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.

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.

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.

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.

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.

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.

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.

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.

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident.

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.

AZD6280, a novel partial γ-aminobutyric acid A receptor modulator, demonstrates a pharmacodynamically selective effect profile in healthy male volunteers.

OBJECTIVE: AZD6280 is a novel γ-aminobutyric acid A receptor modulator with higher in vitro efficacy at the α2,3 subtypes as compared to the α1 and α5 subtypes. This study compared the pharmacodynamic effects of single oral dose AZD6280 10 mg and 40 mg on the central nervous system with 2 mg of lorazepam. METHODS: Sixteen healthy males were enrolled into the double-blind, randomized, 4-way crossover study. Two validated central nervous system test batteries, Neurocart and CogState, were administered to measure drug effects on cognition, neurophysiologic function, and psychomotor and subjective feelings. Statistical analysis was performed using mixed model analysis of variance, with fixed factors of treatment, period, time and treatment by time, and random factors of subject, subject by treatment and subject by time, and the average prevalue as covariate. RESULTS: Most pharmacodynamic parameters were affected by lorazepam. AZD6280 induced dose-dependent smaller-than-lorazepam effects on saccadic peak velocity (SPV) (AZD6280, 10 mg vs. AZD6280, 40 mg vs. lorazepam [deg/s]: -22.6 vs. -50.0 vs. -62.9, P < 0.001), whereas the impacts on adaptive-tracking, body-sway, smooth-pursuit, and the one-card-learning tests were significant but much smaller than lorazepam. Thus, the slopes of regression lines for the ΔLog(Sway)-ΔSPV, ΔTracking-ΔSPV, and ΔSmooth-ΔSPV relations were flatter with AZD6280 than with lorazepam. AZD6280 caused a distinct electroencephalography signature from that of lorazepam. CONCLUSIONS: The SPV responses to AZD6280 suggest potential concentration-related anxiolytic effects, whereas the smaller SPV-normalized effects of AZD6280 on various non-SPV pharmacodynamic parameters suggest a more favorable side effect profile compared to lorazepam. Overall, the pharmacodynamic profile of AZD6280 matches the pharmacological specificity and selectivity of this compound at the α2,3 γ-aminobutyric acid A receptor subtypes.

Synthesis, binding affinity, radiolabeling, and microPET evaluation of 4-(2-substituted-4-substituted)-8-(dialkylamino)-6-methyl-1-substituted-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-ones as ligands for brain corticotropin-releasing factor type-1 (CRF1) receptors.

Compounds 1-14 were synthesized in a search for high-affinity CRF1 receptor ligands that could be radiolabeled with (11)C or (18)F for use as positron emission tomography (PET) radiotracers. Derivatives of 2 were developed which contained amide N-fluoroalkyl substituents. Compounds [(18)F]24 and [(18)F]25 were found to have appropriate lipophilicities of logP7.4=2.2 but microPET imaging with [(18)F]25 demonstrated limited brain uptake.

Species difference in glucuronidation formation kinetics with a selective mTOR inhibitor.

The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct-cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct-catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1- and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure.

Lack of metabolic activation and predominant formation of an excreted metabolite of nontoxic platynecine-type pyrrolizidine alkaloids.

Pyrrolizidine alkaloid (PA) poisoning is well-known because of the intake of PA-containing plant-derived natural products and PA-contaminated foodstuffs. Based on different structures of the necine bases, PAs are classified into three types: retronecine, otonecine, and platynecine type. The former two type PAs possessing an unsaturated necine base with a 1,2-double bond are hepatotoxic due to the P450-mediated metabolic activation to generate reactive pyrrolic ester, which interacts with cellular macromolecules leading to toxicity. With a saturated necine base, platynecine-type PAs are reported to be nontoxic and their nontoxicity was hypothesized to be due to the absence of metabolic activation; however, the metabolic pathway responsible for their nontoxic nature is largely unknown. In the present study, to prove the absence of metabolic activation in nontoxic platynecine-type PAs, hepatic metabolism of platyphylline (PLA), a representative platynecine-type PA, was investigated and directly compared with the representatives of two toxic types of PAs in parallel. By determining the pyrrolic ester-derived glutathione conjugate, our results confirmed that the major metabolic pathway of PLA did not lead to formation of the reactive pyrrolic ester. More interestingly, having a metabolic rate similar to that of toxic PAs, PLA also underwent oxidative metabolisms mediated by P450s, especially P450 3A4, the same enzyme that catalyzes metabolic activation of two toxic types of PAs. However, the predominant oxidative dehydrogenation pathway of PLA formed a novel metabolite, dehydroplatyphylline carboxylic acid, which was water-soluble, readily excreted, and could not interact with cellular macromolecules. In conclusion, our study confirmed that the saturated necine bases determine the absence of metabolic activation and thus govern the metabolic pathway responsible for the nontoxic nature of platynecine-type PAs.

Structural findings of cinnolines as anti-schizophrenic PDE10A inhibitors through comparative chemometric modeling.

Schizophrenia is a complex psychiatric disorder associated with the distortion of striatopallidal neurotransmission of central nervous system. Phosphodiesterase10A (PDE10A) enzyme plays crucial role in cellular signaling pathways in schizophrenia. Inhibition of this enzyme may facilitate better treatment of this disease. 2D-QSAR, HQSAR, pharmacophore mapping, molecular docking, and 3D-QSAR analyses were performed on 81 cinnoline derivatives having PDE10A inhibitory activity. 2D-QSAR models were developed by multiple linear regression and partial least square analyses using both atom based and whole molecular descriptors. The best model, having considerable internal (q(2) = 0.812) and external (R(2)(pred) = 0.691) predictabilities, demonstrated importance of atom-based topological and whole molecular E-state as well as 3D topological indices. The best HQSAR model was also found to be statistically significant (q(2) = 0.664, R(2)(pred) = 0.513) and it highlighted some important structural features. PHASE-based pharmacophore hypothesis showed the importance of three hydrogen bond acceptor and one each of ring aromatic and hydrophobic features for higher activity. 3D-QSAR CoMFA and CoMSIA models were generated on two different types of alignment procedures-(1) pharmacophore (PHASE) based and (2) docking (GLIDE) based. GLIDE-based alignment produced better results for both CoMFA (Q(2) = 0.578; R(2)(pred) = 0.841) and CoMSIA (Q(2) = 0.610; R(2)(pred) = 0.824) methods. Molecular dynamics (MDs) simulations were performed for two ligand-receptor complexes and these simulations explored some crucial factors for higher activity. These findings of MD simulations were consistent with the interpretations obtained from other methods of analyses. The current study may help in designing new PDE10A inhibitors of this class.

Structure-based design, synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors.

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC50=1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC50=5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C]=4%, po, 5mg/kg, once-daily) and COLO205 (T/C=13%, po, 15 mg/kg, once-daily) mouse xenograft models.

Peramine and lolitrem B from endophyte-grass associations cascade up the food chain.

Endophytic fungi in cool-season grass species produce herbivore-toxic alkaloids, which are assumed to harm higher trophic levels along food chains. Previous studies have shown fitness disadvantages for higher trophic levels that feed on aphids that were exclusively reared on perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. However, it is unknown whether the alkaloids produced by the fungus-grass association can be assimilated by plant sap-sucking insects like aphids. Using an ultra high performance liquid chromatography method combined with mass spectrometry, we provide the first evidence that the alkaloids peramine and lolitrem B are present in aphids (Rhopalosiphum padi) and in aphid predators when the aphids are reared on endophyte-infected grass. We conclude that alkaloids can enter the plant sap of the grass and are responsible for longer pupal stages of the ladybird Harmonia axyridis and for fitness disadvantages of aphids and their predators as shown in previous studies.

[Transport of PLGA nanoparticles across Caco-2/HT29-MTX co-cultured cells].

The present study is to establish Caco-2/HT29-MTX co-cultured cells and investigate the transport capability of PLGA nanoparticles with different surface chemical properties across Caco-2/HT29-MTX co-cultured cells. PLGA-NPs, mPEG-PLGA-NPs and chitosan coated PLGA-NPs were prepared by nanoprecipitation method using poly(lactic-co-glycolic acid) as carrier material with surface modified by methoxy poly(ethylene glycol) and chitosan. The particle size and zeta potential of nanoparticles were measured by dynamic light scattering. Coumarin 6 was used as a fluorescent marker in the transport of nanoparticles investigated by confocal laser scanning microscopy. The transport of furanodiene (FDE) loaded nanoparticles was quantitively determined by high performance liquid chromatography. Colchicine and nocodazole were used in the transport study to explore the involved endocytosis mechanisms of nanoparticles. Distribution of the tight junction proteins ZO-1 was also analyzed by immunofluorescence staining. The results showed that the nanoparticles dispersed uniformly. The zeta potential of PLGA-NPs was negative, the mPEG-PLGA-NPs was close to neutral and the CS-PLGA-NPs was positive. The entrapment efficiency of FDE in all nanoparticles was higher than 75%. The transport capability of mPEG-PLGA-NPs across Caco-2/HT29-MTX co-cultured cells was higher than that of PLGA-NPs and CS-PLGA-NPs. Colchicine and nocodazole could significantly decrease the transport amount of nanoparticles. mPEG-PLGA-NPs could obviously reduce the distribution of ZO-1 protein than PLGA-NPs and CS-PLGA-NPs. The transport mechanism of PLGA-NPs and mPEG-PLGA-NPs were indicated to be a combination of endocytosis and paracellular way, while CS-PLGA-NPs mainly relied on the endocytosis way. PEG coating could shield the surface charge and enhance the hydrophilicity of PLGA nanoparticles, which leads mPEG-PLGA-NPs to possess higher anti-adhesion activity. As a result, mPEG-PLGA-NPs could penetrate the mucus layer rapidly and transport across Caco-2/HT29-MTX co-cultured cells.

[Fungi of the genus Penicillium as producers of physiologically active compounds (review)].

Fungi of the genus Penicillium isolated from little studied habitats are able to synthesize both previously known and new physiologically active compounds with diverse structures. They include secondary metabolites of alkaloid nature, i.e., ergot alkaloids, diketopiperazines, quinolines, quinazolines, benzodiazepines, and polyketides. We discuss the use of profiles of secondary metabolites for taxonomy purposes. Studying the physicochemical characteristics of producers of biologically active compounds showed that the biosynthesis of alkaloids is initiated on the first days of cultivation and proceeds simultaneously with growth. The cyclic character of alkaloid accumulation was recorded related to the processes of alkaloid biosynthesis, excretion from cells, degradation in culture fluid, and consumption by cells. Synchronic variations in the concentrations of intracellular tryptophan and alkaloids are necessary for the regulation of the optimal quantity of tryptophan necessary for the culture.