In Vitro and in Silico Methods to Predict Cytochrome P450 Enzyme Inhibition

Cytochrome P450 (CYP) enzymes are important enzymes involved in drug metabolism. The CYP enzymes are a family of heme proteins involved in the metabolism of numerous toxic and pharmacologically active compounds and can cause drug-drug-interactions with co-administered drugs as well as unwanted adverse side effects. Several in vitro methods have been developed and are in widespread use to evaluate main kinetic properties (absorption, distribution, metabolism, excretion) in drug development projects. These studies help in identifying molecules that have favourable kinetic properties well before clinical studies in humans. The most modern technological breakthrough is the use of computational (in silico) methods to predict kinetic properties of lead molecules. In the present study, the overall goal was to develop novel in vitro and in silico methods to assess the metabolic features of xenobiotics. The metabolic focus of this thesis work is inhibition of CYP enzymes. The four aims of the present study were (1) to compare three in vitro inhibition screening tests for CYP enzymes (2) to create new inhibition structureactivity databases for human CYP enzymes (CYP1A2, CYP2B6, and CYP2E1) (3) to construct quantitative structure-activity relationship (QSAR) models of inhibitors of these enzymes and (4) to identify more potent and selective inhibitors for these enzymes. Three assay types, the traditional single substrate assays, the fluorescent probe method with recombinant human CYPs, and a novel n-in-one technique, yielded similar results with the majority of the CYP forms tested. For CYP1A2, CYP2B6 and CYP2E1, new inhibition structure-activity databases were created, which were large and composed of structurally diverse compounds. The created quantitative structure-activity relationship (QSAR) models revealed the key molecular characteristics of inhibitors of the CYP1A2, CYP2B6 and CYP2E1 and were also able to accurately predict inhibitory potencies of the test set of compounds. For CYP2B6, three novel potent inhibitors were discovered, 4-(4chlorobenzylpyridine) (CBP), 4-benzylpyridine (BP) and 4-(4-nitrobenzylpyridine) (NBP). In particular CBP is a suitable inhibitor for in vitro screening studies. In vitro highthroughput screening and in silico studies can provide early prediction of the possible involvement of CYP enzymes in the metabolism of drugs or drug candidates. Therefore these studies could potentially make drug design more effective, less costly and improve drug safety and replace some of the in vivo experiments. National Library of Medical Classification: WH 190, QU 143, QU 120, QV 38 Medical Subject Headings: Cytochrome P450 Enzyme System; Drug Interactions; Enzyme Inhibitors; Xenobiotics/metabolism; Quantitative Structure-Activity Relationship; High-Throughput Screening Assays