Histamine genomics in silico: polymorphisms of the human genes involved in the synthesis,action and degradation of histamine

Background: Histamine is a ubiquitous biogenic amine involved in the regulation of numerous basic physiological and pathophysiological processes. The DNA sequences of the genes encoding proteins (enzymes and receptors) that participate in the synthesis, degradation and cellular binding of histamine are already identified. Objective: We analyzed the in silico available human sequences to find genetic polymorphisms in histamine-related genes (L-histidine decarboxylase, histamine receptors, histamine N-methyl transferase and diamine-oxidase), and compared these data with findings concerning structure-function relationships in order to get information about the possible pathophysiological relevance of these polymorphisms. Methods: Sequence analysis was performed at the National Center for Biotechnology Information Database. The search tool BLAST was applied. Results: Several sequence variations were found, and it is conceivable that some of these genetic polymorphisms may be related to various pathological conditions. Among sequence variations, variants with no amino acid change, variants resulting in amino acid alterations, and many nucleotide changes involving non-coding sequences were revealed. Conclusions: Histamine genomics may provide a new tool for medical prediction and drug design in the future.     

Structure–activity relationship study of growth inhibitory 2-styrylchromones against carcinoma cells

The structure–activity relationship study of 2-styrylchromones against carcinoma cell growth is discussed in the present report. Taking advantage of 2-styrylchromone as a molecular template, a series of structural modifications was carried out and examined on several carcinoma cell lines. Interestingly, AGS cells exhibited more sensitivity in response to methoxy-bearing compounds, of which compound 23 (3,4,5-trimethoxy group on ring B) showed the most potent activity with a GI₅₀ value of 1.3 μM. Surprisingly, as methoxy groups in 12 and 24–27 were demethylated to generate their hydroxyl counterparts 28–32, none of them displayed appreciable activity against all carcinoma cells. We further confirmed the pivotal role of rigidity for growth inhibitory activity between the rigid 12 and its flexible counterpart 33. Taken together, in the present report, we have clearly demonstrated the structure–activity relationship study of 2-styrylchromones targeting carcinoma cell growth.     

Orphan enzymes could be an unexplored reservoir of new drug targets

Despite the immense progress of genomics, and the current availability of several hundreds of thousands of amino acid sequences, >39% of well-defined enzyme activities (as represented by enzyme commission, EC, numbers) are not associated with any sequence. There is an urgent need to explore the 1525 orphan enzymes (enzymes having EC numbers without an associated sequence) to bridge the wide gap that separates knowledge of biochemical function and sequence information. Strikingly, orphan enzymes can even be found among enzymatic activities successfully used as drug targets. Here, knowledge of sequence would help to develop molecular-targeted therapies, suppressing many drug-related side-effects.     

Synthesis of new 1,3,4-benzotriazepin-5-one derivatives and their biological evaluation as antitumor agents

New derivatives of 1,3,4-benzotriazepin-5-one were designed and synthesized as structural analogues to the antitumor agents devazepide and asperlicin. An efficient and novel approach to the synthesis of 2-amino-1,3,4-benzotriazepin-5-one 2 was developed and its structure was confirmed. The newly synthesized derivatives were evaluated for their in vitro antitumor activity on 60 different cell lines. Compounds 8 and 9 displayed the most potent antitumor activity against several cell lines specifically ovarian cancer, renal cancer and prostate cancer, while compounds 5, 10 and 12 showed significant activities against UO-31 renal cancer cell line.     

A polybromodiphenyl ether from an Indonesian marine sponge Lamellodysidea herbacea and its chemical derivatives inhibit protein tyrosine phosphatase 1B, an important target for diabetes treatment

The ethanol extract of an Indonesian marine sponge Lamellodysidea herbacea inhibited the activity of protein tyrosine phosphatase 1B (PTP1B), an important target enzyme for the treatment of type II diabetes. Bioassay-guided isolation yielded a known polybromodiphenyl ether (1) as a sole bioactive component. The structure of 1 was confirmed by spectroscopic data for 1 and its methyl ether derivative (2). Compound 1 markedly inhibited the PTP1B activity (IC₅₀ = 0.85 μM) and showed a moderate cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma) cells. On the other hand, compound 2 maintained potent inhibitory activity against PTP1B (IC₅₀ = 1.7 μM) but did not show apparent cytotoxicity at 18 μM against these cancer cells. Four ester derivatives [acetyl (3), butyryl (4), hexanoyl (5), and benzoyl (6)] were prepared from 1 and their activities evaluated against PTP1B and two cancer cell lines to investigate the structure–activity relationships. Although compounds 3–6 exhibited potent inhibitory effects against PTP1B activity, cytotoxicity against HCT-15 and Jurkat cells was observed as a similar efficacy to that of 1. From these results, compound 2 was found to be the best inhibitor of PTP1B with no apparent cytotoxicity. Therefore, 2 may be a lead compound for making a new type of PTP1B inhibitor. Moreover, compound 2 did not inhibit the cell growth of Huh-7 cells (hepatoma). Hepatocytes are one of the locations of PTP1B, and Huh-7 cells are used to study the mechanism of action of compound 2.     

Searching for functional genetic variants in non-coding DNA

1. The search for DNA sequence variants for complex human polygenic conditions has been a strong focus of recent genetic research. While gene loci have been identified, few variants in the coding sequences of these genes have been found, suggesting that non-coding sequence variation may underlie many complex conditions. 2. Non-coding DNA harbours regulatory elements capable of making changes to gene expression. However, regulatory DNA sequences are currently difficult to recognize and their function is poorly understood, complicating the task of assigning potential functional significance to non-coding variation. 3. Comparative genomics, the study of evolutionary DNA conservation, has enabled the emergent field of non-coding DNA identification in human disease analysis. 4. This brief review will focus on the potential of a relatively high throughput technique based on comparative genomics, that may aid in the identification of functionally important non-coding sequence variation in complex diseases.     

Synthesis of 3,5-disubstituted isoxazolines as protein tyrosine phosphatase 1B inhibitors

The protein tyrosine phosphatases (PTPase) are a group of regulatory enzymes that are critically important to a wide variety of cellular functions. PTPase 1B has recently been implicated in the pathogenesis of diabetes, neuronal disease, and autoimmune diseases. A number of these PTPase that act as negative regulators of the insulin signaling cascade have been identified as novel targets for the therapeutic enhancement of insulin action in insulin-resistant disease states like type II diabetes. Therefore, in the present work we describes a study of the synthesis and structure–activity relationship (SAR) of chromene and 2,4-dimethoxy benzaldehyde-based isoxazolines, which are structurally related to potent PTPase inhibitors. Compounds 5–7 and 10–19 were synthesized via 1,3-dipolar cycloaddition reaction and evaluated against PTPase enzyme in vitro. Compounds 10, 14, and 19 displayed significant inhibitory activity with IC₅₀ values of 69, 88, and 62.7 μM, respectively. Active compounds 10, 11, 14–16, and 19 were also tested in the STZ-S in vivo assay model, and compounds 10, 14, and 19 were found to be active.     

Synthesis,molecular docking studies,and in vitro screening of barbiturates/thiobarbiturates as antibacterial and cholinesterase inhibitors

On the basis of observed biological activity of barbiturates/thiobarbiturates, a set of 13 hydrazinecarboxamide/hydrazinecarbothioamides derivatives were designed and synthesized in good to excellent yield with extensive structural characterization. These compounds were screened for antibacterial and cholinesterase inhibitory activities. Two of the compounds 1 and 2 showed moderate bactericidal activity. Compounds 10 and 4 were found to be the most active acetyl/butyryl cholinesterase inhibitor, respectively (AChEI; 10; IC₅₀ = 40.78 μM and BChEI; 4; IC₅₀ = 3.31 μM). In silico molecular docking studies were carried out to identify active interacting sites of drug and enzyme and to establish structure–activity relationships. When predicted cholinesterase binding energies were compared with the experimentally determined inhibitory concentrations (IC₅₀), most active compounds were also found to be the most favorable for binding. The binding scores of compounds 10 and 4 were ?10.2 and ?9.3 kcal/mol, respectively.     

Evaluation of insecticidal activity of camptothecin analogs against Brontispa longissima

Continuing our search for natural product-based compounds for the control of Brontispa longissima Larvae, 31 camptothecin analogs were first tested for their insecticidal activity against the fifth-instar larvae of B. longissima in vivo. Among them, compounds 6–8, 11, 26, 28, and 29 showed more promising and pronounced insecticidal activity than toosendanin, a commercial insecticide derived from Melia azedarach. The different insecticidal activity ranges of compounds 1–31 indicated that variation of chemical structures in the camptothecin skeleton markedly affected the activity profiles of this compound class, and some important SAR information has been revealed from it. The quantitative structure–activity relationship (QSAR) model was applied to provide insight into the camptothecin analogs’ structural properties that are responsible for their activities. The results obtained from SAR analysis showed good correlation with the QSAR studies, which allowed for the rational design of more potent camptothecin analogs in the development of potential new insecticides.     

Solvent-free, one-pot synthesis and biological evaluation of some new dipyrazolo [3,4-b:4′,3′-e]pyranylquinolones and their precursors

One-pot synthesis of 24 new compounds, belonging to three families; dipyrazolo[3,4-b:4′,3′-e]pyranylquinolones 7a–h and its precursors (pyrazolonylidene)methylquinolones 5a–h and 4,4′-[(quinolinyl)methylene]bispyrazols 6a–h, 8 from each, has been achieved in the presence of catalyst tetrabutylammonium hydrogen sulfate (TBA–HS) in solvent-free conditions. In addition to assuring chromatography-free product isolation, this method had also allowed the reaction to proceed in a regio-selective manner provided the temperature and amount of pyrazolone are varied. At 100 °C, while 1:1 mixture of aldehyde 3 and pyrazolone 4 underwent Knoevenagel condensation, same reactants taken in ratio of 1:2 mainly domino/Knoevenagel–Michael reaction. At 120 °C, however, the domino/Knoevenagel–Michael-adducts converted into cyclized product, highlighting a new domino/Knoevenagel–Michael-cyclization synthetic sequence. The structure of all heterocycles has been confirmed by mass, IR and NMR spectral data. Based on 2D NMR NOESY experiment, it was also confirmed that the formation of only ‘Z’ configuration of Knoevenagel alkene took place in the transformation. All are good antitubercular agents as they were found to be active against M. tuberculosis H37RV, in addition to being found active against three Gram-positive (Streptococcus pneumoniae, Clostridium tetani, Bacillus subtilis) and three Gram-negative (Salmonella typhi, Vibrio cholerae, Escherichia coli) bacteria, respectively.     

Anti-inflammatory and analgesic effects displayed by peptides derived from PKI55 protein, an endogenous protein kinase C inhibitor

We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1–16 (peptide 5), 1–8 (peptide 8) and 1–5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse. The peptides 5, 8 and 9 (0.1 and 10 nmol i.c.v.) were effective in both the parameters chosen to test the anti-inflammatory activity, i.e., the xylene-induced ear edema and the acetic acid-induced infiltration of neutrophils in the peritoneal cavity. In addition, they displayed analgesic effect, evaluated by the acetic acid-induced writhing test. All the peptides’ effects were shared by the reference compounds, dexamethasone and indomethacin (10 mg?kg^?1?i.p.), but not by the 9-scramble peptide (10 nmol i.c.v.). The peptide 9, which represents the shortest active sequence of the PKI55 protein, was tested in the ear edema model even following intraperitoneal (i.p.) administration and proved to be effective in the range doses 3–30 mg?kg^?1. Moreover, an increase in plasma corticosterone levels was detected in mice treated with the peptide 9, but not with the 9-scramble peptide (both at 10 nmol i.c.v.). The anti-inflammatory and analgesic effects of the PKI55-derived synthetic peptides, possibly related both to PKC inhibition and hypothalamic–pituitary–adrenal axis activation, deserve further investigation in view of potential therapeutic exploitation.     

Biotransformation of artemisinin derivatives by Glycyrrhiza glabra,Lavandula officinalis,and Panax quinquefolium

Biotransformation of α-artemether and dihydroartemisinin (DHA) by Glycyrrhiza glabra (Linn.), Lavandula officinalis (L.), and Panax quinquefolium was investigated. Two metabolites: tetrahydrofuran derivative (3) and a 13-carbon ring-rearranged product (4) were produced from α-artemether (1). DHA (2) provided metabolite 4. The structure of the metabolites were characterized by proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) imaging, fourier transform infrared spectroscopy, and mass spectroscopy. This is the first report that G. glabra and L. officinalis have the capability to biotransform α-artemether, and P. quinquefolium to biotransform DHA. Metabolite 3 is a new compound and metabolite 4 is reported here for the first time from artemisinin derivatives 1 and 2. The presence of acetate function in the derivative 3 and hydroxyl and C-12 deoxo groups in 4 obtained in our study make them interesting synthones for further modification into new clinically potent molecules.     

Synthesis and QSAR studies of some novel disubstituted 1,2,4-triazoles as antimicrobial agents

Disubstituted 1,2,4-triazoles 3a–k, 4a–k, and 6a–k have been synthesized from anthranilic acid and nicotinic acid, respectively, through a multi-step reaction sequence via their hydrazides. Synthesized compounds were evaluated for their in vitro antimicrobial activity against two gram-positive bacteria (S. aureus and B. subtilis), three gram-negative bacteria (E. coli, S. typhi, and K. pneumonia) as well as four fungi (A. niger, A. fumigatus, A. flavus, and C. albicans). To explore computational approach, structure–activity relationships were generated statistically using the synthesized compounds and their respective quantitative values of biological activities. These models can be used in future for predicting antimicrobial activity on similar class of compounds.     

Functional Screening of Drug Target Genes

Background and objectives: A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT. Methods: We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells. Results: Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (?pEC50 ± standard deviation) of 5.8 ± 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 ± 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants. Conclusion: The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.     

Anti-HIV-1 screening of (2E)-3-(2-chloro-6-methyl/methoxyquinolin-3-yl)-1-(aryl)prop-2-en-1-ones

Biological studies of two series of 38 quinolinyl chalcones (4a–s and 5a–s) were investigated for their in vitro anti-HIV-1 and cytotoxic activities. Out of 38 compounds, seventeen compounds were observed as good anti-HIV-1 agents with EC₅₀ values less than 20 μM. Compounds 4a, 4l, 4o, 5e, 5h, 5l, 5o, and 5r displayed potent anti-HIV-1 activity with EC₅₀ values less than 5 μM. Among these, compound 5h emerged as the best anti-HIV-1 agent (EC₅₀ = 1.1 μM). Compounds 4d, 4n, 4q, 4r, 4s, 5n, and 5r showed no toxicity in human lymphocytes. Bioassay results show that the type(s) and position(s) of the substituents seem to be critical for their cytotoxic and anti-HIV-1 activities. These results could be useful in the invention of new anti-HIV agents through structural modification.     

Esters of substituted benzoic acids as anti-thrombotic agents

Aliphatic esters of protocatechuic acid (PA,1), vanillic acid (VA,9) and gallic acid (GA,18) were prepared and their anti-thrombotic effects were evaluated in the mouse model of thrombosis. The aliphatic groups included methyl, ethyl,n-propyl,i-propyl,n-butyl,i-butyl,n-amyl and cyclohexyl.n-Amyl ester of PA (7), i-propyl and cyclohexyl esters of VA (13 and17 respectively) and ethyl ester of GA (20) treatment significantly lowered the death rate and increased the recovery from paralysis due to the thrombotic challenge. From the limited analogs available, it was tentatively concluded that the structural conformation, where carboxy oxygen (=O or-O^?) of the carboxyl group (COOH) at C₁ and the oxygen function at C₃ (either OH or OCH₃) are closely situated, is favorable for the esters of PA, VA and GA to be more antithrombotic.     

Synthesis and evaluation of anti-proliferative activity of 1,4-disubstituted phthalazines

A series of new phthalazine derivatives 3a–i and 4a–c were synthesized via the reaction of 1-chlorophthalazine derivative 2 with either N-substituted-piperazines, primary or their secondary amines. The structure of the synthesized, new compounds were characterized by spectral data. The anti-proliferative activity on human breast cancer cell line MCF-7 of the synthesized compounds was determined. The results showed that six of the test compounds (3a, 3g, 3i, and 4a–c) displayed potent cytotoxic activity ranging from 1.4 to 2.3?μmol.     

Anti-inflammatory activities of crocetin derivatives from processed Gardenia jasminoides

This study was designed to investigate changes of anti-oxidant and anti-nitric oxide (NO) production activities of Gardenia jasminoides (Gj) by roast processing, and anti-inflammatory activities of crocetin derivatives isolated from Gj. In order to evaluate anti-oxidant and anti-inflammatory activities, DPPH radical scavenging activities and inhibitory activities against lipopolysaccharide (LPS)-induced NO production were determined. Then we isolated crocin (1), gentiobiosyl glucosyl crocetin (3), and mono-gentiobiosyl crocetin (4) from the fruit of Gj, and crocetin (2) from the processed fruit of Gj (PGj) by column chromatography. Their structures were based on spectroscopic methods including IR, MS, and NMR (1D and 2D). Then we assayed contents of crocetin derivatives by HPLC analysis. These crocetin derivatives were evaluated the inhibitory activities on NO production in LPS-stimulated macrophage RAW 264.7 cells and expressions of protein and m-RNA of iNOS and COX-2 by western blot analysis and RT-PCR experiment. The DPPH radical scavenging activities were increased and NO productions in LPS-stimulated RAW 264.7 cells were decreased dose-dependently by processing. Crocin contents were decreased and crocetin contents were increased by processing in HPLC analysis. Compounds 1, 2, 3 and 4 reduced NO production in a dose-dependent manner with IC₅₀ values of 58.9 μM (1), 29.9 μM (2), 31.1 μM (3), and 37.6 μM (4) respectively. Crocetin (2) showed the most potent anti-inflammatory activity (IC₅₀ = 29.9 μM), and compound 3 and 4 were firstly measured for inhibitory activities on NO production. Their correlation between structure and activity was not clear but the activity of aglycone type showed the most potent activity. They also suppressed the protein and m-RNA expressions of iNOS and COX-2 in LPS-activated macrophage. These results suggest that anti-oxidant and anti-NO production activities of Gj were increased by processing, and increased anti-inflammatory activities of Gj by processing were due to the increase of crocetin, the aglycone that has greater activity than crocin.     

Design, synthesis, and anticonvulsant screening of some substituted piperazine and aniline derivatives of 5-phenyl-oxazolidin-2,4-diones and 5,5-diphenylimidazolidin-2,4 diones

Substituted piperazine and aniline derivatives of oxazolidin-2,4-diones and imidazolidin-2,4-diones were synthesized by N3 alkylation and screened for their anticonvulsant activity by the maximal electroshock (MES) test, and their neurotoxicity was evaluated by the rotarod test. Among all the synthesized derivatives, compounds 4b, 6c, 6d, 10b, 11a, 11b, and 11d were found to exhibit maximum seizure protection in MES test and were devoid of any neurotoxic effects. Furthermore, the functional activity of these compounds were evaluated in vivo for 5-HT_1A receptor affinity by using rectal body temperature and lower lip retraction in rats, while head twitch response in mice was performed for the determination of probable affinity toward 5-HT_2A receptor. The results of these tests demonstrated that compounds 4b, 6c, 6d, 10b, 11a, 11b, and 11d exhibited 5-HT_1A (pre- and postsynaptic) agonist/antagonist features whereas compounds 11a and 11b exhibited antagonist action for 5-HT_2A receptor. From the in vivo studies it was observed that a majority of aniline derivatives (6c, 6d, 11a, 11b, 11d) were found to be more active as compared to their bulky piperazine congeners (4b, 10b). Thus, the overall reduction in the bulkiness of the derivatives without compromising the lipophilicity is well appreciated for providing insights into the structural requirements necessary for development of new effective molecules having anticonvulsant effect.