Molecular dynamics simulation revealed binding of nucleotide inhibitors to ZIKV polymerase over 444 nanoseconds.

In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2' carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti-hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide-range antiviral drug.

Assessment of the clinical cardiac drug-drug interaction associated with the combination of hepatitis C virus nucleotide inhibitors and amiodarone in guinea pigs and rhesus monkeys.

In 2015, European and U.S. health agencies issued warning letters in response to 9 reported clinical cases of severe bradycardia/bradyarrhythmia in hepatitis C virus (HCV)-infected patients treated with sofosbuvir (SOF) in combination with other direct acting antivirals (DAAs) and the antiarrhythmic drug, amiodarone (AMIO). We utilized preclinical in vivo models to better understand this cardiac effect, the potential pharmacological mechanism(s), and to identify a clinically translatable model to assess the drug-drug interaction (DDI) cardiac risk of current and future HCV inhibitors. An anesthetized guinea pig model was used to elicit a SOF+AMIO-dependent bradycardia. Detailed cardiac electrophysiological studies in this species revealed SOF+AMIO-dependent selective nodal dysfunction, with initial, larger effects on the sinoatrial node. Further studies in conscious, rhesus monkeys revealed an emergent bradycardia and bradyarrhythmia in 3 of 4 monkeys administered SOF+AMIO, effects not observed with either agent alone. Morever, bradycardia and bradyarrhythmia were not observed in rhesus monkeys when intravenous infusion of MK-3682 was completed after AMIO pretreatment. CONCLUSIONS: These are the first preclinical in vivo experiments reported to replicate the severe clinical SOF+AMIO cardiac DDI and provide potential in vivo mechanism of action. As such, these data provide a preclinical risk assessment paradigm, including a clinically relevant nonhuman primate model, with which to better understand cardiovascular DDI risk for this therapeutic class. Furthermore, these studies suggest that not all HCV DAAs and, in particular, not all HCV nonstructural protein 5B inhibitors may exhibit this cardiac DDI with amiodarone. Given the selective in vivo cardiac electrophysiological effect, these data enable targeted cellular/molecular mechanistic studies to more precisely identify cell types, receptors, and/or ion channels responsible for the clinical DDI. (Hepatology 2016;64:1430-1441).

Design, synthesis, and evaluation of DNA topoisomerase II-targeted nucleosides.

We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small structural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucleoside analogues containing TBS and 1,3-dithian moieties had potent and selective DNA topoisomerase II inhibitory activities. In further experiments, compound 25b having a beta configuration of the thymine moiety showed relatively strong growth inhibitory activity against cancer cell lines, and was more potent against all cancer cell lines than compound 26b, which carries a thymine moiety in the alpha configuration.

The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability.

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

Balancing antiviral potency and host toxicity: identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase.

Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2',3'-didehydro-2',3'-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4'-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2',3'-didehydro-2',3'-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

Exploring the dNTP -binding site of HIV-1 reverse transcriptase for inhibitor design.

HIV-1 reverse transcriptase (RT) plays a central role in the viral life cycle, and roughly half of the FDA-approved anti-HIV drugs are targeting RT. Nucleoside analogs (NRTIs) require cellular phosphorylation for binding to RT, and to bypass this rate-limiting path, we designed a new series of acyclic nucleoside phosphonate analogs as nucleoside triphosphate mimics, aiming at the chelation of the catalytic Mg2+ ions via a phosphonate and/or a carboxylic acid group. Novel synthetic procedures were developed to access these nucleoside phosphonate analogs. X-ray structures in complex with HIV-1 RT/dsDNA demonstrated that their binding modes are distinct from that of our previously reported compound series. The impact of chain length, chirality and linker atom have been discussed. The detailed structural understanding of these new compounds provides opportunities for designing new class of HIV-1 RT inhibitors.

Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal.

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.

Targeting Metabolism of Extracellular Nucleotides via Inhibition of Ectonucleotidases CD73 and CD39.

In the tumor microenvironment, unusually high concentrations of extracellular adenosine promote tumor proliferation through various immunosuppressive mechanisms. Blocking adenosine production by inhibiting nucleotide-metabolizing enzymes, such as ectonucleotidases CD73 and CD39, represents a promising therapeutic strategy that may synergize with other immuno-oncology mechanisms and chemotherapies. Emerging small-molecule ectonucleotidase inhibitors have recently entered clinical trials. This Perspective will outline challenges, strategies, and recent advancements in targeting this class with small-molecule inhibitors, including AB680, the first small-molecule CD73 inhibitor to enter clinical development. Specific case studies, including structure-based drug design and lead optimization, will be outlined. Preclinical data on these molecules and their ability to enhance antitumor immunity will be discussed.

Molecular docking revealed the binding of nucleotide/side inhibitors to Zika viral polymerase solved structures.

A new Zika virus (ZIKV) outbreak started in 2015. According to the World Health Organization, 84 countries confirmed ZIKV infection. RNA-dependent RNA polymerase (RdRp) was an appealing target for drug designers during the last two decades. Through molecular docking, we screened 16 nucleotide/side inhibitors against ZIKV RdRp. While the mode of interaction with ZIKV is different from that in the hepatitis C virus (HCV), nucleotide/side inhibitors in this study (mostly anti-HCV) showed promising binding affinities (-6.2 to -9.7 kcal/mol calculated by AutoDock Vina) to ZIKV RdRp. Setrobuvir, YAK and, to a lesser extent, IDX-184 reveal promising results compared to other inhibitors in terms of binding ZIKV RdRp. These candidates would be powerful anti-ZIKV drugs.

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity.

Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.

Thrombin-induced increase in intracellular cyclic 3',5'-adenosine monophosphate in human platelets.

The present data disagree with earlier suggestions that thrombin's effect on platelets is to cause a decrease in intracellular cyclic 3',5'-adenosine monophosphate. Washed human platelets or platelet-rich plasma were incubated at 37 degrees C with human thrombin. After centrifugation, the supernates were assayed for nucleotides and calcium released. The platelet pellets, and in some experiments the supernates as well, were assayed by radioimmunoassay for intracellular cyclic AMP. In the washed platelet system, increasing doses of thrombin to 0.5 U/cc induced increasing release of nucleotides and calcium. This was accompanied by an average twofold increase in intracellular cyclic AMP levels. Prostaglandin E(1), which inhibited 30-50% of release, induced a four- to fivefold increase in cyclic AMP levels that was additive to the cyclic AMP-stimulatory effect of thrombin. Theophylline, which inhibited only 20-40% of nucleotide release, was synergistic with thrombin in the intracellular accumulation of cyclic AMP. The time-course of cyclic AMP accumulation in response to thrombin was slower than thrombin-induced nucleotide release. Similar findings were made in the platelet-rich plasma system where thrombin stimulation of nucleotide release also resulted in a marked accumulation of intracellular cyclic AMP. Thrombin did not appear to stimulate the release of intracellular cyclic AMP. The mechanism underlying these observations was not apparent. The thrombin had no measurable inhibitory effect on platelet phosphodiesterase activity in either intact washed cells or the platelet homogenate supernates. Furthermore, thrombin inhibited, rather than stimulated, platelet adenyl cyclase activity in both intact washed cells and washed platelet particulate fractions. Of note, however, was the finding that thrombin did not completely inhibit the adenyl cyclase activity of prostaglandin-stimulated cells. Further work is needed to clarify the significance of this observation.Nonetheless, the accumulation of intracellular cyclic AMP in response to thrombin observed in the present study suggests that the antagonistic actions of various agents on the platelet release reaction, thought to underlie platelet function, may depend upon a mechanism more intricate than a straightforward mediation through directly opposite effects on platelet cyclic AMP.

Protein-drug complexes important for immunoregulation and organ transplantation.

Recently, two structures of the Ser/Thr phosphorylase calcineurin in complex with FK506 and its cognate immunophilin, FKBP12, have been reported, both solved by small pharmaceutical companies focused on structure-based drug design. A realization, however, that the toxicities associated with calcineurin-mediated immunosuppressants might be mechanism based has driven the current interest in alternative approaches to autoimmunity prophylaxis and preventing transplant rejection. Regulatory approval in 1995 of the immunosuppressant prodrug mycophenolate mofetil, whose active metabolite, mycophenolic acid, inhibits inosine monophosphate dehydrogenase, has focused attention on the potential significance of the de novo purine-biosynthesis pathway as a target for immunosuppressive drugs, leading ultimately to the solution of enzyme structure as a drug design target. As this and other clinically relevant targets are discovered, elaborated and refined via the activity of their cognate agents (as was the case for the phosphatase calcineurin via the activity of cyclosporin), a critical opportunity should ensue for structural biology to exert a profound effect on the future development of these therapies.

Gangliosides potentially inhibit extracellular nucleotide metabolism.

Gangliosides are glycolipids that contain sialic acid and they are mainly located on the outer leaflet of the cellular plasma membrane of most vertebrate and some invertebrate cells. Because they have structurally diverse, bulky and negatively charged oligosaccharide moieties, gangliosides endow cell membranes with unique molecular characteristics. Although they are abundant in the central nervous system (CNS), the complete loss of gangliosides in mice does not result in gross morphological abnormalities of the CNS. However, mutant mice develop neurodegenerative diseases and die soon after birth, suggesting that gangliosides are required for the maintenance and development of a stable CNS and are crucial to sustain life. At the cellular level, gangliosides influence cell growth and death, probably because they are involved in the lipid-mediated assembly of signaling molecules such as growth factor receptors or integrins on the membranes. This article addresses the structural similarity between the tandem sialic acid residues of gangliosides and nicotinamide adenine dinucleotide (NAD(+)) determined from biochemical data showing that gangliosides inhibit NAD(+) glycohydrolase activity and theoretical considerations. An essential feature of the structural similarity resides in a negative charge cluster formed by the two carboxyl groups in the tandem sialic acid residues and the diphosphate moiety of NAD(+). The potential physiological role(s) of gangliosides on the regulation of extracellular nucleotide metabolism are discussed.

Direct-acting antiviral agents for hepatitis C: structural and mechanistic insights.

The treatment of HCV infection has evolved at an extremely rapid pace over the past few years. The development of direct-acting antiviral agents, which potently inhibit different stages in the viral life cycle, has led to the replacement of interferon with well-tolerated oral therapies with cure rates of >90% in most patient populations. Understanding the mechanisms of action of the various agents as well as related issues, including the molecular basis for resistance, helps to guide drug development and clinical use. In this Review, we provide a mechanistic description of NS3/4A protease inhibitors, nucleotide and non-nucleotide inhibitors of the NS5B viral polymerase and inhibitors of the NS5A protein, followed by a summary of clinical data from studies of each drug class alone and in combination. Remaining challenges in drug development efforts are also discussed.

Reprogramming of Seed Metabolism Facilitates Pre-harvest Sprouting Resistance of Wheat.

Pre-harvest sprouting (PHS) is a worldwide problem for wheat production and transgene antisense-thioredoxin-s (anti-trx-s) facilitates outstanding resistance. To understand the molecular details of PHS resistance, we analyzed the metabonomes of the transgenic and wild-type (control) wheat seeds at various stages using NMR and GC-FID/MS. 60 metabolites were dominant in these seeds including sugars, organic acids, amino acids, choline metabolites and fatty acids. At day-20 post-anthesis, only malate level in transgenic wheat differed significantly from that in controls whereas at day-30 post-anthesis, levels of amino acids and sucrose were significantly different between these two groups. For mature seeds, most metabolites in glycolysis, TCA cycle, choline metabolism, biosynthesis of proteins, nucleotides and fatty acids had significantly lower levels in transgenic seeds than in controls. After 30-days post-harvest ripening, most metabolites in transgenic seeds had higher levels than in controls including amino acids, sugars, organic acids, fatty acids, choline metabolites and NAD(+). These indicated that anti-trx-s lowered overall metabolic activities of mature seeds eliminating pre-harvest sprouting potential. Post-harvest ripening reactivated the metabolic activities of transgenic seeds to restore their germination vigor. These findings provided essential molecular phenomic information for PHS resistance of anti-trx-s and a credible strategy for future developing PHS resistant crops.

Novel inhibitors of HIV integrase: the discovery of potential anti-HIV therapeutic agents.

The viral enzyme, HIV integrase (MW 32 kDa), is one of the three key enzymes of the pol gene of HIV. HIV integrase is involved in the integration of HIV DNA into host chromosomal DNA. There is apparently no functional equivalent of this enzyme in human cells. Integration of HIV DNA into the host cell genome apparently occurs by a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. It is clear that new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. This review focuses on the major classes of compounds that have been discovered as inhibitors of HIV integrase. Some of these compounds are non-specific inhibitors of the enzyme while evidence suggests that others may possess some specificity. The various classes include nucleotides, oligonucleotides, dinucleotides, and miscellaneous small molecules including heterocyclic systems, natural products, diketo acids and sulfones. A major focus of the review is on discoveries from my laboratory in the area of non-natural, nuclease-resistant dinucleotide inhibitors of HIV integrase.

Nitrosoureido nucleosides as potential inhibitors of nucleotide biosynthesis.

Several nitrosoureido nucleosides (3a, 3b, 5a, 7a, 7c, and 10a) designed as inhibitors of enzymes that metabolize pyrimidine nucleotides have been prepared and their chemical and biological properties studied. The methylnitrosoureas 3a and 3b were not significantly cytotoxic to H.Ep.-2 and L1210 cells in vitro but showed moderate activity in the P388 mouse leukemia screen (79% ILS for 3a and 56% ILS for 3b). The (chloroethyl)nitrosoureas 7a and 7c inhibited proliferation of L1210 cells, were cytotoxic to H.Ep.-2 cells, and demonstrated good activity against P388 in vivo (135% ILS with one 30-day survivor for 7a and 191% ILS with two 30-day survivors for 7c). Overnight exposure of L1210 cells to 7a and 7c resulted in cell enlargement accompanied by cell lysis. Macromolecular synthesis in enlarged cells, particularly RNA and protein synthesis, was markedly increased relative to that in untreated control cells. The half-lives of each of the nitrosoureas in pH 7 buffer was determined and compared with biological activity.

Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells.

1. The effect of extracellular nucleotides on the transepithelial ion transport of Madin Darby canine kidney cells (MDCK) was investigated. Cells were grown up to confluency on permeable supports and the short circuit current (ISC) was measured with an Ussing chamber-like mini-perfusion system. 2. Apical ATP stimulated a biphasic ISC increase consisting of a first rapid and transient peak followed by a broader one. 3. The first peak evoked by ATP was reversibly blocked by basilen blue (BB) in a concentration-dependent fashion, with an EC50 of 7.5 microM. 4. The P2 gamma receptor agonist, 2-methylthioATP (2-MeSATP) caused a single transient ISC increase that was completely blocked by pretreatment with BB. On the contrary, the P2x agonist, alpha, beta-methylene ATP (alpha, beta-meATP) was almost completely ineffective on ISC. UTP essentially induced a monophasic response the time-course of which resembled that of the second peak stimulated by ATP. The agonist potency order was 2-MeSATP > or = ATP >> UTP, alpha, beta-meATP for the first peak and UTP > or = ATP > 2-MeSATP > alpha, beta-meATP for the second peak. 5. Monolayer incubation with the membrane permeable calcium chelator [bis-o-aminophenoxy)-ethane-N,N,N',N',-tetraacetic acid, tetra(acetoximethyl)-ester] (BAPTA/AM) inhibited the ATP-evoked first peak. 6. The non-hydrolyzable ATP analogue, adenosine-5'-O-(3-thio)-trisphosphate (ATP-gamma-S) elicited a biphasic response similar to that of ATP. The P1 receptor agonist, 2-chloroadenosine and CGS-21680, were almost unable to induce an ISC increase.2+ increase. The second induces prostaglandin synthesis probably through a P2U receptor activation.