Omarigliptin/galangin combination mitigates lipopolysaccharide-induced neuroinflammation in rats: Involvement of glucagon-like peptide-1, toll-like receptor-4, apoptosis and Akt/GSK-3ß signaling.

AIMS: The objectives of this work were to assess the possibility of administration of omarigliptin and/or galangin to combat lipopolysaccharide (LPS)-induced neuroinflammation in rats and to explore the possible mechanisms that might contribute to their actions. MATERIALS AND METHODS: In a rat model of LPS-induced neuroinflammation, the changes in the behavioral tests, biochemical parameters, and the histopathological picture were assessed. KEY FINDINGS: Administration of either omarigliptin or galangin to LPS-injected rats was able to significantly improve the behavioral changes with restoration of the oxidant/antioxidant balance, decrement of toll-like receptor-4 levels, and amelioration of the neuroinflammation associated with inhibition of apoptosis and restoration of glucagon-like peptide-1 levels in the cerebral tissues. In addition, omarigliptin and/or galangin significantly reduced the levels of phospho-Akt and glycogen synthase kinase 3 beta (GSK-3ß) and significantly increased the expression of beclin-1 in the cerebral tissues compared versus the group treated with LPS alone. As a result, these changes were positively reflected on the histopathological and the electron microscopic picture of the cerebral tissues. These beneficial effects were maximally evidenced in rats treated with omarigliptin/galangin combination relative to the use of either omarigliptin or galangin alone. SIGNIFICANCE: Omarigliptin/galangin combination might be proposed as a promising therapeutic line for mitigation of the pathophysiologic events of LPS-induced neuroinflammation.

Structure-Based Design and Discovery of Pyridyl-Bearing Fused Bicyclic HIV-1 Inhibitors: Synthesis, Biological Characterization, and Molecular Modeling Studies.

Two series of new pyridyl-bearing fused bicyclic analogues designed to target the dual-tolerant regions of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket were synthesized and evaluated for their anti-HIV activities. Several compounds, such as 6, 14, 15, 21, 30, and 33, were found to be potent inhibitors against the wild-type (WT) HIV-1 strain or multiple NNRTI-resistant strains at low nanomolar levels. Detailed structure-activity relationships were obtained by utilizing the variation of moieties within the corresponding pharmacophores. In vitro metabolic stability profiles and some drug-like properties of selected compounds were assessed, furnishing the preliminary structure-metabolic stability relationships. Furthermore, molecular modeling studies elucidated the binding modes of compounds 6, 15, 21, and 30 in the binding pocket of WT, E138K, K103N, or Y181C HIV-1 RTs. These promising compounds can be used as lead compounds and warrant further structural optimization to yield more active HIV-1 inhibitors.

Repurposing of Omarigliptin as a Neuroprotective Agent Based on Docking with A2A Adenosine and AChE Receptors, Brain GLP-1 Response and Its Brain/Plasma Concentration Ratio after 28 Days Multiple Doses in Rats Using LC-MS/MS.

The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson's disease while AChE plays a major role in Alzheimer's disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats' brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses' study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC-MS/MS method with enhanced liquid-liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats' plasma (10-3100 ng/mL) and rats' brain tissue (15-2900 ng/mL) using liquid-liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats' plasma and rats' brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats' plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases.

Bicyclic Diazepinones as Dual Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels and the Norepinephrine Transporter.

The synthesis and pharmacological activity of a new series of bicyclic diazepinones with dual activity toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) and the norepinephrine transporter (NET) are reported. Exploration of the positions amenable for substitution on a nonaminoacidic Cavα2δ-1 scaffold allowed the identification of favorable positions for the attachment of NET pharmacophores. Among the patterns explored, attachment of the 2-ethylamino-9-methyl-6-phenyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one framework to the meta-position of the phenyl ring of the 3-methylamino-1-phenylpropoxy and 3-methylamino-1-thiophenylpropoxy moieties provided dual compounds with excellent NET functionality. Alternative bicyclic frameworks were also explored, and some lead molecules were identified, which showed a balanced dual profile and exhibited good ADMET properties.

Structural Basis of Inhibitor Selectivity in the BRD7/9 Subfamily of Bromodomains.

Inhibition of the bromodomain containing protein 9 (BRD9) by small molecules is an attractive strategy to target mutated SWI/SNF chromatin-remodeling complexes in cancer. However, reported BRD9 inhibitors also inhibit the closely related bromodomain-containing protein 7 (BRD7), which has different biological functions. The structural basis for differential potency and selectivity of BRD9 inhibitors is largely unknown because of the lack of structural information on BRD7. Here, we biochemically and structurally characterized diverse inhibitors with varying degrees of potency and selectivity for BRD9 over BRD7. Novel cocrystal structures of BRD7 liganded with new and previously reported inhibitors of five different chemical scaffolds were determined alongside BRD9 and BRD4. We also report the discovery of first-in-class dual bromodomain-kinase inhibitors outside the bromodomain and extraterminal family targeting BRD7 and BRD9. Combined, the data provide a new framework for the development of BRD7/9 inhibitors with improved selectivity or additional polypharmacologic properties.

Optimization of Fused Bicyclic Allosteric SHP2 Inhibitors.

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

Photodegradation of fluazaindolizine in water under simulated sunlight irradiation: Identification of transformation products and elucidation of transformation mechanism.

The photodegradation of fluazaindolizine in water was investigated under simulated sunlight irradiation. The effects of solution pH, humic acids (HA), nitrates (NO3-) and Fe(III) ions on photolysis of fluazaindolizine were studied. The results indicated that pH did not significantly affect its photodegradation. At low concentration (up to 5 mg/L), HA slightly facilitated the photodegradation of fluazaindolizine, while at high concentration (10-20 mg/L), HA inhibited its photodegradation. The presence of NO3- (0-10 mg/L) and Fe(III) (0-5 mg/L) noticeably accelerated the photodegradation of fluazaindolizine. Moreover, eleven direct transformation products (TPs) were isolated and identified by liquid chromatography quadrupole time-of-flight mass spectrometry. Density functional theory (DFT) calculation was utilized to characterize molecular property of fluazaindolizine and predict the potentiality of the possible photodegradation reaction. Ultimately, a possible transformation mechanism was proposed based on the identified TPs, degradation profiles and DFT calculation. The predominant photoproduct came from ring opening of imidazole-ring and dechlorination. Other TPs resulted from a series of photochemical reactions involving hydroxyl substitution, ring-opening, cleavage, oxidation and decarboxylation. These results were important in elucidating environmental fate of fluazaindolizine in aquatic system and further environmental risk assessment.

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis.

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Synthesis and biological evaluation of new N-benzylpyridinium-based benzoheterocycles as potential anti-Alzheimer's agents.

A novel series of benzylpyridinium-based benzoheterocycles (benzimidazole, benzoxazole or benzothiazole) were designed as potent acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The title compounds 4a-q were conveniently synthesized via condensation reaction of 1,2-phenylenediamine, 2-aminophenol or 2-aminothiophenol with pyridin-4-carbalehyde, followed by N-benzylation using various benzyl halides. The results of in vitro biological assays revealed that most of them, especially 4c and 4g, had potent anticholinesterase activity comparable or more potent than reference drug, donepezil. The kinetic study demonstrated that the representative compound 4c inhibits AChE in competitive manner. According to the ligand-enzyme docking simulation, compound 4c occupied the active site at the vicinity of catalytic triad. The compounds 4c and 4g were found to be inhibitors of Aß self-aggregation as well as AChE-induced Aß aggregation. Meanwhile, these compounds could significantly protect PC12 cells against H2O2-induced injury and showed no toxicity against HepG2 cells. As multi-targeted structures, compounds 4c and 4g could be considered as promising candidate for further lead developments to treat Alzheimer's disease.

Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi.

Peramine is a non-ribosomal peptide-derived pyrrolopyrazine (PPZ)-containing molecule with anti-insect properties. Peramine is known to be produced by fungi from genus Epichloë, which form mutualistic endophytic associations with cool-season grass hosts. Peramine biosynthesis has been proposed to require only the two-module non-ribosomal peptide synthetase (NRPS) peramine synthetase (PerA), which is encoded by the 8.3 kb gene perA, though this has not been conclusively proven. Until recently, both peramine and perA were thought to be exclusive to fungi of genus Epichloë; however, a putative perA homologue was recently identified in the genome of the insect-pathogenic fungus Metarhizium rileyi. We use a heterologous expression system and a hydrophilic interaction chromatography-based analysis method to confirm that PerA is the only pathway-specific protein required for peramine biosynthesis. The perA homologue from M. rileyi (MR_perA) is shown to encode a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë. Furthermore, perA is part of a larger seven-gene PPZ cluster in M. rileyi, Metarhizium majus and the stalked-cup lichen fungus Cladonia grayi. These PPZ genes encode proteins predicted to derivatize peramine into more complex PPZ metabolites, with the orphaned perA gene of Epichloë spp. representing an example of reductive evolution.

A concise synthesis of peramine, a metabolite of endophytic fungi.

The total synthesis of peramine, a natural product isolated from an endophytic fungi, has been achieved in four steps and 34% overall yield from known compounds. The key step was the one-pot construction of the pyrrolopyrazinone ring from pyrrole amide and propargyl bromide. The preparation of peramine-d4 as an internal standard for quantitative analysis by MS is also described.

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (Lolium perenne L.) with altered expression of fructosyltransferases.

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI- (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI- for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI- compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI-. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte-grass associations. Endophyte-host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.

Development of Bruton's Tyrosine Kinase Inhibitors for Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease and becomes one of the major causes of disability and work force loss. The presence of abnormal B cell and autoantibodies produced by most RA patients, primarily ACPA and RF, indicate that the function of B cell was involved in the development of RA disease. Accordingly, the drug targeting B cell has become a hot spot in the treatment of RA. Studies have shown that Bruton's tyrosine kinase (BTK) is involved in the regulation of B cell proliferation and activation process. Some small molecule BTK inhibitors have shown excellent inhibition in biological activity analysis and animal models. Therefore, this review will briefly introduce BTK and its role in cell signaling and overview recent progress of BTK inhibitors for RA treatment.

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria.

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Discovery and Optimization of Phosphopantetheine Adenylyltransferase Inhibitors with Gram-Negative Antibacterial Activity.

In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.

Late-occurring and Long-circulating Metabolites of GABAAα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance.

AZD7325 [4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide] is a selective GABAAα2,3 receptor modulator intended for the treatment of anxiety disorders through oral administration. An interesting metabolic cyclization and aromatization pathway led to the tricyclic core of M9, i.e., 2-ethyl-7-(2-fluoro-6-methoxyphenyl)pyrimido[5,4-c]cinnolin-4(3H)-one. Further oxidative metabolism generated M10 via O-demethylation and M42 via hydroxylation. An authentic standard of M9 was synthesized to confirm the novel structure of M9 and that of M10 and M42 by liver microsomal incubation of the M9 standard. Metabolites M9, M10, and M42 were either minor or absent in plasma samples after a single dose; however, all became major metabolites in human and preclinical animal plasma after repeated doses and circulated in humans longer than 48 hours after the end of seven repeated doses. The absence of these long circulating metabolites from selected patients' plasma samples was used to demonstrate patient noncompliance as the cause of unexpected lack of drug exposure in some patients during a Phase IIb outpatient clinical study. The observation of late-occurring and long-circulating metabolites demonstrates the need to collect plasma samples at steady state after repeated doses when conducting metabolite analysis for the safety testing of drug metabolites. All 12 major nonconjugate metabolites of AZD7325 observed in human plasma at steady state were also observed in dog, rat, and mouse plasma samples collected from 3-month safety studies and at higher exposures in the animals than humans. This eliminated concern about human specific or disproportional metabolites.

A mimetic of the mSin3-binding helix of NRSF/REST ameliorates abnormal pain behavior in chronic pain models.

The neuron-restrictive silencing factor NRSF/REST binds to neuron-restrictive silencing elements in neuronal genes and recruits corepressors such as mSin3 to inhibit epigenetically neuronal gene expression. Because dysregulation of NRSF/REST is related to neuropathic pain, here, we have designed compounds to target neuropathic pain based on the mSin3-binding helix structure of NRSF/REST and examined their ability to bind to mSin3 by NMR. One compound, mS-11, binds strongly to mSin3 with a binding mode similar to that of NRSF/REST. In a mouse model of neuropathic pain, mS-11 was found to ameliorate abnormal pain behavior and to reverse lost peripheral morphine analgesia. Furthermore, even in the less well epigenetically defined case of fibromyalgia, mS-11 ameliorated symptoms in a mouse model, suggesting that fibromyalgia is related to the dysfunction of NRSF/REST. Taken together, these findings show that the chemically optimized mimetic mS-11 can inhibit mSin3-NRSF/REST binding and successfully reverse lost peripheral and central morphine analgesia in mouse models of pain.

Synthesis and biological evaluation of novel imidazol-1-ylacetic acid derivatives as non-brain penetrant bombesin receptor subtype-3 (BRS-3) agonists.

Novel compounds based on 1a were synthesized with the focus of obtaining agonists acting upon peripheral BRS-3. To identify potent anti-obesity compounds without adverse effects on the central nervous system (CNS), a carboxylic acid moiety and a labile carboxylic ester with an antedrug functionality were introduced. Through the extensive synthetic exploration and the pharmacokinetic studies of intravenous administration in mice, the ester 2b was selected owing to its most suitable pharmacological profile. In the evaluation of food intake suppression in C57BL/6N mice, 2b showed significant in vivo efficacy and no clear adverse effects on blood pressure change in dogs administered the compound by intravenous infusion.

Climate change increases the risk of herbicide-resistant weeds due to enhanced detoxification.

MAIN CONCLUSION: Global warming will increase the incidence of metabolism-based reduced herbicide efficacy on weeds and, therefore, the risk for evolution of non-target site herbicide resistance. Climate changes affect food security both directly and indirectly. Weeds are the major biotic factor limiting crop production worldwide, and herbicides are the most cost-effective way for weed management. Processes associated with climatic changes, such as elevated temperatures, can strongly affect weed control efficiency. Responses of several grass weed populations to herbicides that inhibit acetyl-CoA carboxylase (ACCase) were examined under different temperature regimes. We characterized the mechanism of temperature-dependent sensitivity and the kinetics of pinoxaden detoxification. The products of pinoxaden detoxification were quantified. Decreased sensitivity to ACCase inhibitors was observed under elevated temperatures. Pre-treatment with the cytochrome-P450 inhibitor malathion supports a non-target site metabolism-based mechanism of herbicide resistance. The first 48 h after herbicide application were crucial for pinoxaden detoxification. The levels of the inactive glucose-conjugated pinoxaden product (M5) were found significantly higher under high- than low-temperature regime. Under high temperature, a rapid elevation in the level of the intermediate metabolite (M4) was found only in pinoxaden-resistant plants. Our results highlight the quantitative nature of non-target-site resistance. To the best of our knowledge, this is the first experimental evidence for temperature-dependent herbicide sensitivity based on metabolic detoxification. These findings suggest an increased risk for the evolution of herbicide-resistant weeds under predicted climatic conditions.