Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

Conversion of oxadiazolo[3,4-b]pyrazines to imidazo[4,5-b]pyrazines via a tandem reduction-cyclization sequence generates new mitochondrial uncouplers.

We report new mitochondrial uncouplers derived from the conversion of [1,2,5]oxadiazolo[3,4-b]pyrazines to 1H-imidazo[4,5-b]pyrazines. The in situ Fe-mediated reduction of the oxadiazole fragment followed by cyclization gave access to imidazopyrazines in moderate to good yields. A selection of orthoesters also allowed functionalization on the 2-position of the imidazole ring. This method afforded a variety of imidazopyrazine derivatives with varying substitution on the 2, 5 and 6 positions. Our studies suggest that both a 2-trifluoromethyl group and N-methylation are crucial for mitochondrial uncoupling capacity.

NACHO and 14-3-3 promote expression of distinct subunit stoichiometries of the α4ß2 acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of pentameric ligand-gated ion channels, and in neuronal tissues, are assembled from various types of α- and ß-subunits. Furthermore, the subunits α4 and ß2 assemble in two predominant stoichiometric forms, (α4)2(ß2)3 and (α4)3(ß2)2, forming receptors with dramatically different sensitivity to agonists and allosteric modulators. However, mechanisms by which the two stoichiometric forms are regulated are not known. Here, using heterologous expression in mammalian cells, single-channel patch-clamp electrophysiology, and calcium imaging, we show that the ER-resident protein NACHO selectively promotes the expression of the (α4)2(ß2)3 stoichiometry, whereas the cytosolic molecular chaperone 14-3-3η selectively promotes the expression of the (α4)3(ß2)2 stoichiometry. Thus, NACHO and 14-3-3η are potential physiological regulators of subunit stoichiometry, and are potential drug targets for re-balancing the stoichiometry in pathological conditions involving α4ß2 nAChRs such as nicotine dependence and epilepsy.

Deconstructing the Pharmacological Contribution of Sphingosine-1 Phosphate Receptors to Mouse Models of Multiple Sclerosis Using the Species Selectivity of Ozanimod, a Dual Modulator of Human Sphingosine 1-Phosphate Receptor Subtypes 1 and 5.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator that binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), is approved for the treatment of relapsing multiple sclerosis (MS) in multiple countries. Ozanimod profiling revealed a species difference in its potency for S1P5 in mouse, rat, and canine compared with that for human and monkey. Site-directed mutagenesis identified amino acid alanine at position 120 to be responsible for loss of activity for mouse, rat, and canine S1P5, and mutation back to threonine as in human/monkey S1P5 restored activity. Radioligand binding analysis performed with mouse S1P5 confirmed the potency loss is a consequence of a loss of affinity of ozanimod for mouse S1P5 and was restored with mutation of alanine 120 to threonine. Study of ozanimod in preclinical mouse models of MS can now determine the S1P receptor(s) responsible for observed efficacies with receptor engagement as measured using pharmacokinetic exposures of free drug. Hence, in the experimental autoimmune encephalomyelitis model, ozanimod exposures sufficient to engage S1P1, but not S1P5, resulted in reduced circulating lymphocytes, disease scores, and body weight loss; reduced inflammation, demyelination, and apoptotic cell counts in the spinal cord; and reduced circulating levels of the neuronal degeneration marker, neurofilament light. In the demyelinating cuprizone model, ozanimod prevented axonal degradation and myelin loss during toxin challenge but did not facilitate enhanced remyelination after intoxication. Since free drug levels in this model only engaged S1P1, we concluded that S1P1 activation is neuroprotective but does not appear to affect remyelination. SIGNIFICANCE STATEMENT: Ozanimod, a selective modulator of human sphingisone 1-phosphate receptor subtypes 1 and 5 (S1P1/5), displays reduced potency for rodent and dog S1P5 compared with human, which results from mutation of threonine to alanine at position 120. Ozanimod can thus be used as a selective S1P1 agonist in mouse models of multiple sclerosis to define efficacies driven by S1P1 but not S1P5. Based on readouts for experimental autoimmune encephalomyelitis and cuprizone intoxication, S1P1 modulation is neuroprotective, but S1P5 activity may be required for remyelination.

Absorption, Metabolism, and Excretion, In Vitro Pharmacology, and Clinical Pharmacokinetics of Ozanimod, a Novel Sphingosine 1-Phosphate Receptor Modulator.

Ozanimod is approved for the treatment of relapsing forms of multiple sclerosis. Absorption, metabolism, and excretion of ozanimod were investigated after a single oral dose of 1.0 mg [14C]ozanimod hydrochloride to six healthy subjects. In vitro experiments were conducted to understand the metabolic pathways and enzymes involved in the metabolism of ozanimod and its active metabolites. The total mean recovery of the administered radioactivity was ∼63%, with ∼26% and ∼37% recovered from urine and feces, respectively. Based on exposure, the major circulating components were active metabolite CC112273 and inactive metabolite RP101124, which together accounted for 50% of the circulating total radioactivity exposure, whereas ozanimod accounted for 6.7% of the total radioactive exposure. Ozanimod was extensively metabolized, with 14 metabolites identified, including two major active metabolites (CC112273 and CC1084037) and one major inactive metabolite (RP101124) in circulation. Ozanimod is metabolized by three primary pathways, including aldehyde dehydrogenase and alcohol dehydrogenase, cytochrome P450 isoforms 3A4 and 1A1, and reductive metabolism by gut microflora. The primary metabolite RP101075 is further metabolized to form major active metabolite CC112273 by monoamine oxidase B, which further undergoes reduction by carbonyl reductases to form CC1084037 or CYP2C8-mediated oxidation to form RP101509. CC1084037 is oxidized rapidly to form CC112273 by aldo-keto reductase 1C1/1C2 and/or 3ß- and 11ß-hydroxysteroid dehydrogenase, and this reversible oxidoreduction between two active metabolites favors CC112273. The ozanimod example illustrates the need for conducting timely radiolabeled human absorption, distribution, metabolism, and excretion studies for characterization of disproportionate metabolites and assessment of exposure coverage during drug development. SIGNIFICANCE STATEMENT: Absorption, metabolism, and excretion of ozanimod were characterized in humans, and the enzymes involved in complex metabolism were elucidated. Disproportionate metabolites were identified, and the activity of these metabolites was determined.

Synthesis of novel 5-(2,5-bis(2,2,2-trifluoroethoxy)phenyl)-1,3,4-oxadiazole-2-thiol derivatives as potential glucosidase inhibitors.

BACKGROUND: A hybrid molecule of different biologically active substances can improve affinity and efficiency compared to a standard drug. Hence based on this fact, we predict that a combination of fluorine, oxadiazole, sulfur, etc., may enhance α-glucosidase inhibition activity compared to a standard drug. METHODS: A series of novel 5-(2,5-bis(2,2,2-trifluoroethoxy)phenyl)-1,3,4-oxadiazole-2-thiol derivatives (2a-2i) were synthesized and characterized using spectroscopic techniques such as 1HNMR and LC-MS. In order to evaluate its bioactivity, in vitro α-amylase and α-glycosidase inhibitory activity were performed. In vivo study was carried using a genetic model, Drosophila melanogaster, for assessing the antihyperglycemic effects. RESULTS: The compounds 2a-2i demonstrated α-amylase inhibitory activity in the range of IC50 = 40.00-80.00 µg/ml as compare to standard acarbose (IC50 = 34.71 µg/ml). Compounds 2a-2i demonstrated α-glucosidase inhibitory activity in the range of IC50 = 46.01-81.65 µg/ml as compared to standard acarbose (IC50 = 34.72 µg/ml). Docking studies on a target protein, N-terminal subunit of human Maltase-glucoamylase (PDB:2QMJ) was carried and the compounds were found to dock into the active site of the enzyme (Fig. 1). The predicted binding energies of the compounds were calculated. The in vitro studies indicate that compounds 2b and 2g had better activity among the synthesized compounds. Whereas in vivo study indicates that 2b, 2g, and 2i could lower glucose levels in the Drosophila, but then 17-30% reduced capacity than acarbose and may be overcome by adjusting their dosage. CONCLUSIONS: The in vitro and in vivo studies indicate that compounds 2b and 2g had better activity among the synthesized compounds. This study has recognized that compounds like 2b, 2g, and 2i may be considered potential candidates for further developing a novel class of antidiabetic agents.

GPR119 agonists: Novel therapeutic agents for type 2 diabetes mellitus.

Diabetes mellitus type 2 (T2D) is a group of genetically heterogeneous metabolic disorders whose frequency has gradually risen worldwide. Diabetes mellitus Type 2 (T2D) has started to achieve a pandemic level, and it is estimated that within the next decade, cases of diabetes might get double due to increase in aging population. Diabetes is rightly called the 'silent killer' because it has emerged to be one of the major causes, leading to renal failure, loss of vision; besides cardiac arrest in India. Thus, a clinical requirement for the oral drug molecules monitoring glucose homeostasis appears to be unmet. GPR119 agonist, a family of G-protein coupled receptors, usually noticed in ß-cells of pancreatic as well as intestinal L cells, drew considerable interest for type 2 diabetes mellitus (T2D). GPR119 monitors physiological mechanisms that enhance homeostasis of glucose, such as glucose-like peptide-1, gastrointestinal incretin hormone levels, pancreatic beta cell-dependent insulin secretion and glucose-dependent insulinotropic peptide (GIP). In this manuscript, we have reviewed the work done in the last five years (2015-2020) which gives an approach to design, synthesize, evaluate and study the structural activity relationship of novel GPR119 agonist-based lead compounds. Our article would help the researchers and guide their endeavours in the direction of strategy and development of innovative, effective GPR119 agonist-based compounds for the management of diabetes mellitus type 2.

Tuning NO release of organelle-targeted furoxan derivatives and their cytotoxicity against lung cancer cells.

We herein report a study on a set of hybrid compounds in which 3-R-substituted furoxan moieties (R = CH3, CONH2, CN, SO2C6H5), endowed with varying NO-releasing capacities, are joined to a mitochondrial probe, rhodamine B. Each product has been investigated for its ability to release NO both in physiological solution, in the presence of cysteine, and in A549 lung adenocarcinoma cancer cells. The cytotoxicity of all the products against the aforementioned cancer cells has been assessed, including the structurally related compounds with no mitochondrial targeting, which were taken as a reference. In the case of the models bearing the -CH3 and -CONH2 groups at the 3-position on the furoxan, only the targeted models showed a significant cytotoxic activity, and only at the highest concentrations, in accordance with their weak NO-releasing properties. On the contrary, the presence of the strong electron-withdrawing groups, as -CN and -SO2C6H5, at the 3-position gave rise to anticancer agents, likely because of the high NO-releasing and of their capability of inhibiting cellular proteins by covalent binding. In detail, the rhodamine hybrid containing the 3-SO2C6H5 substituted furoxan moiety emerged as the most interesting product as it showed high cytotoxicity over the entire concentration range tested. This substructure was also linked to a phenothiazine scaffold that is able to accumulate in lysosomes. Nevertheless, mitochondrial targeting for these NO-donor furoxan substructures was found to be the most efficient.

Synthesis, biological evaluation of benzothiazole derivatives bearing a 1,3,4-oxadiazole moiety as potential anti-oxidant and anti-inflammatory agents.

Twenty benzothiazole derivatives bearing a 1,3,4-oxadiazole moiety were synthesized and evaluated for their anti-oxidant and anti-inflammatory activities. Among these compounds, 8h and 8l were appeared to have high radical scavenging efficacies as 0.05 ± 0.02 and 0.07 ± 0.03 mmol/L of IC50 values in ABTS+ bioassay, respectively. In anti-inflammatory tests, compound 8h displayed good activity with 57.35% inhibition after intraperitoneal administration, which was more potent than the reference drug (indomethacin). Molecular modeling studies were performed to investigate the binding mode of the representative compound 8h into COX-2 enzyme. In vitro enzyme study implied that compound 8h exerted its anti-inflammatory activity through COX-2 inhibition.

Design, synthesis, biological evaluation and molecular docking of new uracil analogs-1,2,4-oxadiazole hybrids as potential anticancer agents.

A new series of uracil analogues-1,2,4-oxadiazole hybrid derivatives were synthesized by a new, simple, and efficient method using for the first time HAP-SO3H as an heterogenous acid catalyst for the condensation and cyclization between amidoxime and aldehyde. The new derivatives were characterized by HRMS, FT-IR, 1H NMR, and 13C NMR spectroscopy techniques. The synthesized 1,2,4-oxadiazole hybrids were evaluated for their cytotoxic activity in five human cancer cell lines: melanoma (A-375), fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A-549). Data showed that compounds 22 and 23 were potent cytotoxic agents against HT-1080 and MFC-7 cells with IC50 inferior to 1 µM. The possible mechanism of apoptosis induction by the derivatives was investigated using Annexin V staining, caspase-3/7 activity, mitochondrial membrane potential measurement, and analysis cell cycle progression. The compound 22 induced apoptosis through caspase-3/7 activation and S-phase arrest in HT-1080 and A549 cells. The molecular docking showed that compound 22 activated the caspase-3 by forming a stable protein-ligand complex.

Design, synthesis and antitrypanosomal activity of heteroaryl-based 1,2,4-triazole and 1,3,4-oxadiazole derivatives.

Two series of novel 1,2,4-triazol-3-yl-thioacetamide 3a-b and 4a-b and 5-pyrazin-2-yl-3H-[1,3,4]oxadiazole-2-thiones 9a-h were designed and synthesized. The compounds prepared have been identified using 1H NMR, 13C NMR and elemental analyses. The synthesized compounds 3a, 3b, 4a, 4b, 9a, 9b, 9d-e and 9f have been evaluated with α-difluoromethylornithine (DFMO) as a control drug for their in vitro antitrypanosomal activity against Trypanosoma brucei. Results showed that 3b was the most active compound in general and also more potent than control DFMO. 3b was 8 folds more potent than the reference with IC50 of 0.79 µM and IC90 of 1.35 µM, respectively compared to DFMO (IC50 = 6.10 µM and IC90 of 8.66 µM). The tested compounds showed moderate cytotoxicity with selectivity indices ranging from 12 (9d) to 102 (3b) against L6 cells. Docking study was performed into ten of T. brucei enzymes which have been identified as potential/valid targets for most of the antitrypanosomal agents. The results of the docking study revealed high binding scores toward many of the selected enzymes. A good correlation was observed only between log (IC50) of antitrypanosomal activity of the new compounds and their calculated Ki values against TryR enzyme (R2 = 0.726). Compound 3b, the most active as antitrypanosomal agents exhibited similar binding orientation and interaction to those of WP6 against TryR enzyme. However, in a next round of work, a complementary studies will be carried out to clarify the mechanism of action of these compounds.

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site.

Neuronal nicotinic receptors containing α4 and ß2 subunits assemble in two pentameric stoichiometries, (α4)3(ß2)2 and (α4)2(ß2)3, each with distinct pharmacological signatures; (α4)3(ß2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(ß2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(ß2)2 receptors, we show that mutations at either the principal face of the ß2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the ß2-α4 pseudo-agonist sites, the α4-α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the ß2 subunit at either ß2-α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4-α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the ß2-α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(ß2)2 receptor, the findings have implications for structure-guided drug design.

Design, Synthesis, and Biological Evaluation of 2-(2-Bromo-3-nitrophenyl)-5-phenyl-1,3,4-oxadiazole Derivatives as Possible Anti-Breast Cancer Agents.

Breast Cancer (BCa) is the most often diagnosed cancer among women who were in the late 1940's. Breast cancer growth is largely dependent on the expression of estrogen and progesterone receptor. Breast cancer cells may have one, both, or none of these receptors. The treatment for breast cancer may involve surgery, hormonal therapy (Tamoxifen, an aromatase inhibitor, etc.) and oral chemotherapeutic drugs. The molecular docking technique reported the findings on the potential binding modes of the 2-(2-bromo-3-nitrophenyl)-5-phenyl-1,3,4-oxadiazole derivatives with the estrogen receptor (PDB ID: 3ERT). The 1,3,4-oxadiazole derivatives 4a-4j have been synthesized and described by spectroscopic method. 2-(2-Bromo-6-nitrophenyl)-5-(4-bromophenyl)-1,3,4-oxadiazole (4c) was reconfirmed by single-crystal XRD. All the compounds have been tested in combination with generic Imatinib pharmaceutical drug against breast cancer cell lines isolated from Caucasian woman MCF-7, MDA-MB-453 and MCF-10A non-cancer cell lines. The compounds with the methoxy (in 4c) and methyl (in 4j) substitution were shown to have significant cytotoxicity, with 4c showing dose-dependent activation and decreased cell viability. The mechanism of action was reported by induced apoptosis and tested by a DNA enzyme inhibitor experiment (ELISA) for Methyl Transferase. Molecular dynamics simulations were made for hit molecule 4c to study the stability and interaction of the protein-ligand complex. The toxicity properties of ADME were calculated for all the compounds. All these results provide essential information for further clinical trials.

Metabolism of Strained Rings: Glutathione S-transferase-Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation.

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione-related metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs, including GSTA1, A2-2, M1a, M2-2, T1-1, and GST from human placenta, were incubated with AZD1979. All GSTs tested catalyzed the formation of M12, with GSTA2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

Design, synthesis, in vivo, and in silico evaluation of new coumarin-1,2,4-oxadiazole hybrids as anticonvulsant agents.

A novel series of coumarin-1,2,4-oxadiazole hybrids were designed, synthesized, and evaluated as anticonvulsant agents. The title compounds were easily synthesized from reaction of appropriate coumarins and 3-aryl-5-(chloromethyl)-1,2,4-oxadiazole derivatives. In vivo anticonvulsant activity of the synthesized compounds were determined using pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures confirming that they were more effective against MES test than PTZ test. It should be noted that compounds 3b, 3c, and 3e showed the best activity in MES model which possessed drug-like properties with no neurotoxicity. Anticonvulsant activity of the most potent compound 3b was remarkably reduced after treatment with flumazenil which confirmed the participation of a benzodiazepine mechanism in the anticonvulsant activity. Also, docking study of compound 3b in the BZD-binding site of GABAA receptor confirmed possible binding of 3b to the BZD receptors.

In vivo multiple metabolic pathways for a novel G protein-coupled receptor 119 agonist DS-8500a in rats: involvement of the 1,2,4-oxadiazole ring-opening reductive reaction in livers under anaerobic conditions.

A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated. Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile. DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a. Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions. These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes.

Unraveling amino acid residues critical for allosteric potentiation of (α4)3(ß2)2-type nicotinic acetylcholine receptor responses.

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(ß2)2 but not (α4)2(ß2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(ß2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(ß2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(ß2)2 nAChR.

Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Noncoherent Light.

FLNBD-BAMPEG2k, bearing a nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer ( Mn = 2,000), was designed to fluorescently label cell membranes by docking its hydrophobic oleyl terminus. During laser scanning microscopy in a minimal essential medium (MEM), human hepatocellular carcinoma Hep3B cells labeled with FLNBD-BAMPEG2k appeared to undergo optoporation at their plasma membrane. We confirmed this unprecedented possibility by a series of cellular uptake experiments using negatively charged and therefore membrane-impermeable quantum dots (QDs; Dh = 4.7 nm). Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (1O2), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers FLNBD-Oleyl and FLNBD-BAMPEG8k having shorter or longer hydrophilic spacers between the NBD and oleyl units showed a little or substantially no optoporation. For understanding these results, one must consider the following contradictory factors: (1) The photosensitized 1O2 generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of 1O2 in aqueous media is very short (3.0-3.5 µs). As supported experimentally and computationally, the hydrophilic spacer length of FLNBD-BAMPEG2k is optimal for compromising these factors. Further to note, the optoporation using FLNBD-BAMPEG2k is not accompanied by cytotoxicity.

Soluble guanylyl cyclase is a critical regulator of migraine-associated pain.

Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite - factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.