Insights into the Mutation-Induced HHH Syndrome from Modeling Human Mitochondrial Ornithine Transporter-1

Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109–113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective drugs against the disease

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

Analysis of ginger root essential oil and hydrosol: CYP450 inhibition

Former researches showed ginger extracts could contribute to hepatic protection. Two species of cytochrome P450 enzymes, CYP2E1 and CYP2A6, are both important heme-containing liver enzymes for hepatictoxicology via metabolizing small organic molecules into toxic metabolites. CYP2E1 metabolizes more than 2% of all the oral drugs and is closely associated with liver toxicity. CYP2A6 metabolizes fewer than 5% of all the oral drugs and activates some tobacco procarcinogens. Therefore, inhibitors from natural extracts might be able to provide with preventative therapy to liver toxicity and cancer.CYP2E1 inhibitors may be used to inhibit liver toxicity of CYP2E1 in metabolizing the pain killer acetaminophen into toxic N-acetyl-p-quinoimine (NAPQI) to prevent liver cell necrosis. In addition, CYP2A6 inhibitors may be used to suppress the metabolism of certain pro-carcinogensthat are inhaled from smoking. In this research, two extracts from steam distillation of ginger root, ginger essential oil and ginger hydrosol, were collected. CYP2E1 and CYP2A6 were assayed with presence of the two extracts at a series of concentrations to test the ginger extracts’ dose-dependent effects. The results showed both of the two extractsas CYP2E1 inhibitors but as poor inhibitors to CYP2A6. The major organic contents of ginger essential oil and ginger hydrosol were identified by GC-MS. The results showed both of them contained citral. The major organic components of ginger hydrosol were determined as citral (neral and geranial), 2-heptanol and eucalyptol. The Michaelis-Menten kinetic analysis showed that ginger essential oil, citral, and ginger hydrosol were all competitive inhibitors to CYP2E1 from human liver S9 fraction. The KI for Brazilian ginger essential oil was 27.3mg/L and the KI for Costa Rican ginger essential oil was 29.9 mg/L.The inhibitory constant of citral was KI of 43.0µM. Meanwhile, the KI for Brazilian ginger hydrosol was 4.6% of its original concentration, which corresponded to 45.5 µM citral (neral: geranial=1:1.4).The KI for the Brazilian ginger hydrosol was 3.6% of its original concentration, which corresponded to 44.3µM citral (neral: geranial=1:1.4). Citral was implicated to be the most potent inhibitor from ginger hydrosol to human liver CYP2E1.Ginger hydrosol and citral were shown to be inhibitors to purified human liver CYP2E1 in the metabolism of acetaminophen to NAPQI by analysis with LC-MS. In the presence of the substrate acetaminophen at 800µM, the concentration close to its toxic concentration, Brazilian ginger hydrosol exhibited a 50% inhibition of enzyme activity at the concentration over 25%. This concentration of Brazilian ginger hydrosol corresponds to a dose of citral of around 158 µM. This result implicated that ginger hydrosol and citral could be used as a natural therapeutic for toxicity of acetaminophen

Effects of açaí berry (Euterpe oleracea) extracts on human antioxidant systems and drug metabolism

The source and sink of reactive oxygen species are diverse and so are the control mechanisms to counteract them. Sources may be exogenous or endogenous from normal metabolic pathways. Similarly, the sink could be a simple radical scavenging event by small molecule antioxidants and antioxidant enzymes or complex events involving cellular signaling processes. The effects of reactive oxygen species may precipitate cellular dysfunction from its toxicity, protect from invading microorganisms or perform essential functions through regulation of cell signaling pathways. Excessive Reactive oxygen species leads to oxidative stress which mediates cellular damage and is implicated in several pathological conditions. The current research addresses biological systems that may effect redox balance in humans. One goal of the current research was to establish structure activity relationship for substrate binding to several human drug metabolizing enzymes implicated in reactive oxygen species generation, namely CYP2A6 and CYP2E1. The substrate dynamics of these two enzymes were studied by probing the active site with a series of small chain saturated and 2,3-unsaturated aldehydes using human liver microsomes. The study demonstrated that the aldehydes inhibited both the enzymes in competitive manner with unsaturated aldehydes being more potent than their saturated counterparts. The potential for p-stacking interactions between the phenylalanine rich active site of these enzymes and the double bond at 2-position in unsaturated aldehydes conferred high affinity for these aldehydes. It also confirmed an earlier findings that the active site of CYP2A6 is rigid where as CYP2E1 is flexible due to the presence of extended p-system allowing the expansion of its active site. Another goal was to examine the possible interactions between human cytochrome P450 enzymes of pharmacological and toxicological importance with a natural product called açaí. Açaí (Euterpe oleracea) is a Brazilian palm tree that has emerged from traditional medicinal plant to a recent super-fruit status. The assumption that it is safe to consume açaí currently lacks evidence from its interactions with drug metabolizing cytochrome P450 enzymes. The interaction between the crude extracts of açaí and major cytochrome P450 enzymes involved in drug metabolism and toxicology demonstrated the potential for chloroform extract to inhibit the isoforms CYP1A1, CYP2B6 and CYP2C8. The Michaelis-Menten Kinetics studies indicated mixed mode of inhibition of these enzymes by crude chloroform extract of açaí with low KI and KI’ values for CYP2C8 followed by CYP2B6 and CYP1A1. In addition, the study was extended to identification of inhibitors for toxicologically important CYP2A6 and CYP2E1 using a collaborative bioassay-guided fractionation approach. Although the crude chloroform extract of açaí showed considerable inhibition of these enzymes, specific inhibitors were not identified. Finally, the regulation of a signal transduction pathway namely Nrf2/ARE signaling pathway by açaí constituents was also studied as a potential strategy to prevent oxidative damage. The nuclear factor erythoid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense to counteract oxidative stress. Activation of this pathway increases the expression of a battery of antioxidant genes. This was achieved by monitoring the activation of a cis¬-acting DNA sequence referred to as Antioxidant Response Element (ARE) contained in a luciferase-containing promoter vector in cultured HepG2 cells. A high-throughput analysis of fractions generated using bioassay-guided fractionation of açaí has resulted in the identification of a class of compounds known as Pheophorbides as the inducers of ARE-luciferase. Dose response analysis using pure compounds demonstrated significant induction of ARE-luciferase at concentrations as low as 8.2 µM and 16.9µM for Pheophorbide a methyl ester and Pheophorbide a, respectively

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

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Computational modelling of supramolecular human and animal structures: applications to enzymes relevant in comparative physiological studies

Currently, there is a need for improved drugs to treat diseases such as diabetes and cancer. This study used different computational methods to investigate a range of scientific problems that require rapid solutions to improve health and wellbeing. Ligand docking, in silico ADMET, protein-ligand binding affinity, density functional theory (DFT), and analytical techniques were applied to study the allosteric binding pocket of the GLP-1 receptor, the interactions between flavonoids and cytochrome P450s, the binding of phorbol diesters to the CYP19A1 enzyme and nuclear magnetic resonance (NMR) shielding. Findings from the study on ligand binding to the glucagon-like peptide-1 receptor GLP1-R) showed that the allosteric binding pocket of the GLP-1R is located near the transmembrane (TM) domain 6 of the receptor. This finding would enable the development of new allosteric modulators which can target the allosteric binding pocket of GLP-1R identified from this study. The next study which explored the binding of different ligands into cytochrome P450s showed that specific amino acid residues in the cytochrome P450s (CYP1A1 and CYP1B1) interact with different EROD flavonoids (Asp 313 and Phe 224 upon docking into CYP1A1 and Ala 330 upon docking into CYP1B1). The flavonoids isorhamnetin and pedalitin had the lowest binding energy upon docking into the crystal structures of 6DWM and 6IQ5. The results suggest the flavonoids isorhamnetin and pedalitin as potential precursors for natural productderived therapeutics. Additionally, the study also explored the interactions between phorbol diesters and CYP19A1. The results showed that the phorbol diesters had higher binding energy than commercial aromatase inhibitors. This suggests that phorbol diesters have the potential to modify the activity of the aromatase enzyme. A study investigating the lack of rotational invariance of some density functional theory (DFT) grids on DFT computed NMR spectra of a low vibrational frequency reaction showed that the lack of rotational invariance in some DFT grids does not impact DFT-calculated NMR spectra of low vibrational frequency reactions

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

A BIOPHYSICAL INVESTIGATION OF STABILITY, LIGAND BINDING, AND IRON STATE OF CYP102A1

Cytochrome P450s (CYPs) are cysteine ligated Fe-heme monooxygenases that are found in all domains of life. In mammals, they have a role in xenobiotic metabolism and steroid synthesis, making them a fundamental requirement for survival. In addition, their ability to perform a variety of chemical reactions on an array of substrates makes CYPs highly sought for biotechnical applications such as wastewater remediation, production of potential drug candidates, and creation of drug metabolites. By mutating specific amino acids, these enzymes can be engineered to change their substrate binding profiles and achieve stereo- and regio-specific chemistry. While these mutations are essential to change CYP activity, the major drawback to using them on an industrial scale is a decrease in stability of the enzyme. This work elaborated how CYP stability is effected by mutations, binding of native and non-native substrates, and changes in iron oxidation state. Cytochrome P450BM3 (BM3, or CYP102A1), a bacterial enzyme, was used as a model system. In contrast to membrane associated human CYPs, BM3 is soluble and has efficient turnover due to the fusion of the reductase partner the heme domain. BM3 is naturally selective, but mutations can be incorporated to make it promiscuous, similar to CYPs responsible for xenobiotic breakdown. This allowed for the comparison of a selective vs. a promiscuous CYP while conserving the greatest possible sequence identity. An approach was used combining experimental solution phase data, x-ray crystallography, and molecular dynamic simulations. The results showed that mutations resulted in an cumulative decrease in stability as promiscuity increased. This reduction in stability was due to a decrease in the number of salt bridges and disruption of hydrophobic contacts. Regions of P450BM3 were found that could be targeted through mutation to increase the stability of a highly promiscuous and active variant known as the pentuple mutant (PM). Further investigations demonstrated the impact of native and non-native substrate binding. The Gibbs free energy of binding (ΔGb°) was determined for a small library of molecules and was rationalized computationally, concluding that attractive dispersion forces negated the impact of electrostatic and repulsive forces. In addition, the impact of the iron-heme charge state on CYP stability was examined as a function of promiscuity. In general, there was an association between promiscuity and similarities in the stability of the Fe(III) and Fe(II) states. This is consistent with a model where the promiscuous variants of the enzyme are in a more “reduction-ready” state, and can undergo catalysis with greater ease than the wild type enzyme. These findings have implications for the role of CYPs in human health and for biotechnical applications