Onur Cil

Family History of Renal Disease Severity Predicts the Mutated Gene in ADPKD

Mutations of PKD1 and PKD2 account for 85 and 15% of cases of autosomal dominant polycystic kidney disease (ADPKD), respectively. Clinically, PKD1 is more severe than PKD2, with a median age at ESRD of 53.4 versus 72.7 yr. In this study, we explored whether a family history of renal disease severity predicts the mutated gene in ADPKD. We examined the renal function (estimated GFR and age at ESRD) of 484 affected members from 90 families who had ADPKD and whose underlying genotype was known. We found that the presence of at least one affected family member who developed ESRD at age < or =55 was highly predictive of a PKD1 mutation (positive predictive value 100%; sensitivity 72%). In contrast, the presence of at least one affected family member who continued to have sufficient renal function or developed ESRD at age >70 was highly predictive of a PKD2 mutation (positive predictive value 100%; sensitivity 74%). These data suggest that close attention to the family history of renal disease severity in ADPKD may provide a simple means of predicting the mutated gene, which has prognostic implications.

CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation.

Background & Aims Constipation is a common clinical problem that negatively impacts quality of life and is associated with significant health care costs. Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the primary pathway that drives fluid secretion in the intestine, which maintains lubrication of luminal contents. We hypothesized that direct activation of CFTR would cause fluid secretion and reverse the excessive dehydration of stool found in constipation. Methods A cell-based, high-throughput screen was performed for 120,000 drug-like, synthetic small molecules. Active compounds were characterized for mechanism of action and one lead compound was tested in a loperamide-induced constipation model in mice. Results Several classes of novel CFTR activators were identified, one of which, the phenylquinoxalinone CFTRact-J027, fully activated CFTR chloride conductance with an half-maximal effective concentration (EC50) of approximately 200 nmol/L, without causing an increase of cytoplasmic cyclic adenosine monophosphate. Orally administered CFTRact-J027 normalized stool output and water content in a loperamide-induced mouse model of constipation with a 50% effective dose of approximately 0.5 mg/kg; CFTRact-J027 was without effect in cystic fibrosis mice lacking functional CFTR. Short-circuit current, fluid secretion, and motility measurements in mouse intestine indicated a prosecretory action of CFTRact-J027 without direct stimulation of intestinal motility. Oral administration of 10 mg/kg CFTRact-J027 showed minimal bioavailability, rapid hepatic metabolism, and blood levels less than 200 nmol/L, and without apparent toxicity after chronic administration. Conclusions CFTRact-J027 or alternative small-molecule CFTR-targeted activators may be efficacious for the treatment of constipation.

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors

Urea transport (UT) proteins of the UT‐A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high‐throughput assay to identify UT‐A inhibitors, a screen of 50,000 synthetic small molecules identified UT‐A inhibitors of aryl‐thiazole, γ‐sultambenzosulfonamide, aminocarbonitrile butene, and 4‐isoxazolamide chemical classes. Structure‐activity analysis identified compounds that inhibited UT‐A selectively by a noncompetitive mechanism with IC50 down to ~1 μM. Molecular modeling identified putative inhibitor binding sites on rat UTA. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ‐sultambenzosulfonamide at 20 mg/kg increased urine output by 3‐5‐fold and reduced urine osmolality by ~ 2‐fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1‐deamino‐8‐D‐arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT‐A‐selective inhibitors. UT‐A inhibitors are first in their class salt‐sparing diuretics with potential clinical indications in volume‐overload edemas and high‐vasopressin‐associated hyponatremias.—Esteva‐Font, C., Cil, O., Phuan, P.‐W., Su, T., Lee, S., Anderson, M. O., Verkman, A. S. Diuresis and reduced urinary osmolality in rats produced by small‐molecule UT‐A‐ selective urea transport inhibitors. FASEB J. 28, 3878‐3890 (2014). www.fasebj.org

Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins

Secretory diarrheas caused by bacterial enterotoxins, including cholera and travelers diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido‐pyrrolooxazinedione (R)‐BPO‐27 inhibits CFTR chloride conductance with low‐nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)‐BPO‐27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)‐BPO‐27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat‐stable enterotoxin of E. coli (STa toxin), with IC50 down to ~5 nM. (R)‐BPO‐27 prevented cholera toxin and STa toxin‐induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 μg/kg. (R)‐BPO‐27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7‐d administration at 5 μg/kg/d. As evidence to support efficacy in human diarrheas, (R)‐BPO‐27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)‐BPO‐27 for therapy of CFTR‐mediated secretory diarrheas.—Cil, O., Phuan, P.‐W., Gillespie, A.M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N.C., Lin, R., Donowitz, M., Verkman, A.S. Benzopyrimido‐pyrrolo‐oxazine‐dione CFTR inhibitor (R)‐BPO‐27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J. 31, 751–760 (2017). http://www.fasebj.org

Small-Molecule Inhibitors of Pendrin Potentiate the Diuretic Action of Furosemide

Pendrin is a Cl-/HCO3- exchanger expressed in type B and non-A, non-B intercalated cells in the distal nephron, where it facilitates Cl- absorption and is involved in Na+ absorption and acid-base balance. Pendrin-knockout mice show no fluid-electrolyte abnormalities under baseline conditions, although mice with double knockout of pendrin and the Na+/Cl- cotransporter (NCC) manifest profound salt wasting. Thus, pendrin may attenuate diuretic-induced salt loss, but this function remains unconfirmed. To clarify the physiologic role of pendrin under conditions not confounded by gene knockout, and to test the potential utility of pendrin inhibitors for diuretic therapy, we tested in mice a small-molecule pendrin inhibitor identified from a high-throughput screen. In vitro, a pyrazole-thiophenesulfonamide, PDSinh-C01, inhibited Cl-/anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of 1-3 µM, without affecting other major kidney tubule transporters. Administration of PDSinh-C01 to mice at predicted therapeutic doses, determined from serum and urine pharmacokinetics, did not affect urine output, osmolality, salt excretion, or acid-base balance. However, in mice treated acutely with furosemide, administration of PDSinh-C01 produced a 30% increase in urine output, with increased Na+ and Cl- excretion. In mice treated long term with furosemide, in which renal pendrin is upregulated, PDSinh-C01 produced a 60% increase in urine output. Our findings clarify the role of pendrin in kidney function and suggest pendrin inhibition as a novel approach to potentiate the action of loop diuretics. Such combination therapy might enhance diuresis and salt excretion for treatment of hypertension and edema, perhaps including diuretic-resistant edema.

High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activators

We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

&NA; Constipation is a common condition for which current treatments can have limited efficacy. By high‐throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid‐induced constipation. Here, we report phenylquinoxalinone structure‐activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure‐activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact‐J027, activated CFTR with EC50 ˜ 200 nM, with patch‐clamp analysis showing a linear CFTR current‐voltage relationship with direct CFTR activation. CFTRact‐J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact‐J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact‐J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact‐J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure‐activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10–20 μM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2–3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 hours, and a urine concentration of 20–40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Nanomolar-Potency 1,2,4-Triazoloquinoxaline Inhibitors of the Kidney Urea Transporter UT-A1

Urea transporter A (UT-A) isoforms encoded by the Slc14a2 gene are expressed in kidney tubule epithelial cells, where they facilitate urinary concentration. UT-A1 inhibition is predicted to produce a unique salt-sparing diuretic action in edema and hyponatremia. Here we report the discovery of 1,2,4-triazoloquinoxalines and the analysis of 37 synthesized analogues. The most potent compound, 8ay, containing 1,2,4-triazolo[4,3- a]quinoxaline-substituted benzenesulfonamide linked by an aryl ether, rapidly and reversibly inhibited UT-A1 urea transport by a noncompetitive mechanism with IC50 ≈ 150 nM; the IC50 was ∼2 μM for the related urea transporter UT-B encoded by the Slc14a1 gene. Molecular modeling suggested a putative binding site on the UT-A1 cytoplasmic domain. In vitro metabolism showing quinoxaline ring oxidation prompted the synthesis of metabolically stable 7,8-difluoroquinoxaline analogue 8bl, which when administered to rats produced marked diuresis and reduced urinary osmolality. 8bl has substantially improved UT-A1 inhibition potency and metabolic stability compared with prior compounds.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.