Ethacrynic Acid Research Articles

Investigating insecticide resistance and knock-down resistance (kdr) mutation in Dielmo, Senegal, an area under long lasting insecticidal-treated nets universal coverage for 10\xa0years

BackgroundThe use of insecticides, through indoor residual spraying and long-lasting insecticide-treated nets (LLINs), is essential to control malaria vectors. However, the sustainability of these tools is challenged by the spread of insecticide resistance in Anopheles mosquitoes. This study was conducted to assess the susceptibility to insecticides and to determine the resistance mechanisms in malaria vectors in Dielmo, a rural area of western Senegal where LLINs were introduced a decade ago.MethodsCDC bottle bioassays were used to determine the susceptibility of 2–5 day-old unfed Anopheles gambiae s.l. females to alphacypermethrin (12.5 µg/bottle), deltamethrin (12.5 µg/bottle), etofenprox (12.5 µg/bottle), lambdacyhalothrin (12.5 µg/bottle), permethrin (21.5 µg/bottle), DDT (100 µg/bottle), bendiocarb (12.5 µg/bottle), pirimiphos-methyl (20 µg/bottle) and fenitrothion (50 µg/bottle). The involvement of glutathione-S-transferases (GSTs) in insecticide resistance was assessed using a synergist, etacrynic acid (EA, 80 µg/bottle). Polymerase chain reaction (PCR) was used to investigate the presence of ‘knock-down resistance (kdr)’ mutation and to identify sibling species within the An. gambiae complex.ResultsCDC bottle bioassays showed that mosquitoes were fully susceptible to lambdacyhalothrin, bendiocarb and fenitrothion. Overall, mortality rates of 97, 94.6, 93.5, 92.1, and 90.1% were, respectively, observed for permethrin, deltamethrin, pirimiphos-methyl, etofenprox and alphacypermethrin. Resistance to DDT was observed, with a mortality rate of 62%. The use of EA significantly improved the susceptibility of An. gambiae s.l. to DDT by inhibiting GSTs (p = 0.03). PCR revealed that Anopheles arabiensis was the predominant species (91.3%; IC 95 86.6–94%) within An. gambiae complex from Dielmo, followed by Anopheles coluzzii (5.4%; IC 95 2.7–8.1%) and Anopheles gambiae s.s. (3.3%; IC 95 0.6–5.9%). Both 1014F and 1014S alleles were found in An. arabiensis population with frequencies of 0.08 and 0.361, respectively, and 0.233 and 0.133, respectively in An. coluzzii. In An. gambiae s.s. population, only kdr L1014F mutation was detected, with a frequency of 0.167. It was observed that some individual mosquitoes carried both alleles, with 19 specimens recorded for An. arabiensis and 2 for An. coluzzii. The presence of L1014F and L1014S alleles were not associated with resistance to pyrethroids and DDT in An. arabiensis.ConclusionsThe co-occurrence of 1014F and 1014S alleles and the probable involvement of GSTs enzymes in insecticide resistance in An. gambiae s.l. should prompt the local vector programme to implement non-pyrethroid/DDT insecticides alternatives.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H2O2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H2O2-, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H2O2, but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H2O2, were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na+-K+ ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na+-K+ ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H2O2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators.

Repurposing ethacrynic acid for the treatment of bladder cancer.

521 Background: Bladder cancer is a common cancer in the US. Approximately seventy-five percent of new cases present as non-muscle invasive bladder cancer (NMIBC) with a high recurrence rate. Our group has demonstrated in well-characterized human NMIBC cell lines that ethacrynic acid (EA) suppresses growth by inhibiting proliferation, clonogenicity, spheroid formation and inducing apoptosis. Additionally, EA affects markers of stemness and may work through the NOTCH signaling pathway. We present a clinical trial characterizing the safety and urinary tract exposure to EA and metabolites following oral administration in patients undergoing transurethral resection of bladder tumor (TURBT). Methods: Institutional review board approval (#3674) was obtained for a Pilot trial in patients with presumed non-muscle invasive bladder cancer undergoing TURBT. All participants were given a single, 50 mg oral dose of EA in the pre-operative bay. Urine was collected at baseline, upon insertion of cystoscope (30-60 minutes post dose), and post-operatively. Urine concentrations of EA and metabolites were determined using a fully-validated UPLC-MS/MS-based analytical method. Participants were monitored for adverse events. Results: Twelve participants participated in the trial between August 2016 and February 2017. Eleven male and one female patient (median age 70.5 years) were enrolled. Final pathology demonstrated urothelial carcinoma in 5/12 (42%) subjects, three with high grade T1 and two with low-grade Ta tumors. Urine concentrations of EA and conjugates observed 30-60 minutes post-dose were as follows: EA 0.1 ug/mL, EA-glutathione 0.2 ug/mL, EA-cysteine 3 ug/mL, and EA-mercapturate 2 ug/mL. No drug-related adverse events were observed following oral EA administration. Conclusions: Despite EA receiving FDA approval over 40 years ago, our pilot trial describes for the first time, urinary tract exposure of EA and its active metabolites in bladder cancer patients after a single oral dose. These data are guiding ongoing preclinical proof of principle studies evaluating EA alone and in combination with standard of care agents in bladder cancer models. These studies warrant further studies of EA in patients with non-muscle invasive bladder cancer. Clinical trial information: NCT02852564.

Acute blockade of inner ear marginal and dark cell K+ secretion: Effects on gravity receptor function

Specific pharmacological blockade of KCNQ (Kv7) channels with XE991 rapidly (within 20 min) and profoundly alters inner ear gravity receptor responses to head motion (Lee et al., 2017). We hypothesized that these effects were attributable to the suppression of K+ secretion following blockade of KCNQ1-KCNE1 channels in vestibular dark cells and marginal cells. To test this hypothesis, K+ secretion was independently inhibited by blocking the Na+-K+-2Cl- cotransporter (NKCC1, Slc12a2) rather than KCNQ1-KCNE1 channels. Acute blockade of NKCC1 with ethacrynic acid (40 mg/kg) eliminated auditory responses (ABRs) within approximately 70 min of injection, but had no effect on vestibular gravity receptor function (VsEPs) over a period of 2 h in the same animals. These findings show that, vestibular gravity receptors are highly resistant to acute disruption of endolymph secretion unlike the auditory system. Based on this we argue that acute suppression of K+ secretion alone does not likely account for the rapid profound effects of XE991 on gravity receptors. Instead the effects of XE991 likely require additional action at KCNQ channels located within the sensory epithelium itself.

Ethacrynic acid decreases expression of proinflammatory intestinal wall cytokines and ameliorates gastrointestinal stasis in murine postoperative ileus

OBJECTIVES:Several compounds characterized by an olefin linkage conjugated to a carbonyl group have anti-inflammatory properties. The diuretic ethacrynic acid (EA) is a compound of this type. Herein, we tested the hypothesis that ethacrynic acid can modulate the development of ileus after bowel manipulation.METHODS:Groups (n=9) of male C57Bl/6 mice underwent surgical manipulation of the small intestine using a pair of cotton-tipped applicators (MAN). Control animals (CONT) did not undergo any surgical intervention or receive treatment. MAN mice were pre- and post-treated with four intraperitoneal doses of phosphate buffered saline (PBS), EA1 (1mg/kg per dose), or EA10 (10mg/kg per dose). Gastrointestinal transit of non-absorbable FITC-labeled dextran was assessed by gavaging the mice with the tracer 24h after operation and assessing FD70 concentration 120 min later in the bowel contents from the stomach, 10 equally long segments of small intestine, cecum, and two equally long segments of colon. The geometric center for the tracer was calculated for each animal. Expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) transcripts in the ileal muscularis propria was assessed using semiquantitative reverse transcriptase-polymerase chain reaction.RESULTS:In control animals, the mean (±SE) geometric center for the transit marker was 9.89±0.47, whereas it was 4.59±0.59 for PBS-treated animals (p<0.05 vs CONT). The geometric center for pre- post treatment with low (1mg/kg) and high (10mg/kg) doses of ethacrynic acid were 7.23±0.97 and 5.15±0.57, respectively. Compared to PBS, treatment with ethacrynic acid (1mg/kg) significantly decreased manipulation-induced IL-6 and iNOS mRNA expression in the wall of the small bowel.CONCLUSIONS:Pre- and post-treatment with ethacrynic acid ameliorates ileus and modulates inflammation in the gut wall induced by bowel manipulation.

Encouraging Effects of Ethacrynic Acid Derivatives Possessing a Privileged α, β-Unsaturated Carbonyl Structure Scaffold

α, β-unsaturated carbonyl unit has remarkable utility in the development of biologically active and therapeutically relevant compounds in medicinal chemistry. In recent years, a number of Ethacrynic acid (EA) derivatives containing α, β-unsaturated carbonyl group were reported, which displayed versatile biological activities, such as antitumor activity, antimalarial effects, antiviral activity and reducing intraocular pressure. In this review, we summed up a number of derivatives regarding Ethacrynic acid as the lead and α, β-unsaturated carbonyl moiety as the functional group, researching on structure-activity relationship and evaluating their various biological activities in recent years. By summarizing the structure-activity relationships of these derivatives, we aimed to demonstrate α, β-unsaturated carbonyl group is a privileged scaffold in these structures and we should pay more attention to α, β-unsaturated carbonyl group to the development of more effectively bioactive new compounds.'

Synthesis and molecular modeling of new benzimidazoles as glutathione S-transferase inhibitors and anticancer agents.

Synthesis of novel glutathione S-transferases (GSTs) inhibitors constitutes a promising strategy in cancer treatment. Results & methodology: A new set of benzimidazoles clubbed with various heterocycles as GST inhibitors and anticancer agents were synthesized. The biological results proved the potential of the new compounds as GST inhibitors, specifically compounds 7 and 14 which produced more potency than ethacrynic acid by three- and tenfold, respectively. Most compounds exhibited promising cytotoxic activity against breast and colon cancer cell lines. Molecular modeling studies revealed that compounds 7 and 14 showed good binding with the amino acids of the GST protein. Both compounds 7 and 14 fulfilled the Lipinski's rule of five suggesting them as new promising GST inhibitors and anticancer agents.

Enzymatic characterization of two epsilon-class glutathione S-transferases of Spodoptera litura.

Two cDNAs encoding glutathione S-transferase (GST) of the tobacco cutworm, Spodoptera litura, were cloned by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequences of the resulting clones revealed 32-51% identities to the epsilon-class GSTs from other organisms. The recombinant proteins were functionally overexpressed in Escherichia coli cells in soluble form and were purified to homogeneity. The enzymes were capable of catalyzing the bioconjugation of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)-propane, and ethacrynic acid. A competition assay revealed that the GST activity was inhibited by insecticides, suggesting that it could be conducive to insecticide tolerance in the tobacco cutworm.

Synthesis and spectroscopic/DFT structural characterization of coordination compounds of Nb(V) and Ti(IV) with bioactive carboxylic acids

The reactions are reported of NbX5 (X = Cl, Br), TiCl4 and Ti(OiPr)4 with a selection of carboxylic acids exhibiting a known biological role, in a chlorinated solvent. The reactions of NbX5 with acetylsalicylic acid (aspirin) proceeded with selective deacetylation of the organic reactant and formation of the salicylate complexes NbX4(C7H5O3) (1a, X = Cl; 1b, X = Br) in 60–65% yields. NbCl5 reacted with diclofenac and ethacrynic acid (EA-CO2H) to give NbCl3[κ3O,O,N-O2CCH2(C6H4)NC6H3Cl2], 2 (80% yield), and NbCl4(O2C-EA), 3 (72% yield), respectively. Ti(OiPr)4 reacted with ethacrynic acid giving Ti(OiPr)2(O2C-EA)2, 4, in 74% yield, as a mixture of two isomers. All the products were characterized by means of analytical and spectroscopic methods, moreover DFT studies were carried out to give insight into structural features.

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma.

KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Six pairs of KCNJ5-mutated, high and low aldosterone-secreting APAs, five non-KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells.

Enhancement of diuresis with metolazone in infant paediatric cardiac intensive care patients.

Few data are available regarding the use of metolazone in infants in cardiac intensive care. Researchers need to carry out further evaluation to characterise the effects of this treatment in this population. This is a descriptive, retrospective study carried out in patients less than a year old. These infants had received metolazone over a 2-year period in the paediatric cardiac intensive care unit at our institution. The primary goal was to measure the change in urine output from 24 hours before the start of metolazone therapy to 24 hours after. Patient demographic variables, laboratory data, and fluid-balance data were analysed. The study identified 97 infants with a mean age of 0.32±0.25 years. Their mean weight was 4.9±1.5 kg, and 58% of the participants were male. An overall 63% of them had undergone cardiovascular surgery. The baseline estimated creatinine clearance was 93±37 ml/minute/1.73 m2. Initially, the participants had received a metolazone dose of 0.27±0.10 mg/kg/day, the maximum dose being 0.43 mg/kg/day. They had also received other diuretics during metolazone initiation, such as furosemide (87.6%), spironolactone (58.8%), acetazolamide (11.3%), bumetanide (7.2%), and ethacrynic acid (1%). The median change in urine output after metolazone was 0.9 ml/kg/hour (interquartile range 0.15-1.9). The study categorised a total of 66 patients (68.0%) as responders. Multivariable analysis identified acetazolamide use (p=0.002) and increased fluid input in the 24 hours after metolazone initiation (p0.05). Metolazone increased urine output in a select group of patients. Efficacy can be maximised by strategic selection of patients.

Organometallic Glutathione S-Transferase Inhibitors

A new family of organometallic p-cymene ruthenium(II) and osmium(II) complexes conjugated to ethacrynic acid, a glutathione transferase (GST) inhibitor, is reported. The ethacrynic acid moiety (either one or two) is tethered to the arene ruthenium(II) and osmium(II) fragments via strongly coordinating modified bipyridine ligands. The solid-state structure of one of the complexes, i.e. [Os(η6-p-cymene)Cl][(4′-methyl-[2,2′-bipyridin]-4-yl)methyl-2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetate]Cl, was established by single-crystal X-ray diffraction, corroborating the expected structure. The complexes are efficient inhibitors of GST P1-1, an enzyme expressed in cancer cells and implicated in drug resistance, and are cytotoxic to the GST-overexpressing chemoresistant A2780cisR ovarian cancer cell line.

Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs) with ethacraplatin micelles in vitro and in vivo

Platinum-based DNA-adducting agents are used extensively in the clinic for cancer chemotherapy. However, the anti-tumor efficacy of these drugs is severely limited by cisplatin resistance, and this can lead to the failure of chemotherapy. One of cisplatin resistance mechanisms is associated with overexpression of glutathione S-transferases (GSTs), which would accelerate the deactivation of cisplatin and decrease its antitumor efficiency. Nanoscale micelles encapsulating ethacraplatin, a conjugate of cisplatin and ethacrynic acid (an effective GSTs inhibitor), can enhance the accumulation of active cisplatin in cancer cells by inhibiting the activity of GSTs and circumventing deactivation of cisplatin. Invitro and invivo results provide strong evidence that GSTs inhibitor-modified cisplatin prodrug combined with nanoparticle encapsulation favor high effective platinum accumulation, significantly enhanced antitumor efficacy against cisplatin-resistant cancer and decreased system toxicity. It is believed that these ethacraplatin-loaded micelles have the ability of overcoming resistance of cancers toward cisplatin and will improve the prospects for chemotherapy of cisplatin-resistant cancers in the near future.

Varying the metal to ethacrynic acid ratio in ruthenium(ii)/osmium(ii)-p-cymene conjugates

Following the identification of a ruthenium(II)-arene complex with an ethacrynic acid-modified imidazole ligand, which inhibits glutathione transferase (GST) and is cytotoxic to chemo-resistant cancer cells, a series of structurally related ruthenium(II)- and osmium(II)-p-cymene compounds have been prepared. In these complexes the ethacrynic acid is linked to the metals via appropriately modified pyridine ligands. The influence of the metal center and the metal:ethacrynic acid ratio on the cytotoxicity of the compounds was evaluated with the derivatives with one metal center and two ethacrynic acid moieties being the most potent against chemo-resistant A2780cisR cells (human ovarian cancer cells with acquired resistance to cisplatin). Moreover, compared to a complex with an ethacrynic acid-modified imidazole ligand (RAIMID-EA, Figure 2), these complexes display a significant degree of cancer cell specificity.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection.

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus.

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors.

Modulation of hepatic glutathione transferases isoenzymes in three bivalve species exposed to purified microcystin-LR and Microcystis extracts

This study compares the role of hepatic cytosolic glutathione transferases (cGST) isoforms of three different bivalve species to a Microcystis aeruginosa extract and purified MC-LR exposure (both at 150 μg MC-LR L−1) for 24 h. Characterization and alterations of the cytosolic GST activities in Mytilus galloprovincialis, Ruditapes philippinarum and Corbicula fluminea were measured using four class-specific substrates and changes in individual GST isoforms expression were achieved by a subsequent two-dimensional electrophoresis analysis. Evaluation of cGST activity basal levels using the four class-specific substrates denoted quantitative differences between the three bivalves. Purified MC-LR did not induce any significant response from bivalves. On the other hand, cell extracts caused significant alterations according to bivalves and substrates. Among the three bivalves, only R. philippinarum showed a significant induction of cGST activity using generic 1-chloro-2,4-dinitrobenzene (CDNB) substrate. However, no significant alterations were detected in these clams by cell extracts using the other specific substrates. In contrast, C. fluminea revealed significant induction of cGST activity when using 3,4-dichloronitrobenzene (DCNB) and ethacrynic acid (EA). In M. galloprovincialis, cell extracts promoted a significant decrease of cGST activity when using EA substrate. Altered protein expression was quantitatively detected upon exposure to cell extracts for one spot in R. philippinarum and another for C. fluminea, both upregulated (2.0 and 8.5-fold, respectively) and identified as a sigma1-class GST in the case of the first. The results showed that the three bivalves presented specific adaptive biotransformation responses to MCs and other cyanobacteria compounds supported by the modulation of distinct cGST classes.

Abstract 185: Development of a novel non-diuretic brain-penetrating ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models

Abstract The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy of these cancers. A number of factors including the inefficient drug entry through the BBB, intratumoral heterogeneity, overexpression of MGMT repair protein and bone marrow toxicity due to alkylating regimens have impeded successful therapy of these cancers. There is, therefore, an urgent need to design new, more effective brain-penetrating drugs that can effectively eliminate brain tumors. We are highly interested in exploiting the elevated oxidative stress present in gliomas and have synthesized a hydrophobic, non-diuretic analog of ethacrynic acid (EA) called KSS72 [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] by removing the carboxylate side chain. KSS72 was selectively cytotoxic to cancer cells including gliomas. Compared to EA which does not cross the BBB, Pharmacokinetics following intravenous or oral administrations in mice showed its excellent penetrance through the blood brain-barrier, KSS72 accumulating at levels equivalent to TMZ (25% of plasma levels) in the brain In vitro assays measuring protein carbonyl content, GSH content, ROS generation, GSTpi enzyme activity and others showed that KSS72 triggers a redox imbalance by inhibiting GSTpi and by lowering the antioxidant (GSH) and reducing equivalent (NADPH) levels, leading to a significant elevation of ROS levels. We also showed the upregulation of endoplasmic reticulum (ER) stress-responsive proteins, activation of MAPK, autophagy and apoptotic pathways by KSS72 in several cell lines. KSS72-induced autophagy was a post event of redox perturbation that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. In view of its excellent brain penetrance and multiple cytocidal events mediated by ROS and autophagy, we tested the antitumor efficacy of KSS72 in human GBM cell lines and intracranial GBM xenografts. SF-188 human GBM cells expressing firefly luciferase were injected into the brain just left of bregma for the development of orthotopic GBM in nude mice. These mice were given 25 mg/kg/day of KSS72 intraperitoneally for 2 weeks. The animals when imaged by IVIS after luciferin injections showed a complete lack of intracranial tumors in all KSS72 administered animals. H&amp;E staining of mouse brain sections confirmed the total elimination of GBM by KSS72. KSS72 did not exert any toxicity on host tissues and serum ALT and AST levels were not altered. In summary, we conclude that KSS72 acting through multiple pathways of oxidative stress is a hugely promising non-toxic anti-glioma drug with potential to enter clinical trials (supported by CPRIT grants RP130266 and RP170207 to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Development of a novel non-diuretic brain-penetrating ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 185. doi:10.1158/1538-7445.AM2017-185

Abstract 2570: Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant

Abstract p53 gene mutations in 50% of human cancers drive the emergence of oncogenic genomes and confer resistance to anticancer drugs &amp; apoptosis. Therefore, a pharmacological reactivation of p53 mutant proteins to their wild-type forms has emerged as an intense research area. We have shown that induction of oxidative protein modifications primarily underlie the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). R248W/Q DNA contact mutations in the p53 core are highly frequent and cause functional inactivation. No small molecules reactivating this p53 mutation are available. Here, we describe the design and synthesis of a small molecule KSS72 using a cell-based screening and its potency in reactivating the R248 p53 mutants. KSS72 is a non-diuretic hydrophobic analog [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] of ethacrynic acid (Edecrin) obtained by removing the carboxylate side chain. KSS-72 retained the ability to bind with GSH, inhibit GSTs and induce marked redox-imbalance in tumor cells. KSS72 displayed preferential cytotoxicity (3-4 fold more cell killing) against cancer cells expressing the allele specific R248W mutation (MiaPaca-2) in comparison with the wt p53 or p53-null cancer cell lines. Further, we developed isogenic cell lines expressing the R248W and R248Q mutants in the p53-null H1299 lung cancer cells and evaluated their sensitivity towards KSS72; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of KSS72 stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that KSS72 treatment of R248W or R248Q p53-cells results in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed. Other data showed that KSS72 restores the wt structure and function by covalently binding the p53R248 protein. Pharmacokinetics showed that KSS72 has excellent oral bioavailability with a half-life of 3.5 h. In MiaPaca-2 xenografts developed in nude mice, there was a marked tumor growth suppression with KSS72 as a single drug. In conclusion, KSS72 is an attractive lead compound for further development because p53 reactivation occurred without discernible organ toxicities after oral doses and a wide therapeutic window. The redox-sensitive cysteines involved in the p53 reactivation are being identified by mass spectrometry (supported by CPRIT grants RP130266 &amp; RP170207 to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2017-2570

Ethacrynic Acid Decreases Expression of Proinflammatory Cytokines in Intestinal Wall and Ameliorates Gastrointestinal Motility after Small Bowel Manipulation

Background: Gastrointestinal dysmotility is frequently observed after visceral transplantation. The development of postoperative GI dysmotility is multifactorial with an intricate interaction between inflammatory, hormonal, neurogenic, and humoral components. Several studies have shown an increased expression of proinflammatory cytokines in the gut wall as well as infiltration by polymorphonuclear leukocytes into the muscularis propria after SB manipulation. A number of compounds characterized by an olefin linkage conjugated to a carbonyl group have anti-inflammatory properties. The diuretic, ethacrynic acid (EA), is a compound of this type. Herein, we tested the hypothesis that EA can modulate the development of GI dysmotility after bowel manipulation. Methods: Groups (n=9) of male C57Bl/6 mice underwent surgical manipulation of the small intestine using a pair of cotton-tipped applicators (MAN). Control animals (CONT) did not undergo any surgery or receive treatment. MAN mice were pre- and post-treated with four i.p. doses of PBS (phosphate buffered saline), EA1 (1mg/kg per dose) or EA10 (10mg/kg per dose). Gastrointestinal transit of non-absorbable FITC-labeled dextran (MW 70 kDA; FD70) was assessed by gavaging the mice with the tracer 24 h after operation and assessing FD70 concentration 120 min later in bowel contents from the stomach, 10 equally long segments of small intestine, cecum, and two equally long segments of colon. The geometric center (GC) for the tracer was calculated for each animal. Expression of interleukin-6 and inducible nitric oxide synthase transcripts in ileal muscularis propria was assessed using the semiquantitative reverse transcriptase-polymerase chain reaction. Results: In control animals, the mean (±SE) geometric center for the transit marker was 9.89±0.47, whereas for PBS treated animals was 4.59±0.59 (p<0.05 vs CONT). The GC for pre- post treatment with low (1mg/kg) and high (10mg/kg) doses of ethacrynic acid were 7.23±0.97 and 5.15±0.57, respectively. Compared to PBS, treatment with EA (1mg/kg) significantly decreased manipulation-induced IL-6 and iNOS mRNA expression in the wall of the small bowel. Conclusions: Pre- and post-treatment with EA ameliorates ileus and modulate inflammation in the gut wall induced by bowel manipulation.