The contribution of heme electrostatics and active-site conformational dynamics to CYP3A4-Mediated dehydrogenation of raloxifene and 4-Hydroxy-Tamoxifen

The characterization of novel and reactive Phase I metabolites of xenobiotics, such as those frequently produced by P450 enzymes, is an area of interest that has led to increased research efforts during preclinical drug-testing and development. A key interest is improving our understanding of factors that contribute to competing Phase I reaction mechanisms, some of which produce stable products that can be further metabolized and excreted, and others that produce reactive metabolites capable of causing toxicities. Due to the highenergy nature of the P450 catalytic oxyferryl heme species, Compound I, P450 enzymes can also catalyze different oxidation reaction mechanisms, including dehydrogenation reactions. Dehydrogenation reactions are more difficult to predict than the more common P450 oxygenation and dealkylation reactions. Moreover, dehydrogenation mechanisms can compete with hydroxylation mechanisms to produce unstable desaturated electrophilic metabolites capable of forming potentially toxic biomolecular adducts. The work presented here focuses on improving existing computational tools for the prediction of P450 metabolism of two model substrates, raloxifene and 4-hydroxy-tamoxifen. These two compounds are FDA-approved selective estrogen receptor modulators currently used in the treatment of breast cancer. In Chapter 2 the development, iv testing and refinement of molecular mechanics parameters for key species of the heme prosthetic group during the P450 catalytic cycle is presented. It is shown that the assignment of atomic partial charges for key heme species improves the identification of the sites of metabolism of raloxifene by CYP3A4. Building on this work, in Chapter 3 it is shown that despite using these new heme parameters, extensive quantum mechanics calculations to probe substrate reactivity, molecular dynamics of the enzyme structure to find representative active site conformations makes the greatest improvement in the identification of the sites of metabolism for 4-hydroxy-tamoxifen. In summary, this work identifies that heme electrostatics and enzyme conformational dynamics play important roles in enzyme function and that the ability to predict sites of metabolism for P450- substrates requires the integration of both for the improvement of future in silico tools.