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

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dc.contributor.advisorBlackburn, Jonathanen_ZA
dc.contributor.authorArendse, Lauren Bethen_ZA
dc.date.accessioned2015-05-04T07:06:37Z
dc.date.available2015-05-04T07:06:37Z
dc.date.issued2014en_ZA
dc.descriptionIncludes bibliographical references.en_ZA
dc.description.abstractCytochrome 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.en_ZA
dc.identifier.apacitationArendse, L. B. (2014). <i>Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Department of Clinical Laboratory Sciences. Retrieved from http://hdl.handle.net/11427/12711en_ZA
dc.identifier.chicagocitationArendse, Lauren Beth. <i>"Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Department of Clinical Laboratory Sciences, 2014. http://hdl.handle.net/11427/12711en_ZA
dc.identifier.citationArendse, L. 2014. Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Arendse, Lauren Beth AB - 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. DA - 2014 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2014 T1 - Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro TI - Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro UR - http://hdl.handle.net/11427/12711 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/12711
dc.identifier.vancouvercitationArendse LB. Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Department of Clinical Laboratory Sciences, 2014 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/12711en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Clinical Laboratory Sciencesen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.titleExploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitroen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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