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 IC 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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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 IC value of 5 nM. It displayed excellent selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 (IC >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 IC 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 ICs 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.