Cytochrome P450s catalyze the metabolism of a wide variety of drugs and other xenobiotics.  In this process, they may form reactive intermediates which covalently bind to cellular macromolecules, potentially leading to toxicity.  Compounds undergoing P450 metabolism may also form highly reactive intermediates that can inactivate the proteins forming covalent adducts with the heme or the apoprotein, a process known as mechanism-based inactivation.  These mechanism-based inactivators can be used to probe the active sites of P450s in their catalytically functional conformations.  The identification of the site of modification can provide valuable information regarding the three-dimensional structure of the active site.  We have investigated a variety of mechanism-based inactivators and determined the structural aspects and functional groups leading to mechanism-based inactivation. This presentation will focus on studies on mechanism-based inactivation of P450s, trapping and identification of the reactive intermediates responsible for inactivation, determination of the sites of modification on the protein responsible for the loss of activity, mechanisms of inactivation, and the use of combined structural and computational analytical methods to obtain insights into the three-dimensional structures of the active sites and how the modified P450s may show changes in their product profiles while retaining partial metabolic activity.  Clopidogrel, a widely prescribed antiplatelet agent, requires metabolic biotransformation to its pharmacologically active form by human P450s.  The biologically active metabolite (AM) also causes mechanism-based inactivation of CYP2B6.  The AM can be trapped as a stable mixed disulfide conjugate which still exhibits full antiplatelet activity and has potential as a therapeutic for overcoming the interindividual variability that plagues clopidogrel therapy.  (Supported in part by NIH Grant CA 16954).

Cytochrome P-450-catalyzed formation of reactive intermediates: The perspective makes the poison

248th National Meeting (2014)

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