A high-specificity aniline-based mass tag for aldehyde detection.

RATIONALE: We studied an aldehyde-labeling reagent, N-{2-[(4-aminophenoxy)methyl]benzyl}-N,N-diethylethanaminium bromide (CAX-A), containing an aniline functional group for the detection of aldehydes with high specificity. METHODS: Six standard aldehydes were labeled by CAX-A and analyzed using LC-ESI-Orbitrap-MS. The aldehydes (each 40 nmol) were derivatized with CAX-A in the presence of sodium cyanoborohydride at room temperature overnight. The labeling reaction was applied to two urine samples for the detection of putative aldehydes. RESULTS: All six standard CAX-aldehyde derivatives were detected as precursor ions by dilution to 830 fmol/injection (signal-to-noise [S/N] ratio 587-1573). A total of 2184 MS1 features were detected overall in urine and blanks, of which 14 were putative aldehydes found only in urine. CONCLUSIONS: CAX-A can provide three levels of specificity for aldehyde detection. First is the known labeling specificity of the aniline functional group for aldehydes, which we confirmed here by observing a significant peak only from the aldehyde (S/N = 3388) when a mixture of an aldehyde, a ketone (no peak), and a quinone (S/N = 2.3) was tested. Second is the ease of formation of an analyte-characteristic first product ion (via anchimeric-assisted loss of triethylamine as a neutral) in MS2 from a CAX-labeled analyte. Third is the formation of a characteristic second product ion via loss of CO in MS3. CAX-A enables the specific, convenient detection of putative aldehydes in urine.

Inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse vulnerability in rats.

Glutamate signalling through the N-methyl-d-aspartate receptor (NMDAR) activates the enzyme neuronal nitric oxide synthase (nNOS) to produce the signalling molecule nitric oxide (NO). We hypothesized that disruption of the protein-protein interaction between nNOS and the scaffolding protein postsynaptic density 95 kDa (PSD95) would block NMDAR-dependent NO signalling and represent a viable therapeutic route to decrease opioid reward and relapse-like behaviour without the unwanted side effects of NMDAR antagonists. We used a conditioned place preference (CPP) paradigm to evaluate the impact of two small-molecule PSD95-nNOS inhibitors, IC87201 and ZL006, on the rewarding effects of morphine. Both IC87201 and ZL006 blocked morphine-induced CPP at doses that lacked intrinsic rewarding or aversive properties. Furthermore, in vivo fast-scan cyclic voltammetry (FSCV) was used to ascertain the impact of ZL006 on morphine-induced increases in dopamine (DA) efflux in the nucleus accumbens shell (NAc shell) evoked by electrical stimulation of the medial forebrain bundle (MFB). ZL006 attenuated morphine-induced increases in DA efflux at a dose that did not have intrinsic effects on DA transmission. We also employed multiple intravenous drug self-administration approaches to examine the impact of ZL006 on the reinforcing effects of morphine. Interestingly, ZL006 did not alter acquisition or maintenance of morphine self-administration, but reduced lever pressing in a morphine relapse test after forced abstinence. Our results provide behavioural and neurochemical support for the hypothesis that inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse-like behaviour, highlighting a previously unreported application for these novel therapeutics in the treatment of opioid addiction.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Distinct Subpopulations of Intravalvular Methicillin-Resistant Staphylococcus aureus with Variable Susceptibility to Daptomycin in Tricuspid Valve Endocarditis.

We present a case of endocarditis wherein organisms cultured from different valve leaflets yielded different daptomycin susceptibilities from each other and from organisms obtained from peripheral blood culture. Genomic analyses showed mutations in mprF, purR, and agrA Pharmacokinetic simulations showed consistent activity of daptomycin plus beta-lactam against all subpopulations. This represents an opportunity to understand S. aureus evolution and fitness in vivo on daptomycin therapy and the role of beta-lactams to prevent the selection of daptomycin-resistant subpopulations.

Focused structure-activity relationship profiling around the 2-phenylindole scaffold of a cannabinoid type-1 receptor agonist-positive allosteric modulator: site-III aromatic-ring congeners with enhanced activity and solubility.

Specific tuning of cannabinoid 1 receptor (CB1R) activity by small-molecule allosteric modulators is a therapeutic modality with multiple properties inherently advantageous to therapeutic applications. We previously generated a library of unique CB1R positive allosteric modulators (PAMs) derived from GAT211, which has three pharmacophoric sites critical to its ago-PAM activity. To elaborate our CB1R PAM library, we report the rational design and molecular-pharmacology profiling of several 2-phenylindole analogs modified at the "site-III" aromatic ring. The comprehensive structure-activity relationship (SAR) investigation demonstrates that attaching small lipophilic functional groups on the ortho-position of the GAT211 site-III phenyl ring could markedly enhance CB1R ago-PAM activity. Select site-III modifications also improved GAT211's water solubility. The SAR reported both extends the structural diversity of this compound class and demonstrates the utility of GAT211's site-III for improving the parent compound's drug-like properties of potency and/or aqueous solubility.

Allosteric Cannabinoid Receptor 1 (CB1) Ligands Reduce Ocular Pain and Inflammation.

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2-/-) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ8-tetrahydrocannabinol (Δ8-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2-/- mice. Two percent GAT228, or the combination of 0.2% Δ8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.

Enantiomer-specific positive allosteric modulation of CB1 signaling in autaptic hippocampal neurons.

The cannabinoid signaling system is found throughout the CNS and its involvement in several pathological processes makes it an attractive therapeutic target. Because orthosteric CB1 cannabinoid receptor ligands have undesirable adverse effects there has been great interest in the development of allosteric modulators - both negative (NAMs) and positive (PAMs) - of these receptors. NAMs of CB1 appeared first on the scene, followed more recently by PAMs. Because allosteric modulation can vary depending on the orthosteric ligand it is important to study their function in a system that employs endogenous cannabinoids. We have recently surveyed first generation NAMs using cultured autaptic hippocampal neurons. These neurons express depolarization induced suppression of excitation (DSE), a form of synaptic plasticity that is mediated by CB1 and 2-arachidonoyl glycerol (2-AG); they are therefore an excellent neuronal model of endogenous cannabinoid signaling in which to test CB1 modulators. In this study we find that while two related compounds, GAT211 and ZCZ011, each show PAM-like responses in autaptic hippocampal neurons, they also exhibit complex pharmacology. Notably we were able to separate the PAM- and agonist-like responses of GAT211 by examining the enantiomers of this racemic compound: GAT228 and GAT229. We find that GAT229 exhibits PAM-like behavior while GAT228 appears to directly activate the CB1 receptor. Both GAT229 and ZCZ011 represent the first PAMs that we have found to be effective in using this 2-AG utilizing neuronal model system. Because these compounds may exhibit both probe selectivity and biased signaling it will be important to test them with anandamide as well as other signaling pathways.

Development of acridine derivatives as selective Mycobacterium tuberculosis DNA gyrase inhibitors.

In this study we have designed p-phenylene diamine linked acridine derivative from our earlier reported quinoline-aminopiperidine hybrid MTB DNA gyrase inhibitors with aiming more potency and less cardiotoxicity. We synthesized thirty six compounds using four step synthesis from 2-chloro benzoic acid. Among them compound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)benzenesulphonamide (6) was found to be more potent with MTB DNA gyrase super coiling IC50 of 5.21±0.51µM; MTB MIC of 6.59µM and no zHERG cardiotoxicity at 30µM and 11.78% inhibition at 50µM against mouse macrophage cell line RAW 264.7.

Replacement of cardiotoxic aminopiperidine linker with piperazine moiety reduces cardiotoxicity? Mycobacterium tuberculosis novel bacterial topoisomerase inhibitors.

Recently numerous non-fluoroquinolone-based bacterial type II topoisomerase inhibitors from both the GyrA and GyrB classes have been reported as antibacterial agents. Inhibitors of the GyrA class include aminopiperidine-based novel bacterial type II topoisomerase inhibitors (NBTIs). However, inhibition of the cardiac ion channel remains a serious liability for the aminopiperidine based NBTIs. In this paper we replaced central aminopiperidine linker with piperazine moiety and tested for its biological activity. We developed a series of twenty four compounds with a piperazine linker 1-(2-(piperazin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one, by following a multistep protocol. Among them compound 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-N-(4-nitrophenyl)piperazine-1-carboxamide (11) was the most promising inhibitor with Mycobacterium tuberculosis (MTB) DNA gyrase enzyme supercoiling IC50 of 0.29±0.22µM, with a good MTB MIC of 3.45µM. These kind of compounds retains good potency and showed reduced cardiotoxicity compared to aminopiperidines.

5-Benzylidene-2,4-thiazolidenedione derivatives: Design, synthesis and evaluation as inhibitors of angiogenesis targeting VEGR-2.

A series of novel 5-benzylidene-2,4-thiazolidinediones were designed as inhibitors of angiogenesis targeting VEGFR-2. In docking study, molecules showed similar way of binding with VEGFR-2 as that of the co-crystallized ligand. Compounds were then synthesized, purified and characterized by spectroscopic techniques. Compounds 3f and 3i were found to be most active in the series showing good inhibition of angiogenesis in both CAM and in zebrafish embryo assays. Compound 3i also exhibited IC50 of 0.5µM against VEGFR-2.

Enabling the (3 + 2) cycloaddition reaction in assembling newer anti-tubercular lead acting through the inhibition of the gyrase ATPase domain: lead optimization and structure activity profiling.

DNA gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis, is absent in humans and is a well validated target for anti-tubercular drug discovery. In this study, a moderately active inhibitor of Mycobacterium tuberculosis GyrB, the pharmaceutically unexploited domain of DNA gyrase, was reengineered using a combination of molecular docking and medicinal chemistry strategies to obtain a lead series displaying considerable in vitro enzyme efficacy and bacterial kill against the Mycobacterium tuberculosis H37Rv strain. Biophysical investigations using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain. Furthermore, the molecules were completely devoid of hERG toxicity up to 30 µM, as evaluated in a zebra fish model with a good selectivity index, and from a pharmaceutical point of view, turned out as potential candidates against TB.

Design and synthesis of novel quinoline-aminopiperidine hybrid analogues as Mycobacterium tuberculosis DNA gyraseB inhibitors.

Antibiotics with good therapeutic value and novel mechanism of action are becoming increasingly important in today's battle against bacterial resistance. One of the popular targets being DNA gyrase, is currently becoming well-established and clinically validated for the development of novel antibacterials. In the present work, a series of forty eight quinoline-aminopiperidine based urea and thiourea derivatives were synthesized as pharmacophoric hybrids and evaluated for their biological activity. Compound, 1-(4-chlorophenyl)-3-(1-(6-methoxy-2-methylquinolin-4-yl)piperidin-4-yl)thiourea (45) was found to exhibit promising in vitro Mycobacterium smegmatis GyrB IC50 of 0.95 ± 0.12 µM and a well correlated Mycobacterium tuberculosis (MTB) DNA gyrase supercoiling IC50 of 0.62 ± 0.16 µM. Further, compound 45 also exhibited commendable MTB MIC, safe eukaryotic cytotoxic profile with no signs of cardiotoxicity in zebrafish ether-a-go-go-related gene (zERG).

Exploring the gyrase ATPase domain for tailoring newer anti-tubercular drugs: hit to lead optimization of a novel class of thiazole inhibitors.

Gyrase ATPase domain, the pharmaceutical underexploited segment of DNA gyrase, the sole Type II topoisomerase present in Mycobacterium tuberculosis represents an attractive target for anti-tubercular drug discovery. Here we report, the development of a novel series of MTB DNA gyraseB inhibitor identified through a medium throughput screening (MTS) of BITS in-house chemical library (3000 compounds). The MTS hit was further remodeled by chemical synthesis to identify the most potent analogue 27 exhibiting an in vitro gyrB inhibitory IC50 of 0.15 µM. The series also demonstrated well correlating gyrase super coiling activity and in vitro anti-mycobacterial potency against MTB H37Rv strain. Furthermore the compounds displayed good safety profile in their subsequent cytotoxicity and hERG toxicity evaluations, to be worked out from a pharmaceutical point of view as potential anti-tubercular agents.

NaSH in the construction of thiophene ring fused with N-heterocycles: a rapid and inexpensive synthesis of novel small molecules as potential inducers of apoptosis.

A facile construction of a thiophene ring fused with N-heterocycles has been achieved via the reaction of NaSH with 2-chloro-3-alkynyl quinoxalines/pyrazines leading to novel 2-substituted thieno[2,3-b]pyrazine/quinoxaline derivatives as potential inducers of apoptosis. Some of them showed encouraging pharmacological properties when tested in zebrafish.

Ligand-free MCR for linking quinoxaline framework with a benzimidazole nucleus: a new strategy for the identification of novel hybrid molecules as potential inducers of apoptosis.

We report a true MCR involving the reaction of N-(prop-2-ynyl)quinoxalin-2-amine derivatives with 2-iodoanilines and tosyl azide in the presence of 10 mol% of CuI and Et3N in DMSO to afford the pre-designed hybrid molecules containing quinoxaline framework linked with a benzimidazole nucleus. The MCR proceeds in the absence of any ligand and/or lateral addition of the catalyst/base affording products within 30 min in good yields, some of which showed encouraging apoptosis inducing properties in zebrafish.

A Pd-based regioselective strategy to indole-1,2-fused 8- and 9-membered rings: their evaluation as potential scaffolds for apoptosis in zebrafish.

A strategy based on Pd-mediated ring closure of 1,2-disubstituted indoles containing an unactivated olefin leading to indole-1,2-fused 8- and 9-membered rings has been developed for the identification of new and potential scaffolds for apoptosis. A large number of fused indole derivatives containing an endocyclic double bond were synthesized using this robust methodology. A representative compound showed promising apoptotic properties in zebrafish embryos.

Isovanillin derived N-(un)substituted hydroxylamines possessing an ortho-allylic group: valuable precursors to bioactive N-heterocycles.

The intramolecular 1,3-dipolar cycloaddition of isovanillin derived N-aryl hydroxylamines possessing ortho-allylic dipolarophiles affords novel benzo analogues of tricyclic isoxazolidines that can be readily transformed into functionalized lactams, γ-aminoalcohols and oxazepines. The corresponding N-unsubstituted hydroxylamines give rise to tetrahydroisoquinolines. Anxiogenic properties of these compounds are tested in zebra fish.

Structure-guided design and development of novel benzimidazole class of compounds targeting DNA gyraseB enzyme of Staphylococcus aureus.

The gyraseB subunit of Staphylococcus aureus DNA gyrase is a well-established and validated target though less explored for the development of novel antimicrobial agents. Starting from the available structural information in PDB (3TTZ), we identified a novel series of benzimidazole used as inhibitors of DNA gyraseB with low micromolar inhibitory activity by employing structure-based drug design strategy. Subsequently, this chemical class of DNA gyrase inhibitors was extensively investigated biologically through in vitro assays, biofilm inhibition assays, cytotoxicity, and in vivo studies. The binding affinity of the most potent inhibitor 10 was further ascertained biophysically through differential scanning fluorimetry. Further, the most potent analogues did not show any signs of cardiotoxicity in Zebra fish ether-a-go-go-related gene (zERG), a major breakthrough among the previously reported cardiotoxic gyraseB inhibitors.

Transition metal free hydrolysis/cyclization strategy in a single pot: synthesis of fused furo N-heterocycles of pharmacological interest.

A transition metal free tandem two-step strategy has been developed involving hydrolysis of 2-chloro-3-alkynyl quinoxalines/pyrazines followed by in situ cyclization of the corresponding 2-hydroxy-3-alkynyl intermediates in a single pot leading to fused furo N-heterocycles as potential inhibitors of sirtuins. A representative compound showed promising pharmacological properties in vitro and in vivo.

Novel N-indolylmethyl substituted olanzapine derivatives: their design, synthesis and evaluation as PDE4B inhibitors.

A new strategy for converting antipsychotic drug olanzapine into PDE4 inhibitors is described via the design and Pd/C mediated synthesis of novel N-indolylmethyl olanzapine derivatives. One compound showed good inhibition (IC50 1.1 µM) and >10 fold selectivity towards PDE4B over D that was supported by docking studies. This compound also showed significant inhibition of TNF-α and no major toxicities in cell lines and a zebrafish embryo model except the teratogenic effects to be re-assessed in rodents.