutral red) and MB (methylene blue) as intercalator probes showed that triamterene can be replaced by these probes. The viscosity data also confirmed that triamterene binds to calf-thymus DNA through intercalation binding mode. Binding properties of triamterene with HSA in the presence of warfarin and ibuprofen showed that triamterene competes with warfarin for the site I of human serum albumin (HSA). In addition, the binding modes of triamterene with DNA and HSA were verified by molecular docking technique. Abbreviations ct-DNA calf thymus DNA CV cyclic voltammetry DNA deoxyribonucleic acid DPV differential pulse voltammetry FBS fetal bovine serum HSA human serum albumin NR neutral red MB methylene blue MTT 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide Communicated by Ramaswamy H. Sarma.

Investigation of triamterene as an inhibitor of the TGR5 receptor: identification in cells and animals.

BACKGROUND: G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5. METHODS: We transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo. RESULTS: Triamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na+/Ca2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats. CONCLUSION: Based on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.

Drug-Repositioning Screens Identify Triamterene as a Selective Drug for the Treatment of DNA Mismatch Repair Deficient Cells.

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial.Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivoResults: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks.Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880-90. ©2016 AACR.

Chemometrics-assisted determination of amiloride and triamterene in biological fluids with overlapped peaks and unknown interferences.

BACKGROUND: Amiloride (AMI) and triamterene (TRI) are both potassium-saving diuretics, which are ordinarily used as doping to enhance the performance of athletes in sports. For the similar structures and complex matrices existence, chromatography and extraction are commonly employed to realize the determination of AMI and TRI in biological fluids, which are very time-consuming and laborious. RESULTS: A novel method is presented to simultaneous interference-free determination of AMI and TRI in complex biological fluids samples using excitation-emission matrix fluorescence coupled with second-order calibration method based on alternating normalization-weight error algorithm. CONCLUSION: The proposed method can obtain accurate qualitative and quantitative information of the analytes, even in the presence of the interference from complex biological fluids, which requires few prior purification and separation procedures.

Impact of mineralocorticoid receptor polymorphisms on urinary electrolyte excretion with and without diuretic drugs.

AIM: Polymorphisms in the mineralocorticoid receptor may affect urinary sodium and potassium excretion. We investigated polymorphisms in the MR gene in relation to urinary electrolyte excretion in two separate studies. PATIENTS & METHODS: The genotype-phenotype association was studied in healthy volunteers after single doses of bumetanide, furosemide, torsemide, hydrochlorothiazide, triamterene and after NaCl restriction. RESULTS: High potassium excretion under all conditions except torsemide, and high NaCl excretion after bumetanide and furosemide were associated with the A allele of the intron-3 polymorphism (rs3857080). This polymorphism explained 5-10% of the functional variation and in vitro, rs3857080 affected DNA binding of the transcription factor LHX4. CONCLUSION: rs3857080 may be a promising new candidate for research in cardiac and renal disorders and on antialdosteronergic drugs like spironolactone.

Amiloride off-target effect inhibits podocyte urokinase receptor expression and reduces proteinuria.

The urokinase receptor (uPAR) and its soluble form play a key role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). The modification of uPAR pathological actions on podocytes will become an important task for the development of improved nephroprotective therapeutics. Here we show that podocyte uPAR expression can be reduced using amiloride. Amiloride has a significant role in the reduction of podocyte cell motility in vitro and proteinuria in mice. Amiloride inhibited the induction of uPAR protein and PLAUR messenger RNA (encoding uPAR) and with that it reduced uPAR-mediated ß3 integrin activation in lipopolysaccharide (LPS)-treated podocytes. Transwell migration assay and wound healing assay showed that directed and random podocyte motility of LPS-treated podocytes were increased and substantially reduced by amiloride. The off-target effect of amiloride was independent of its function as epithelial sodium channel blocker and different from triamterene. Amiloride was also effective in the LPS mouse model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model (NTX) by significantly inhibiting podocyte uPAR induction, reducing proteinuria. In addition, amiloride attenuated glomerulosclerosis, as determined by glomerulosclerotic index. Thus, our observations show that amiloride inhibits podocyte uPAR induction and reduces proteinuria in NTX rats and LPS mice. Given the pathological relevance of the uPAR-ß3 integrin signaling axis in FSGS, amiloride may be utilized in patients with FSGS.

Responses to diuretic treatment in gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1.

BACKGROUND/AIMS: Serum- and glucocorticoid-inducible kinase 1 (SGK1) stimulates the epithelial sodium channel (ENaC), renal outer medullary K(+) channel 1, Na(+)/K(+)-ATPase and presumably the Na(+)-Cl(-) cotransporter (NCC). SGK1-deficient mice (sgk(-/-)) show a compensated salt-losing phenotype with secondary hyperaldosteronism. The present experiments explored the role of SGK1 in the response to diuretics. METHODS: sgk1(-/-) mice and their wild-type littermates (sgk1(+/+)) were treated with the ENaC blocker triamterene (200 mg/l), the Na(+)-K(+)-2Cl(-) cotransport inhibitor furosemide (125 mg/l), the NCC blocker hydrochlorothiazide (400 mg/l) and the mineralocorticoid receptor blocker canrenoate (800 mg/l) for 8 days. Renal SGK1 expression was studied using quantitative RT-PCR and immunofluorescence. RESULTS: Diuretic treatment increased SGK1 mRNA and protein expression in the kidney of wild-type sgk1(+/+) mice. The responses to furosemide, hydrochlorothiazide or canrenoate were not different between sgk1(+/+) and sgk1(-/-) mice, and were accompanied by moderate increases in plasma aldosterone and urea concentrations. However, treatment with triamterene in sgk1(-/-) mice (but not in sgk1(+/+) mice) led to severe, eventually lethal, body weight loss as well as increases in plasma aldosterone, urea and K(+) concentrations. CONCLUSIONS: SGK1 is required for diuretic tolerance to triamterene. The observations confirm the impaired kaliuretic potency of sgk1(-/-) mice and point to a role of SGK1 in renal Na(+) reabsorption by mechanisms other than ENaC.

Rate-limiting biotransformation of triamterene is mediated by CYP1A2.

OBJECTIVE: Triamterene (TA), a potassium-sparing diuretic, is extensively metabolized by hydroxylation in 4'-position and subsequent conjugation by cytosolic sulfotransferases. To identify the cytochrome P450 enzyme(s) catalyzing hydroxylation of triamterene (the rate-limiting step in the formation of the sulfate ester (STA)), in vitro incubation studies were performed with human liver microsomes. METHODS: Initial rates of TA hydroxylation (0 - 300 microM) were determined during a ten-minute-incubation period with liver microsomes of two donors. The role of individual CYP enzymes was determined by pre-incubation with selective inhibitors/alternative substrates. Vice versa, the effect of TA (0 - 500 microM) on 3-demethylation of caffeine (0 - 1,000 microM) was assessed. Metabolite concentrations were estimated by reversed-phase HPLC methods. RESULTS: TA Km values without inhibitors were 60 and 142 microM, Vmax was 177 and 220 pmol/min/mg protein, respectively. Mean inhibitor induced changes of 4'-hydroxy-TA formation were as follows: Furafylline 25 microM (CYP1A2), complete inhibition (-100%); omeprazole 250 microM (CYP1A2 inhibitor/CYP2C 19 substrate), -30%; coumarin 25 microM (CYP2A6), -11%; quinidine 25 microM (CYP2D6), -9%; ketoconazole 25 microM (CYP3A), -18%; and erythromycin 250 microM (CYP3A), -8%. In the reverse inhibition studies, TA competitively inhibited caffeine 3-demethylation with Ki values of 65 and 111 microM, respectively. CONCLUSION: 4'-hydroxylation of TA in humans appears to be mediated exclusively by CYP1A2. Inhibition or induction of CYP1A2 will change the time course of both TA and its active phase-II metabolite. The net pharmacodynamic effect of such changes is difficult to predict and needs to be evaluated in clinical studies.

Attenuation of the kaluretic properties of furosemide by triamterene (Dyrenium) in healthy volunteers.

OBJECTIVE: To examine if concomitant administration of furosemide, a loop diuretic, with the potassium- and magnesium-sparing diuretic triamterene would decrease loss of potassium and magnesium while improving diuresis. METHODS: In this open-label, three-way crossover study, healthy subjects were randomized to receive treatment with 40 mg furosemide, with 150 mg triamterene, or treatment with 40 mg furosemide and 150 mg triamterene. Urine samples were collected 24 hours before dosing and between 0 - 1, 1 - 2, 2 - 3, 3 - 4, 4 - 6, 6 - 8, 8 - 12, and 12 - 24 hours post-dosing. Sodium and potassium levels were measured by an ion-selective electrode method. Magnesium was measured colorimetrically using a xylidyl blue reaction. RESULTS: Co-administration of furosemide with triamterene resulted in enhanced diuresis, particularly in the first 0 - 12 hours post-dose, compared with either furosemide or triamterene alone. Compared to individual treatments, combination therapy significantly increased urinary sodium excretion (p = 0.0001) while significantly decreasing urinary potassium excretion (p = 0.0001); importantly, the magnesium-sparing characteristic of triamterene was retained with furosemide co-administration. CONCLUSION: Triamterene, when used in combination with the loop diuretic, furosemide, preserves intracellular potassium and magnesium while enhancing the natriuretic effect of furosemide.

The G-tolerance after pharmacological hypohydration.

A group of 18 healthy male subjects (25-45 y.o.) participated in the studies of +8.3 Gx tolerance (profile of ballistic reentry) and +Gz tolerance (up to physiological limit) before and after pharmacological hypohydration of organism. Moderate hypohydration was carried out by one 40 mg Furosemid (Lasix) dose, or by pharmacological complex: Furosemid (40 mg) + Hypothiazide (25 mg) + Triamteren (50 mg). Subjects removed with urine about 2 l of water, that was accompanied by increase excretion of electrolytes and caused reduction of the body mass by 2.0 +/- 0.2% and diminution of the plasma volume by 13.5% +/- 2.0%. Pharmacological hypohydration does not lead to a substantial loss in +Gx tolerance. No pathological signs, limiting +Gx tolerance up to 8.3 g were found. The longitudinal +Gz tolerance was less, than +Gx one. The limit of achieved +Gz loads after diuretics reduced by 0.6 g. The scientific and applied significance of this research is that diuretics can be safety used in pre-launch period to prevent the negative effects associated with initial phase of space flight. Besides the preliminary intake of diuretics promotes to diminish of discomfort of cosmonauts, reducing urination in a period of prelaunch waiting in spacecraft.

[The pharmacodynamic and pharmacokinetic characteristics of furosemide and furesis]. / Osobennosti farmakodinamiki i farmakokinetiki furosemida i furezisa.

The object of the work was comparative study of the special features of the pharmacodynamic and pharmacokinetic properties of the diuretics furosemide and furesis in an ambulant regimen with the subject lying in an antiorthostatic position. Six practically healthy males were examined. They were given per os either 40 mg furosemide or one tablet of furesis (49 mg furosemide and 50 mg triamterine). Blood from the vein and urine were repeatedly tested.

Terbutaline increases open channel density of epithelial sodium channel (ENaC) in distal lung.

Neonatal and adult vertebrate respiration is facilitated by alveolar fluid and sodium (Na+) absorption driven by apical sodium channels (ENaC). ENaC are characterized in Xenopus laevis lung (XLL) epithelia using voltage clamping and fluctuation analysis to non-invasively examine macroscopic transepithelial current and resistance (I(SC), R(T)), single channel current (i(Na)) and total channel density (N(T)) responses to a beta adrenergic agonist (Terbutaline). Terbutaline addition to the basolateral bath of XLL increased Na entry to > 200% of control reflecting a doubling of open channel density (N(o). These data are consistent with the notion that XLL can serve as a useful model for investigation of distal lung ENaC response to agents of physiological interest.