Browsing by Author "Joshi, Prashant"

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    Biotransformation of chrysin to baicalein: Selective C6- hydroxylation of 5,7-dihydroxyflavone using whole yeast cells stably expressing human CYP1A1 enzyme
    (ACS Publications, 2017-08-07) Williams, Ibidapo Steven; Chib, Shifali; Nuthakki, V.; Gatchie, Linda; Joshi, Prashant; Narkhede, N.; Vishwakarma, R. A.; Bharate, Sandip B.; Saran, S.; Chaudhuri, Bhabatosh
    Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multi-step chemical approaches. Here we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme, that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7- trihydroxyflavone (baicalein). Molecular modelling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell-based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds
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    Biphenyl urea derivatives as selective CYP1B1 inhibitors
    (The Royal Society of Chemistry, 2016-09-04) Siddique, Mohd Usman Mohd; Sonawane, Vinay; Horley, Neill; Williams, Ibidapo Steven; Joshi, Prashant; Bharate, Sandip B.; Jayaprakash,Venkatesan; Sinha, Barij N.; Chaudhuri, Bhabatosh; McCann, Glen J. P.
    Highly selective CYP1B1 inhibitors have potential in the treatment of hormone-induced breast and prostate cancers. Mimicry of potent and selective CYP1B1 inhibitors, α-naphthoflavone and stilbenes, revealed that two sets of hydrophobic clusters suitably linked via a polar linker could be implanted into a new scaffold ‘biphenyl ureas’ to create potentially a new class of CYP1B1 inhibitors. A series of sixteen biphenyl ureas were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™, yeast-derived recombinant microsomal enzymes. The most active human CYP1B1 inhibitors were further studied for their selectivity against human CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. The meta-chloro-substituted biphenyl urea 5h was the most potent inhibitor of CYP1B1 with IC50 value of 5 nM. It displayed excellent selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 (IC50 >10 μM in the four CYP assays, indicating >2000-fold selectivity). Similarly, two methoxy-substituted biphenyl ureas 5d and 5e also displayed potent and selective inhibition of CYP1B1 with IC50 values of 69 and 58 nM, respectively, showing >62 and >98-fold selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. In order to probe if the relatively insoluble biphenyl ureas were cell permeable and if they could at all be used for future cellular studies, their CYP1B1 inhibition was investigated in live recombinant human and yeast cells. Compound 5d displayed the most potent inhibition with IC50s of 20 nM and 235 nM, respectively, in the two cell-based assays. The most potent and selective CYP1B1 inhibitor (compound 5h) from Sacchrosomes, also displayed potent inhibition in live cell assays. Molecular modeling was performed to understand the trends in potency and selectivity observed in the panel of five CYP isoenzymes used for the in vitro studies.
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    Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines
    (Elsevier, 2017-02-09) Horley, Neill; Beresford, Kenneth J. M.; Chawla, Tarun; McCann, Glen J. P.; Ruparelia, K. C.; Gatchie, Linda; Sonawane, Vinay; Williams, Vinay R.; Tan, Hoon Leong; Joshi, Prashant; Bharate, Sonali S.; Kumar, Vikas; Bharate, Sandip B.; Chaudhuri, Bhabatosh
    The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC50 values of 10 and 9 nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within ‘live’ recombinant yeast and human HEK293 kidney cells with IC50 values of 63, 65, and 4, 4 nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatineresistance in HEK293 and A2780 cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.
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    (E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocyclic chalconeis a potent and selectiveCYP1A1 inhibitor and cancerchemopreventative agent
    (Elsevier, 2017-11-06) Horley, Neill; Beresford, Kenneth J. M.; Kaduskar, S.; Joshi, Prashant; McCann, Glen J. P.; Ruparelia, K. C.; Williams, Ibidapo Steven; Gatchie, Linda; Sonawane, Vinay; Bharate, Sandip B.; Chaudhuri, Bhabatosh
    The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in SacchrosomesTM and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on nonheterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC50 values of 58 and 65 nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10 fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 subfamily and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.
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    Identification of karanjin isolated from the Indian beech tree as a potent CYP1 enzyme inhibitor with cellular efficacy via screening of a natural product repository
    (Medicinal Chemistry Communications, 2018-02-01) Joshi, Prashant; Sonawane, Vinay; Williams, Ibidapo Steven; McCann, Glen J. P.; Gatchie, Linda; Sharma, Rajni; Satti, Naresh; Chaudhuri, Bhabatosh; Bharate, Sandip B.
    CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 μM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40–97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC50 values of the ‘hits’, belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the ‘hits’ were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC50, 20 nM) and karanjin (IN-195; IC50, 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.
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    Identification of Potent and Selective CYP1A1 Inhibitors via Combined Ligand and Structure-Based Virtual Screening and Their in Vitro Validation in Sacchrosomes and Live Human Cells.
    (ACS Publications, 2017-05-10) Joshi, Prashant; McCann, Glen J. P.; Sonawane, V. R.; Vishwakarma, R. A.; Chaudhuri, B.; Bharate, S. B.
    Target structure-guided virtual screening (VS) is a versatile, powerful, and inexpensive alternative to experimental high-throughput screening (HTS). To discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercial library of 50 000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structure-based VS. These 300 compounds were screened, at 10 μM concentration, for in vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-de-ethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 μM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ∼95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity toward inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e., there was no detectable inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 at 10 μM). It was further investigated in live CYP-expressing human cell system, which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC50 values of 269, 30, and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 μM. The compound 119 rescued CYP1A1 overexpressing HEK293 cells from CYP1A1 mediated benzo[a]pyrene (B[a]P) toxicity and also overcame cisplatin resistance in CYP1B1 overexpressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully be used to identify promising CYP1A1 inhibitors, which may have potential in the development of novel cancer chemo-preventive agents.
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    Quinazoline derivatives as selective CYP1B1 inhibitors
    (Elsevier, 2017-02-16) Siddique, Mohd Usman Mohd; McCann, Glen J. P.; Sonawane, Vinay; Horley, Neill; Gatchie, Linda; Joshi, Prashant; Bharate, Sandip B.; Jayaprakash,Venkatesan; Sinha, Barij N.; Chaudhuri, B.
    CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid 'O' atom with 'N' atom led to the prediction that a 'quinazoline' scaffold could be the basis for designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™. IC50 determinations of six compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best candidates for CYP1B1 inhibition, with IC50 values in the nM range. Further selectivity studies with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed that the compounds are likely to be free from critical drug-drug interaction liability. Molecular modelling studies were performed to rationalize the observed enzymatic inhibitions. Further biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have illustrated the most potent compounds' cellular permeability and capability of potently inhibiting CYP1B1 enzyme expressed within live cells.
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    Synthesis and biological evaluation of pyrrole-based chalcones as CYP1 enzyme inhibitors, for possible prevention of cancer and overcoming cisplatin resistance
    (Elsevier, 2017-07-04) Williams, Ibidapo Steven; Joshi, Prashant; Gatchie, Linda; Sharma, M.; Satti, N. K.; Vishwakarma, R. A.; Chaudhuri, Bhabatosh; Bharate, Sandip B.
    Inhibitors of CYP1 enzymes may play vital roles in the prevention of cancer and overcoming chemo-resistance to anticancer drugs. In this letter, we report synthesis of twenty-three pyrrole based heterocyclic chalcones which were screened for inhibition of CYP1 isoforms. Compound 3n potently inhibited CYP1B1 with an IC50 of 0.2 lM in SacchrosomesTM and CYP1B1-expressing live human cells. However, compound 3j which inhibited both CYP1A1 and CYP1B1 with an IC50 of 0.9 mM, using the same systems, also potently antagonized B[a]P-mediated induction of AhR signaling in yeast (IC50, 1.5 mM), fully protected human cells from B[a]P toxicity and completely reversed cisplatin resistance in human cells that overexpress CYP1B1 by restoring cisplatin’s cytotoxicity. Molecular modeling studies were performed to rationalize the observed potency and selectivity of enzyme inhibition by compounds 3j and 3n.