Novel Natural Inhibitors of CYP1A2 Identified by in Silico and in Vitro Screening

Inhibition of cytochrome P450 (CYP) is a major cause of herb–drug interactions. The CYP1A2 enzyme plays a major role in the metabolism of drugs in humans. Its broad substrate specificity, as well as its inhibition by a vast array of structurally diverse herbal active ingredients, has indicated the possibility of metabolic herb–drug interactions. Therefore nowadays searching inhibitors for CYP1A2 from herbal medicines are drawing much more attention by biological, chemical and pharmological scientists. In our work, a pharmacophore model as well as the docking technology is proposed to screen inhibitors from herbal ingredients data. Firstly different pharmaphore models were constructed and then validated and modified by 202 herbal ingredients. Secondly the best pharmaphore model was chosen to virtually screen the herbal data (a curated database of 989 herbal compounds). Then the hits (147 herbal compounds) were continued to be filtered by a docking process, and were tested in vitro successively. Finally, five of eighteen candidate compounds (272, 284, 300, 616 and 817) were found to have inhibition of CYP1A2 activity. The model developed in our study is efficient for in silico screening of large herbal databases in the identification of CYP1A2 inhibitors. It will play an important role to prevent the risk of herb–drug interactions at an early stage of the drug development process

Exploration of Catalytic Properties of CYP2D6 and CYP3A4 Through Metabolic Studies of Levorphanol and Levallorphan □ S

ABSTRACT: CYP2D6 and CYP3A4, two members of the cytochrome P450 superfamily of monooxygenases, mediate the biotransformation of a variety of xenobiotics. The two enzymes differ in substrate specificity and size and characteristics of the active site cavity. The aim of this study was to determine whether the catalytic properties of these isoforms, reflected by the differences observed from crystal structures and homology models, could be confirmed with experimental data. Detailed metabolite identification, reversible inhibition, and time-dependent inhibition were examined for levorphanol and levallorphan with CYP2D6 and CYP3A4. The studies were designed to provide a comparison of the orientations of substrates, the catalytic sites of the two enzymes, and the subsequent outcomes on metabolism and inhibition. The metabolite identification revealed that CYP3A4 catalyzed the formation of a variety of metabolites as a result of presenting different parts of the substrates to the heme. CYP2D6 was a poorer catalyst that led to a more limited number of metabolites that were interpreted in terms to two orientations of the substrates. The inhibition studies showed evidence for strong reversible inhibition of CYP2D6 but not for CYP3A4. Levallorphan acted as a time-dependent inhibitor on CYP3A4, indicating a productive binding mode with this enzyme not observed with CYP2D6 that presumably resulted from close interactions of the N-allyl moiety oriented toward the heme. All the results are in agreement with the large and flexible active site of CYP3A4 and the more restricted active site of CYP2D6

Review of QSAR Models and Software Tools for predicting Biokinetic Properties

In the assessment of industrial chemicals, cosmetic ingredients, and active substances in pesticides and biocides, metabolites and degradates are rarely tested for their toxicologcal effects in mammals. In the interests of animal welfare and cost-effectiveness, alternatives to animal testing are needed in the evaluation of these types of chemicals. In this report we review the current status of various types of in silico estimation methods for Absorption, Distribution, Metabolism and Excretion (ADME) properties, which are often important in discriminating between the toxicological profiles of parent compounds and their metabolites/degradation products. The review was performed in a broad sense, with emphasis on QSARs and rule-based approaches and their applicability to estimation of oral bioavailability, human intestinal absorption, blood-brain barrier penetration, plasma protein binding, metabolism and. This revealed a vast and rapidly growing literature and a range of software tools. While it is difficult to give firm conclusions on the applicability of such tools, it is clear that many have been developed with pharmaceutical applications in mind, and as such may not be applicable to other types of chemicals (this would require further research investigation). On the other hand, a range of predictive methodologies have been explored and found promising, so there is merit in pursuing their applicability in the assessment of other types of chemicals and products. Many of the software tools are not transparent in terms of their predictive algorithms or underlying datasets. However, the literature identifies a set of commonly used descriptors that have been found useful in ADME prediction, so further research and model development activities could be based on such studies.JRC.DG.I.6-Systems toxicolog

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

Theoretical investigation of substrate specificity for cytochromes p450 IA2, p450 IID6 and p450 IIIA4

Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds

Allelic variation and multigenic metabolic activity of cytochrome P450s confer insecticide resistance in field populations of anopheles funestus s.s., a major malaria vector in Africa

Malaria control relies heavily on the use of insecticides, especially the pyrethroids, for control interventions such as Long Lasting Insecticide Nets (LLINs) and Indoor Residual Spraying (ITNs). However, widespread resistance to insecticides in major malaria vectors, such as An. funestus is threatening to derail these control tools. To design and implement suitable resistance management strategies which will ensure the continued effectiveness of these control tools it is necessary to elucidate the molecular basis of the resistance. In An. funestus, resistance is mainly metabolic with the duplicated P450s CYP6P9a and CYP6P9b implicated as the major pyrethroid resistance genes. Despite the detection of these key resistance genes the detailed molecular mechanisms through which they confer pyrethroid resistance remain uncharacterised. Because CYP6P9a and CYP6P9b were shown to exhibit significant allelic variation between resistant and susceptible mosquitoes, we hypothesised that this allelic variation is potentially a key mechanism conferring pyrethroid resistance. Here, I characterised the role of these genes in the resistance to pyrethroids and identified other candidate genes which confer cross-resistance to non-pyrethroid insecticides. The role of allelic variation in pyrethroid resistance was investigated using polymorphism survery and in silico prediction of activity. Metabolic activities and efficiencies of allelic variants of CYP6P9a and CYP6P9b were investigated using fluorescent probes, metabolism assays and transgenic expression in D. melanogaster system. Pyrethroid resistance causative mutations were detected using the site-directed mutagenesis. Other candidate P450s that confer cross-resistance to pyrethroids carbamates and organochlorines were identified and characterised. This study revealed that CYP6P9a and CYP6P9b from resistant populations of An. funestus are undergoing directional selection with reduced genetic diversity and beneficial mutations selected, compared to the alleles from susceptible strain (FANG), which exhibited high genetic variation. Modelling and docking simulations predicted the alleles of CYP6P9a and CYP6P9b from the resistant strains all across Africa to metabolise pyrethroids with high efficiency while the susceptible alleles FANGCYP6P9a and FANGCYP6P9b were predicted to have low activity toward pyrethroids. Validation of the docking predictions with probes and metabolism assays established that the resistant alleles of CYP6P9a and CYP6P9b possess high activities toward pyrethroids with kinetic profiles significantly different (high affinity and catalytic efficiency) from those obtained from the FANG, indicating that allelic variation is playing a major role in pyrethroid resistance. These findings were further strengthened by results from transgenic expression with GAL4/UAS technology showing that flies expressing the resistant alleles of both genes were significantly more resistant to pyrethroids than those expressing the susceptible alleles. Using mutagenesis, three key residues (Val109, Asp335 and Asn384) from the resistant allele of CYP6P9b were established as the important amino acid changes responsible for resistance with impact on substrate channelling, possible enhancement of interaction with redox partners and inter-molecular hydrogen bonding interactions, respectively conferring high metabolic efficiency. The finding of these resistance markers make it possible to design a diagnostic tool that can allow detection and tracking of the resistant alleles in the field population of An. funestus across Africa. Other up-regulated P450s in multiple resistant populations from southern Africa were also characterised, revealing that pyrethroid resistance is mediated by other P450s as well: CYP6M7, CYP6Z1, CYP9J11 and CYP6AA4 all of which metabolise pyrethroids. CYP6Z1 and CYP9J11 are cross-resistance genes which metabolise bendiocarb, while CYP6Z1 metabolise DDT in addition. In conclusion, allelic variation is a key mechanism conferring pyrethroid resistance in An. funestus s.s. from sub-Saharan Africa. Key amino acid changes control pyrethroid resistance factors and these molecular markers can be used to design DNA-based diagnostic tests which will allow tracking of the resistance alleles in the field. Pyrethroid resistance is multi-genic in the field populations of An. funestus with other P450s involved apart from CYP6P9a and CYP6P9b. The finding of cross-resistance P450s is of concern to resistance management and should be taken into account when designing resistance management strategies