Cytochrome P450 family 4 enzymes in cancer: Leveraging bioactivation for therapeutic potential

Abstract

Cytochrome P450 (CYP) enzymes are a critical family of hemoproteins that are involved in the metabolism of both xenobiotic and endogenous molecules. The CYP4 family constitutes thirteen enzymes in humans that are typically involved in fatty acid and eicosanoid oxidation. CYP4 enzymes are known to play roles in hypertension, stroke, and cancer so modulation of the enzyme activity may be of therapeutic benefit. CYP4B1 and CYP4Z1 are two orphan isozymes in the family, with relatively unknown endogenous substrate selectivities. Therefore, these two enzymes potentially offer new paradigms as CYP drug targets, particularly with regard to anticancer applications, which is a common theme of the research presented herein. The CYP-mediated metabolism of certain substrates can result in the formation of electrophilic, and highly reactive, metabolites. This process is termed bioactivation, and while the resultant species can wreak havoc on biological systems, under certain circumstances the chemistry can be harnessed to achieve a desired beneficial effect. Consequently, the unique catalytic abilities of CYP4B1 and CYP4Z1 were manipulated to design, synthesize, and characterize pro-toxicants and mechanism-based inhibitors for these enzymes, thus leveraging bioactivation for therapeutic potential. Over the past 20 years, CYP4B1 has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new pro-toxicant substrates for CYP4B1. We examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells that express CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1 - C8). Compounds with C3 – C6 carbon chain lengths showed similar potency to IPO (LD50 ~5 M). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative ene-dial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased -hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here. Mammary tissue restricted CYP4Z1 has garnered interest for its potential role in breast cancer progression. CYP4Z1-dependent metabolism of arachidonic acid preferentially generates 14,15-epoxyeicosatrienoic acid (14,15-EET), a metabolite known to influence cellular proliferation, migration, and angiogenesis. We developed mechanism-based inhibitors (MBIs) of CYP4Z1 designed as fatty acid mimetics linked to the bioactivatable pharmacophore, 1-aminobenzotriazole (ABT). The most potent analog, 8-[(1H-benzotriazol-1-yl)amino]octanoic acid (8-BOA), showed a 60-fold lower shifted IC50 for CYP4Z1 compared to ABT, efficient mechanism-based inactivation of the enzyme evidenced by a KI = 2.2 μM, kinact = 0.15 min-1 and a partition ratio of 14. Furthermore, 8-BOA exhibited low off-target inhibition of other CYP isozymes. Finally, low micromolar concentrations of 8-BOA inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation, selective MBI will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its association with breast cancer. Due to the favorable inhibitory activity of 8-BOA in vitro, an in vivo characterization of this inhibitor was performed. The pharmacokinetics and metabolism of 8-BOA in rats was examined after a single IV bolus dose of 10 mg/kg. A biphasic time-concentration profile indicated relatively low clearance and distribution to a peripheral compartment. The major circulating metabolites identified in plasma were products of β-oxidation; bis- and tetra-demethylenated congeners accounted for >50% of metabolites by peak area. The bis-demethylenated product was characterized previously as a CYP4Z1 MBI and so represents an active metabolite that may contribute to the desired pharmacological effect. Ex vivo analysis of total CYP content in rat liver and kidney microsomes showed that off-target CYP inactivation was minimal; liver microsomal probe substrate metabolism also demonstrated low off-target inactivation. Standard clinical chemistries provided no indication of acute toxicity and in silico simulations using the free concentration of 8-BOA in plasma suggested that the in vivo dose used here may effectively inactivate CYP4Z1 in a xenografted tumor. As such, the studies reported in the Chapters of this dissertation provide two excellent examples of leveraging the bioactivation potential of human CYP4 enzymes for use in anticancer settings.