FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 39KINETIC AND MOLECULAR DYNAMICS CORRELATIONS IN CYTOCHROME P450(0023)TYPE:Research Subproject%IDeA $: 5.000% IDeA $: 172,385INVESTIGATOR, DEGREEAguilar, Jarrett PHDGannett, Peter PHDDEPARTMENTNatural Science &MathematicsBasic Pharmaceut SciNON-HOST INSTITUTION: STATE,COUNTRYWest Liberty University, Wv UsaWest Virginia University, Wv UsaTotal # human subjects expected for entire study: 0Total # human subjects enrolled to date: 0SUBPROJECT DESCRIPTIONThe cytochrome P450 enzymes mediate the metabolism of various xenobiotic and endogenous compoundsand can bioactivate pro-carcinogens such as benzo-[a]-pyrene. Many drug-drug interactions are caused bythe effect of one of the drugs on the activity of the P450 isoforms involved in the metabolism of the seconddrug. Some isoforms demonstrate atypical kinetics for the metabolism of certain substrates. We and othershave suggested that simultaneous binding of two substrates in the active site (a two-site model) isresponsible for most atypical kinetic profiles. Dapsone and structurally related substrates, have been shownto activate CYP2C9 metabolism of flurbiprofen, naproxen, and piroxicam. The kinetic data suggest bothsubstrate and activator are present in the active site. CYP2C9 polymorphisms result in reduced enzymecatalytic activity and greater activation by effector molecules as compared to wild-type protein, with themechanism(s) for these changes in activity not fully elucidated. The molecular dynamics for severalmutations involving key sites for possible catalytic activity will be studied by molecular dynamics and theresulting kinetics data will be compared to determine if these key sites are responsible for the changes inactivity between various mutants of CYP 2C9. The mutations will be prepared via site directed mutagenesis,and the enzyme expressed and purified for kinetic analysis. Kinetic analysis will be conducted using anHPLC with a fluorescence detector. The data obtained will be compared to that obtained from the moleculardynamics data for same mutations. The distance between the active H of the substrate and the heme ironappears to be important to substrate metabolism. The kinetics for these mutations will be compared tothese distance changes and correlations between the two studied. The mutants are R108I and N204I asthey are responsible for substrate binding within the active site. Other mutants E300I, S209A, T304A, andN474I will be studied as they may determine binding orientation of the effector. This will provide informationthat would allow one to control the kinetics by proper choice of substrate and effector and provide a greaterunderstanding of the active site mechanism of cytochrome P450 2C9.SUBPROJECT PROGRESSWe first had an issue with our site-directed mutagenesis and our plasmid became smaller than thatof our wild type protein. We attempted many measures to circumvent this problem and determinedto sequence the plasmid. It was noted after many trials that we were missing a Lac I repressor. Thiswas decided would not effect our project as our soul purpose for the plasmids was to produceprotein. The missing LacI only meant the expression of CYP2C9 was always on without control.This is no longer an issue and site directed mutagenesis has been used successfully to generate allof the mutants that are presented in this proposal.The expression of mutants has been an even greater time consumer. The expression of mutatedprotein is a 3 day process in which the protein content must be constantly monitored. It is an issuethat many 3 day trials are unsuccessful at producing the mutated proteins. The most viable reasonthat we can deduce is that many mutations of proteins can be toxic to the cells. In many cases thismeans we may not be able to express these mutations. We have recently taken steps to usechaperones to assist in protein folding as to minimize the toxicity of the mutated proteins. We havebeen able to express several of the mutations and working on getting the chaperones into thosePHS 2590 (Rev. 06/09)Continuation Format Page