Combined Docking and Quantum Chemical Study on CYP-mediated Metabolism of Estrogens in Man

Long-term exposure to estrogens seriously increases the incidence of various diseases including breast cancer. Experimental studies indicate that cytochrome P450 (CYP) enzymes catalyze the bioactivation of estrogens to catechols, which can exert their harmful effects via various routes. It has been shown that the 4-hydroxylation pathway of estrogens is the most malign, while 2-hydroxylation is considered a benign pathway. It is also known experimentally that with increasing unsaturation of ring B of estrogens the prevalence of the 4-hydroxylation pathway significantly increases. In this study, we used a combination of structural analysis, docking, and quantum chemical calculations at the B3LYP/6-311+G* level to investigate the factors that influence the regioselectivity of estrogen metabolism in man. We studied the structure of human estrogen metabolizing enzymes (CYP1A1, CYP1A2, CYP1B1, and CYP3A4) in complex with estrone using docking and investigated the susceptibility of estrone, equilin, and equilenin (which only differ in the unsaturation of ring B) to undergo 2- and 4-hydroxylation using several models of CYP enzymes (Compound I, methoxy, and phenoxy radical). We found that even the simplest models could account for the experimental difference between the 2- and 4- hydroxylation pathways and thus might be used for fast screening purposes. We also show that reactivity indices, specifically in this case the radical and nucleophilic condensed Fukui functions, also correctly predict the likeliness of estrogen derivatives to undergo 2- or 4-hydroxylation

Review of Ligand Specificity Factors for CYP1A Subfamily Enzymes from Molecular Modeling Studies Reported to-Date

The cytochrome P450 (CYP) family 1A enzymes, CYP1A1 and CYP1A2, are two of the most important enzymes implicated in the metabolism of endogenous and exogenous compounds through oxidation. These enzymes are also known to metabolize environmental procarcinogens into carcinogenic species, leading to the advent of several types of cancer. The development of selective inhibitors for these P450 enzymes, mitigating procarcinogenic oxidative effects, has been the focus of many studies in recent years. CYP1A1 is mainly found in extrahepatic tissues while CYP1A2 is the major CYP enzyme in human liver. Many molecules have been found to be metabolized by both of these enzymes, with varying rates and/or positions of oxidation. A complete understanding of the factors that govern the specificity and potency for the two CYP 1A enzymes is critical to the development of effective inhibitors. Computational molecular modeling tools have been used by several research groups to decipher the specificity and potency factors of the CYP1A1 and CYP1A2 substrates. In this review, we perform a thorough analysis of the computational studies that are ligand-based and protein-ligand complex-based to catalog the various factors that govern the specificity/potency toward these two enzymes

Review of Ligand Specificity Factors for CYP1A Subfamily Enzymes from Molecular Modeling Studies Reported to-Date

The cytochrome P450 (CYP) family 1A enzymes, CYP1A1 and CYP1A2, are two of the most important enzymes implicated in the metabolism of endogenous and exogenous compounds through oxidation. These enzymes are also known to metabolize environmental procarcinogens into carcinogenic species, leading to the advent of several types of cancer. The development of selective inhibitors for these P450 enzymes, mitigating procarcinogenic oxidative effects, has been the focus of many studies in recent years. CYP1A1 is mainly found in extrahepatic tissues while CYP1A2 is the major CYP enzyme in human liver. Many molecules have been found to be metabolized by both of these enzymes, with varying rates and/or positions of oxidation. A complete understanding of the factors that govern the specificity and potency for the two CYP 1A enzymes is critical to the development of effective inhibitors. Computational molecular modeling tools have been used by several research groups to decipher the specificity and potency factors of the CYP1A1 and CYP1A2 substrates. In this review, we perform a thorough analysis of the computational studies that are ligand-based and protein-ligand complex-based to catalog the various factors that govern the specificity/potency toward these two enzymes

Population Analysis and Protein Stability Assays Illustrate Xenobiotic Metabolizing Enzymes Have No Detectable Effect on Breast Cancer Development and Progression

Xenobiotic Metabolizing Pathway works to detoxify the cell from numerous carcinogenic, mutagenic, and toxic hydrophobic compounds. As a member of the phase I enzymes (the first phase in the Xenobiotic Metabolizing Pathway) the Cytochrome P-450 Family 1 Sub-family B Protein 1 (Cyp1B1) works to attach an oxygen molecule to its hydrophobic substrate. In performing this reaction, Cyp1B1 often increases the reactivity of the xenobiotic compound. If these reactive Cyp1B1 products migrate into the nucleus and they can cause damage by reacting with DNA. However, the Glutathione S-Transferase Theta 1 (GSTT1) and Glutathione S-Transferase Mu 1 (GSTM1), members of the phase II xenobiotic metabolizing pathway, are able to inactive these reactive Cyp1B1 products through the addition of a glutathione molecule. Previous studies have shown that four single nucleotide polymorphisms, which lead to amino acid substitutions, in the cyp1B1 gene and gene deletions in gstt1 and gstm1 genes lead to differences in cancer susceptibility. However, our analysis genotyped 473 European Americans and 177 African Americans at each locus and found no correlation between genotype and any of 13 tumor characteristics and breast cancer risk factors that impact breast cancer progression or development. Due to high substrate overlap between xenobiotic metabolizing enzymes, we hypothesize the cell can compensate for differences in protein levels and enzymatic rates can by increased expression of highly related enzymes. We have also investigated the role of four cyp1B1 polymorphisms on protein stability using endogenous Cyp1B1 variant proteins in human cell lines. We determined that an Asparagine to Serine amino acid substitution at amino acid position 453 decreases stability by 20% compared to our ancestral control. This result was modest when compared to previously published data, which used African Green Monkey cells with overexpressed Cyp1B1 proteins. Furthermore, we showed that Arginine to Glycine substitutions at amino acid position 48 in conjunction with Alanine to Serine substitution at amino acid position 119 increased stability by 50%. We hypothesize that these differences in protein stability have little effect on the production of carcinogenic compounds and thus cancer development and progression

Metabolism, transport, and physiologically based pharmacokinetic modelling of novel tacrine derivatives

Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly population, and its burden is rapidly growing both in Canada and worldwide. As a result, there is a substantial need for more effective treatments. The first drug that was approved for the management of AD symptoms was tacrine, a dual cholinesterase inhibitor. However, tacrine has since been discontinued after signs of hepatotoxicity were observed in a considerable proportion of patients. This toxicity has been linked to certain metabolites of tacrine formed by oxidation via the hepatic enzyme CYP1A2. Despite this issue, tacrine has remained a popular scaffold for the design of novel anti-Alzheimer’s agents. While tacrine is an example of the “one drug, one target” approach, a popular strategy involves functionalizing tacrine into a multi-targeted compound to target several pathways in the complex pathology of AD. In this regard, a library of tacrine derivatives was developed that exhibited both potent cholinesterase inhibition and the ability to inhibit the formation of the characteristic beta-amyloid plaques. Out of 25 starting compounds, nine compounds were examined further using in vitro and in silico techniques to investigate binding interactions with CYP1A2 and CYP3A4 (to assess potential for hepatotoxicity) and P-gp (to predict central nervous system permeability). Three of the remaining nine compounds displayed the desired properties and further experiments were conducted with these compounds to determine metabolic clearance. These results were incorporated into a physiologically based pharmacokinetic model that was used to predict the dose needed to reach target brain concentrations in a preclinical study