RCMI Pilot Project

January 1, 2020 – December 31, 2020

 

PI:  Maryam Foroozesh, Ph.D., Jayalakshmi Sridhar, Ph.D., Dr. Goyal

 

Title: Developing New Families of Inhibitors to Investigate Polymorphism in Cytochrome P450 2A6 and Associated Changes in Enzyme Structure and Catalytic Activity 

 

Abstract:

Cytochrome P450 2A6 (CYP2A6) is an important member of the cytochrome P450 superfamily of heme-containing monooxygenases involved in the phase I metabolism of environmental toxicants, such as nicotine and other cigarette smoke components, into less toxic (detoxification) or more toxic (metabolic activation) metabolites (1). P450-dependent catalytic reactions are susceptible to mechanism-based inhibition by appropriate reactive “psuedosubstrates” (2). Selective mechanism-based inhibitors are valuable experimental tools for studying the active sites and mechanisms of action of P450 enzymes (2-4).

Tobacco use has been identified as the most preventable cause of cancer (5). Tobacco smoke components have been linked to 18 different types of cancer, including lung cancer. In addition, in smoker cancer patients, decreased response to treatment and increased rate of recurrence and treatment side-effects have been observed (6, 7). In humans, hepatic CYP2A6 accounts for approximately 85-95% of the metabolism of (S)-nicotine to cotinine (8, 9). CYP2A6 is also capable of catalyzing the oxidative conversion of the tobacco-specific procarcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL) to their electrophilic DNA alkylating carcinogenic metabolites (10-12). Selective mechanism-based inhibitors for CYP2A6 are useful experimental agents to study the importance of this enzyme in this carcinogenesis process. Development of such inhibitors is a major goal of this project.

CYP2A6 is highly polymorphic leading to interindividual differences in substrate metabolism, including nicotine metabolism. Interestingly, the various polymorphs can show increased or decreased metabolism of specific substrates. It has been shown that existence of certain polymorphs is ethnic/race-related. Three alleles are chosen for the present studies- *7, occurs more in East Asians; and *17 & *35, occur predominantly in African North Americans and Africans (13, 14). The comparison of the changes in binding pocket’s characteristics between the alleles and the WT (wild-type) CYP2A6 enzymes in the presence and absence of the in-house inhibitors will provide a greater insight into the design of better performing inhibitors. Structural features in the binding pocket of the alleles *17 and *35 that have lower metabolic rates for nicotine will provide us with strategies for targeting specific sites in the binding pocket of CYP2A6. To achieve this, an in-depth study of the dynamic nature of the binding pocket and the changes to the ingress/egress channels with changing structural features of the developed CYP2A6 inhibitors will be performed. These studies are expected to provide an insight into the flexibility of the binding pocket, the accessibility of the binding pocket through the ingress channel for compounds of varied structural features, and the changes to the egress channel upon ligand binding to the enzyme.   

The overall goal of this proposed study is to design, synthesize, and evaluate novel selective mechanism-based inhibitors for P450 2A6, in order to study the mechanisms of action of this enzyme and differences observed due to its polymorphism.