Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro

Includes bibliographical references.Cytochrome P450s are highly polymorphic enzymes responsible for the Phase I metabolism of over 80% of pharmaceutical drugs. Polymorphic variation can result in altered drug efficacy as well as adverse drug reactions so the lack of understanding of the effects of single amino acid substitutions on cytochrome P450 drug metabolism is a major problem for drug development. In order to begin to address this problem, this thesis describes an in silico analysis of over 300 nonsynonymous single nucleotide polymorphisms found across nine of the major human drug metabolising cytochrome P450 isoforms. Information from functional studies - in which regions of the cytochrome P450 structure important for substrate recognition, substrate and product access and egress and interaction with the cytochrome P450 reductase were delineated - was combined with in silico calculations on the effect of mutations on protein stability in order to establish the likely causes of altered drug metabolism observed for cytochrome P450 variants in functional assays carried out to date. This study revealed that 75% of all cytochrome P450 mutations showing altered activity in vitro are either predicted to be damaging to protein structure or are found within regions predicted to be important for catalytic activity. Furthermore, this study showed that 70% of the mutations that showed similar activity to the wild-type enzyme in in vitro studies lie outside of functional regions important for catalytic activity and are predicted to have no effect on protein stability. Based on these results, a cytochrome P450 polymorphic variant map was created that should find utility in predicting the functional effect of uncharacterised variants on drug metabolism. To further test the accuracy of the in silico predictions, in vitro assays were performed on a panel of CYP3A4 and CYP2C9 variants heterogeneously expressed in E.coli. All mutations predicted to alter protein function by stabilising or destabilising the apo-protein structure in silico were found to significantly alter the thermostability of the holo-protein in solution. Thermostability assays also suggest that other mutations may affect stability by disrupting haem binding, changing protein conformation or altering oligomer formation. The utility of a fluorescence-based functional P450 protein microarray platform, previously developed in our laboratory, for generating kinetic data for multiple CYP450 variants in parallel was also examined. Since the microarray platform in its current stage of development was found to be unsuitable for this purpose, kinetic data for the full panel of CYP3A4 and CYP2C9 variants was generated using solution phase assays, revealing several variants with altered catalytic turnover and/or binding affinity for fluorescent substrates

Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0

This book, "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity", presents five papers on human cytochrome P450 (CYP) and P450 reductase, three reviews on the role of CYPs in humans and their use as biomarkers, six papers on CYPs in microorganisms, and one study on CYP in insects. The first paper reports the in silico modeling of human CYP3A4 access channels. The second uses structural methods to explain the mechanism-based inactivation of CYP3A4 by mibefradil, 6,7-dihydroxy-bergamottin, and azamulin. The third article compares electron transfer in CYP2C9 and CYP2C19 using structural and biochemical methods, and the fourth uses kinetic methods to study electron transfer to CYP2C8 allelic mutants. The fifth article characterizes electron transfer between the reductase and CYP using in silico and in vitro methods, focusing on the conformations of the reductase. Then, two reviews describe clinical implications in cardiology and oncology and the role of fatty acid metabolism in cardiology and skin diseases. The second review is on the potential use of circulating extracellular vesicles as biomarkers. Five papers analyze the CYPomes of diverse microorganisms: the Bacillus genus, Mycobacteria, the fungi Tremellomycetes, Cyanobacteria, and Streptomyces. The sixth focuses on a specific Mycobacterium CYP, CYP128, and its importance in M. tuberculosis. The subject of the last paper is CYP in Sogatella furcifera, a plant pest, and its resistance to the insecticide sulfoxaflor

P450 biochips : development of a protein microarray platform for investigating cytochrome P450 clinical drug metabolism

Includes bibliographical references (leaves 224-243).This thesis describes the development of a novel cytochrome P450 array format, the P450 Biochip that allows quantitative and truly high-throughput measurement of cytochrome P450-mediated turnover reactions in sub-nanolitre volumes

Multiscale Simulations of Cytochrome P450 Systems

Cytochrome P450 enzymes (CYP, P450) play a crucial role in drug metabolism and sterol biosynthesis. CYP51 catalyzes the 14α-demethylation of sterol-like molecules. CYP51 has been shown to be a good target for antiparasitic drug design. Understanding the mechanism of P450s not only helps to invesigate drug metabolism but also to design parasite specific inhibitors that do not inhibit human orthologs. P450s are heme-containing enzymes. The active site is buried in the protein and thus, substrates need to enter the active site and products need to exit from the active site via ligand tunnels. P450s are membrane-bound proteins. They anchor in the membrane with a single transmembrane helix. For the catalytic reaction of P450s, two electrons need to be transferred from their redox partners. In eukaryotes, the most often used redox partner of P450s is cytochrome P450 reductase (CPR). CPR is also a membrane-bound protein. The association between P450 and CPR is driven by electrostatics. Current experimental evidence does not provide a full understanding of dynamics of P450s, differences between P450s, of how P450s embed in the membrane, of how ligands can enter and exit from the active site and of how P450s interact with CPR. A multiscale computational approach, including all-atom molecular dynamics simulations, coarse-grain molecular dynamics simulations and Brownian dynamics simulations was performed to tackle these problems. A model of membrane-bound T. brucei CYP51 was built and the model was shown to be consistent with existing experimental data. Simulations using models of CYP51 were compared with those of CYP2C9 and CYP2E1. The binding site residues of both T. brucei CYP51 and human CYP51 are more rigid than those of CYP2C9 and CYP2E1. Differences between the active site residues of T. brucei and human CYP51s may be key for designing T. brucei specific inhibitors. The ligand tunnels in both T. brucei and human CYP51 were also studied. Tunnel 2f serves as the predominant ligand tunnel in both proteins, but T. brucei CYP51 often uses the solvent (S) tunnel, tunnel 1 and the water (W) tunnel as the second predominant tunnel whereas human CYP51 uses tunnel 1. The difference in the use of ligand tunnels may be important for ligand specificity of the two proteins. Interactions of P450 and cytochrome P450 reductase were studied using Brownian dynamics simulations and molecular dynamics simulations. Complexes of soluble forms of different P450s, including CYP51, CYP2B4, CYP1A2, CYP2A6, CYP2C9, CYP2D6 and CYP2E1 were investigated. The P450s bind to the reductase using a similar interface, the positively charged proximal side. P450s bind to CPR with different affinities and these affinities can be inferred from the computed binding energy of the complexes. A model of the membrane-bound complex of T. brucei CYP51 and human CPR was built and simulated. This model was built using the membrane-bound model of T. brucei CYP51 and the encounter complexes of P450s and CPR generated by the Simulation of Diffusional Association (SDA 7) software, which was used for the Brownian dynamics simulation. SDA 7 is a useful software package for performing Brownian dynamics simulations of macromolecules. A webserver for SDA 7 (webSDA) was built to improve the user-friendliness of SDA 7 and automate the procedure for preparing and running SDA jobs. The webserver will not only help new users to become familiar with the SDA software but also experienced users to set up their simulations easily

Exhaustive computational search of ionic-charge clusters that mediate interactions between mammalian cytochrome P450 (CYP) and P450-oxidoreductase (POR) proteins

In this work, a model for the interaction between CYP2B4 and theFMNdomain of rat P450-oxidoreductase is built using as template the structure of a bacterial redox complex. Amino acid residues identified in the literature as cytochrome P450 (CYP)–redox partner interfacial residues map to the interface in our model. Our model supports the view that the bacterial template represents a specific electron transfer complex and moreover provides a structural framework for explaining previous experimental data. We have used our model in an exhaustive search for complementary pairs of mammalian CYP and P450-oxidoreductase (POR) charge clusters. We quantitatively show that among the previously defined basic clusters, the 433K–434R cluster is the most dominant (32.3% of interactions) and among the acidic clusters, the 207D–208D–209D cluster is the most dominant (29%). Our analysis also reveals the previously not described basic cluster 343R–345K (16.1% of interactions) and 373K (3.2%) and the acidic clusters 113D–115E–116E (25.8%), 92E–93E (12.9%), 101D (3.2%) and 179E (3.2%). Cluster pairings among the previously defined charge clusters include the pairing of cluster 421K–422R to cluster 207D–208D–209D. Moreover, 433K–434R and 207D–208D–209D, respectively the dominant positively and negatively charged clusters, are uncorrelated. Instead our analysis suggests that the newly identified cluster 113D–115E–116E is the main partner of the 433K–434R cluster while the newly described cluster 343R–345K is correlated to the cluster 207D–208D–209D

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds