Structure and ligand based design for identification of highly potent molecules against 5-LOX.

We report here small molecules consisting of dichlorophenyl substituted oxindole that is further tagged with pyrrole/indole moieties. These molecules were designed on the basis of the analysis of binding mode of 5-LOX with arachidonic acid and zileuton. The molecules traverse the active site pocket of the enzyme that otherwise hosts AA and zileuton. Moreover, with a provision of derivatization at pyrrole/indole-N, the physico-chemical properties of the molecules can be adjusted. Appreciable 5-LOX inhibitory activities of the compounds in sub-micromolar range were observed and their aqueous solubility, binding with human serum albumin and stability in blood plasma and liver microsomes were checked. The Michaelis-Menten constants obtained during the binding of the compounds with 5-LOX indicated competitive binding of the compounds with the enzyme. Overall, the combination of molecular modelling and experimental studies identified promising molecules against inflammatory diseases.

Proline based rationally designed peptide esters against dipeptidyl peptidase-4: Highly potent anti-diabetic agents.

With the target to develop small molecules based anti-diabetic agents, we, herein, report the design, synthesis and biological studies on Lys-Pro and Gly-Pro esters, and a Phe-Pro-Phe tripeptide inhibiting the activity of glycoprotein dipeptidyl peptidase-4 (DPP-4). Since DPP-4 cleaves the glucagon like peptide (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) hormones which are responsible for inducing insulin secretion, the results of present studies could be significant in making control over glycemia. The structural analysis of DPP-4 and its binding mode with the substrate as well as the reported inhibitors provided the background for the design of new molecules. Among the 17 compounds screened against DPP-4, 14 compounds displayed IC50 better than the known drug Sitagliptin. Collectively, a highly encouraging set of molecules was identified that may prove as the clinical candidates for the treatment of diabetes.

Inflammation: Biochemistry, cellular targets, anti-inflammatory agents and challenges with special emphasis on cyclooxygenase-2.

Keeping in view the involvement of inflammation in the pathogenesis of several diseases including cancer, diabetes, neurodegenerative disorders and rheumatoid arthritis, herein, we review the processes for the initiation of inflammation and the treatment measures. While focusing on the cyclooxygenase mediated arachidonic acid metabolic pathways, biochemistry of inflammatory prostaglandins is discussed. The data corresponding to efficacy, pharmacokinetic profile and the side effects of the available natural and synthetic anti-inflammatory drugs is reviewed. Moreover, the given information for the drug-based design of new anti-inflammatory agents may help in the development of more potent and safe molecules.

Epoxides: Developability as active pharmaceutical ingredients and biochemical probes.

Epoxide functional group is a part of several natural as well as synthetic compounds. Irrespective of the role of epoxide group in drug efficacy and the use of epoxide compounds as tool molecules, uncertainty prevails over concerns associated with the reactivity of this functional group. Herein, we compile information about epoxide-based medicinal compounds and biochemical probes and look into the related advantages and challenges of the epoxide functional group. As a whole, this study is focussed on analyzing the strategies which have been adopted for the successful development of epoxide-based compounds within drug discovery programs.

Rationally designed TNF-α inhibitors: Identification of promising cytotoxic agents.

Design and synthesis of new indole derivatives as tumor growth inhibiting agents via inhibiting the TNF-α is described. The preliminary results showed the inhibition of LPS induced production of NO, TNF-α and IL-6 by these compounds out of which compounds 2d and 2g exhibited appreciable cytotoxicity against the 60 cell lines panel of human cancer. The rationale behind the design of the molecules and the results of their biological studies are presented. 2009 Elsevier Ltd. All rights reserved.

Modification of the lead molecule: Tryptophan and piperidine appended triazines reversing inflammation and hyeperalgesia in rats.

The structural optimization of the molecules making them to fit into the active site pocket of COX-2 occupying the same space as covered by the natural substrate arachidonic acid helped in the emergence of compound 10 as an efficacious anti-inflammatory agent. Selective for COX-2 over COX-1, compound 10 exhibited IC50 0.02 µM for COX-2 and reversed acetic acid induced inflammation in rats by 73% when used at 10 mg kg-1 dose and the same dose of the compound also rescued the animals from inflammatory phase of formalin induced hyperalgesia. As evidenced by the results of molecular modeling studies supported by the nuclear Overhauser enhancement data, the appropriate geometry of the molecule in the active site pocket of COX-2 contributing to its H-bond/hydrophobic interactions with Ser530, Trp387 and Tyr385 seems responsible for the enzyme inhibitory activity of the compound.

Anti-nociceptive and anti-inflammatory effect of imperatorin: evidences for involvement of COX-2, iNOS, NFκB and inflammatory cytokines.

Aim: The objective of the current investigation was to explore the analgesic effect of naturally occurring furanocoumarin, imperatorin and the involvement of inducible cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), NFκB and cytokines in the observed effect.Materials and methods: Anti-nociceptive effect was explored by inducing chemical hyperalgesia using acetic acid and formalin in mice. ED50 of imperatorin was calculated in acetic acid model. Modulation of cyclooxygenase and nitric oxide pathway by imperatorin was examined by stimulator/precursor challenge with substance P and L-arginine, respectively and quantification of COX-2, iNOS and NFκB expression by immunohistochemical analysis in spinal tissues. Involvement of inflammatory cytokines TNF-α and IL-1ß was investigated using LPS challenge and subsequent ELISA analysis of these inflammatory mediators in serum. Carrageenan inflicted paw edema was employed to explore the anti-inflammatory activity of imperatorin.Results: A significant reduction in the nociceptive behaviour was observed with imperatorin treatment in acetic acid and formalin test. ED50 of imperatorin was found to be 4.53 mg/kg. Pre-treatment with substance P and L-arginine significantly attenuated the anti-nociceptive activity of imperatorin in formalin test. Immunohistochemical findings revealed marked decrease in spinal COX-2, iNOS and NFκB expression. Imperatorin administration significantly reduced LPS induced rise in level of TNF-α and IL-1ß dose dependently. In carrageenan-induced paw edema test, maximum possible anti-inflammatory effect of imperatorin was evident after 240 min of carrageenan administration.Conclusion: Current investigation revealed that anti-nociceptive and anti-inflammatory potential of imperatorin is probably mediated through the attenuation of COX-2, iNOS, NFκB activity and reduction in circulatory cytokines.

Targeting tyrosine kinase: Development of acridone - pyrrole - oxindole hybrids against human breast cancer.

Based on the molecular modelling studies, a rational modification of the lead molecule was made to develop highly potent compounds showing anti-cancer activity against human breast cancer cell lines MCF 7, MDA-MB-468 and T-47D. The most potent compounds have Log P and total polar surface area 4.4-5.4 and 59.8 Å, respectively and they also exhibited promising ADME profile.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 µM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.

Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity.

Cytochrome c oxidase (CcOX) containing binuclear heme a3-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX - heme a3-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN- was 2.3 × 105 M-1 and its ED50 for restoring the cyanide bound CcOX activity in 10 min was 16 µM. The compound interacted with CN- over the pH range 5-10. The comparison of the loss of enzymatic activity in the presence of CN- and resumption of enzymatic activity by compound 2 mediated removal of CN- indicated the efficacy of the compound as antidote of cyanide.

Bergapten inhibits chemically induced nociceptive behavior and inflammation in mice by decreasing the expression of spinal PARP, iNOS, COX-2 and inflammatory cytokines.

In continuation with our previous studies on osthole, bergapten, a closely related furanocoumarin was investigated for its ameliorative effect on chemically induced neurogenic and inflammatory hyperalgesia and inflammation in mice. Chemical hyperalgesia and inflammation was induced by administration of formalin (intraplantar), acetic acid (intraperitoneal) and carrageenan (intraplantar) to different groups of animals. Pain responses were quantified and median effective dose (ED50) of bergapten was calculated. Lipopolysaccharide challenge was administered to study inflammatory cytokines which were analyzed in plasma using ELISA. The expression of poly ADP-ribose polymerase (PARP), cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) was quantified by immnofluorescence staining. Bergapten was found to ameliorate both neurogenic and inflammatory hyperalgesia precipitated by formalin, acetic acid induced writhing and carrageenan induced paw inflammation with ED50 dose of 2.96 mg/kg. Bergapten also significantly decreased the levels of TNF-α and IL-6 and the expression of PARP, COX-2 and iNOS in the spine. It is concluded that bergapten is an interesting molecule with significant analgesic and anti-inflammatory activity emanating through the modulation of multiple pain mediating pathways.

Osthole ameliorates neurogenic and inflammatory hyperalgesia by modulation of iNOS, COX-2, and inflammatory cytokines in mice.

BACKGROUND: Osthole is a bioactive component reported in medicinal plants such as Angelica pubescens and Cnidium monnieri, known for analgesic activity. However, the toxicity, median effective dose (ED50), and dual modulation of nitric oxide and cyclooxygenase pathways along with inflammatory cytokines of osthole are yet to be determined. METHODS: The animals (mice) were assessed for general behaviour and mortality in varying doses (50, 300, and 2000 mg kg-1) of osthole for acute toxicity over 14 days. The analgesic activity was investigated using acetic acid and formalin-induced hyperalgesia, and anti-inflammatory activity was explored in carrageenan-induced paw oedema. ED50 of osthole was calculated using Design Expert software. Involvement of nitric oxide and cyclooxygenase pathways was investigated by agonist challenges with L-arginine and substance P, respectively. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was determined in spinal sections by immunohistochemical analysis. Lipopolysaccharide (LPS) challenge was used to assess in vivo effect on inflammatory cytokines (TNFα and IL-6). RESULTS: Acute toxicity studies revealed no behavioural abnormality or mortality on osthole treatment and unremarkable histological findings. Osthole was found to significantly decrease acetic acid and formalin-induced hyperalgesia (ED50 = 5.43 mg kg-1) and carrageenan-induced paw oedema with no toxicity symptoms. Osthole produced a marked decrease in iNOS and COX-2 expression as well as TNFα and IL-6. The findings corroborate to modulation of iNOS and COX-2 and inflammatory cytokines by osthole. This study provides promising insights and prospects for application of osthole in pain management.

Rational modification of semaxanib and sunitinib for developing a tumor growth inhibitor targeting ATP binding site of tyrosine kinase.

Analysis of the crystal structure of tyrosine kinase in complexation with an ATP analogue, supplemented with the molecular docking studies of semaxanib and sunitinib in the ATP binding site of the enzyme enabled us to make design of a series of tyrosine kinase inhibitors. The combination of pyrrole and indolinone in one molecule and placement of appropriate substituent thereof made the molecule compatible for the hydrophobic sub-pocket of the enzyme. Screening of the compounds over 60 cell line panel of human tumor cell lines identified compound 3a that exhibited GI50 35 nM and 63 nM against MCF7 and MDA-MB-468 cell lines of breast cancer.

A bisubstrate reagent orchestrating adenosine triphosphate and l-tyrosine and making tyrosyl adenylate: partial mimicking of tyrosyl-tRNA synthetase.

We report the development of a bisubstrate reagent that, similar to tyrosyl t-RNA synthetase (TyrTS), provides a surface for ATP and l-Tyr to render a pseudo-intramolecular reaction forming 5'-tyrosyl adenylate (tyrAd). The presence of the reagent in solution with TyrTS marred the enzymatic reaction and, noticeably, tyrAd formed under the catalytic mode of the biomodel reagent was not picked up by TyrTS and hence was not transferred to tRNA. A potential application of this reagent, which doesn't allow the formation of tyrosyl tRNA, may lie in an emerging therapeutic targeting the translation machinery of cells without inhibiting the normal workings of enzymes.

Benzoflavones as cholesterol esterase inhibitors: Synthesis, biological evaluation and docking studies.

A library of forty 7,8-benzoflavone derivatives was synthesized and evaluated for their inhibitory potential against cholesterol esterase (CEase). Among all the synthesized compounds seven benzoflavone derivatives (A-7, A-8, A-10, A-11, A-12, A-13, A-15) exhibited significant inhibition against CEase in in vitro enzymatic assay. Compound A-12 showed the most promising activity with IC50 value of 0.78nM against cholesterol esterase. Enzyme kinetic studies carried out for A-12, revealed its mixed-type inhibition approach. Molecular protein-ligand docking studies were also performed to figure out the key binding interactions of A-12 with the amino acid residues of the enzyme's active site. The A-12 fits well at the catalytic site and is stabilized by hydrophobic interactions. It completely blocks the catalytic assembly of CEase and prevents it to participate in ester hydrolysis mechanism. The favorable binding conformation of A-12 suggests its prevailing role as CEase inhibitor.

Rational modification of the lead molecule: Enhancement in the anticancer and dihydrofolate reductase inhibitory activity.

By using molecular docking studies, the practice of fragment based drug discovery is conceptualized by introducing oxindole and iso-propanol moieties in our previous lead molecule 1. The resulting compound 2 exhibited competitive inhibition and favorable Ka and Ki for hDHFR. The screening of compound 2 at 60 cell line panel of human tumor cell lines showed its considerably better efficacy than compound 1 and hence put the candidature of 2 on stronghold for further studies.

Formation of amino acids from NH3 /NO2, CO2 and H2O: implications for the prebiotic origin of biomolecules.

RATIONALE: The search for the conditions which must have prevailed in the long-distant past during the conversion of inanimate matter into animate matter is a fascinating area of research and it continues to draw the attention of the scientific community. The initiation of life on this planet must have been preceded by the development of biomolecules, amongst which amino acids have unique importance. Formation of amino acids under a certain set of conditions is shown in the present experiments. METHODS: Solutions of ammonium carboxylates or the mixture of two such salts were prepared in 3:6.9:0.1 (v/v/v) acetonitrile/water/formic acid at a concentration of 50 µM. The studies were performed using a quadrupole time-of-flight (QqTOF) mass spectrometer. The formation of different amino acids was detected with high-resolution mass spectrometry. RESULTS: Here, we show the formation of amino acids when a solution of ammonium salts was injected into an electrospray ionization (ESI)-QqTOF-MS instrument. The ammonium salts were the source of NH3 and CO2 and H2 O was available in the medium. It seems that the combination of NH3 , CO2 , and H2 O leads to the formation of amino acids. CONCLUSIONS: Further to the literature reports of formation of amino acids under the reduced atmosphere represented by gases such as NH3 , CH4 , H2 and H2 O, here we demonstrate the formation of amino acids by the combination of NH3 /NO2 , CO2 and water vapours in the ESI source of the mass spectrometer.

Identification of amino acid appended acridines as potential leads to anti-cancer drugs.

In order to develop the amino acid appended acridines as potential leads for anticancer drugs, they were subjected to preliminary investigations. Screening through MTT assay as well as the phase contrast micrographs and Confocal images of immunostained C6 Glioma cells for markers such as α-tubulin, GFAP, mortalin and HSP-70 cells indicated that the compounds possess significant antiproliferative activity. The compounds also arrested cells in G0/G1 phase of the cell cycle as indicated by flow cytometry results. Moreover, the upregulation of the senescence markers such as mortalin and HSP70 in the presence of compounds 8, 9 and 12 indicate their senescence inducing potential.

Strategically designed biomodel: engineering C3-C4 cleavage of D-fructose.

Amongst a library of aldolase inspired, rationally designed compounds, the acridine derivative carrying a (S)-Tyr-Gly-(S)-Lys tripeptide selectively effected C3-C4 scissoring of D-fructose and produced D-glyceraldehyde and dihydroxyacetone.

Transformation of gas-phase amino acid clusters to dipeptides: a nice approach to demonstrate the formation of prebiotic peptides.

RATIONALE: Exploring prebiotic developments is a fascinating area of research which is continually drawing the attention of the scientific community. It is probable that first the biomolecules were formed and then they became aggregated to generate life. Formation of one such biomolecules (peptide ions) is shown in the present experiments. METHODS: All amino acid solutions for recording mass spectra were prepared in 3:6.9:0.1 (v/v/v) acetonitrile/water/formic acid at a concentration of 50 µM. The studies were performed using a Bruker MicroTOF QII mass spectrometer. Before carrying out experiments in the collision cell, atmospheric pressure in-source fragmentations were also performed. The formation of different chemical species was detected with high-resolution mass spectrometry. RESULTS: Here, we show experimentally the formation of amino acid cluster ions of varied populations, when a solution of an amino acid was injected into an electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometer. During in-source fragmentation/collision cell fragmentation, the non-covalent interaction between two identical amino acids forms either the [M2 + H](+) dimer cluster ion and/or the [M2 + K](+) adduct ion which, by elimination of one molecule of water, form the covalent linked dipeptide. CONCLUSIONS: After the formation of the amino acid cluster, it was established that the creation of the dipeptides, by a covalent bond resulting from the loss of a water molecule, was the initial step towards the formation of the primordial peptides.