Design and Characterization of the First Selective and Potent Mechanism-Based Inhibitor of Cytochrome P450 4Z1

Abstract

Mammary-tissue-restricted cytochrome P450 4Z1 (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. In this study, we developed time-dependent inhibitors of CYP4Z1 designed as fatty acid mimetics linked to the bioactivatable pharmacophore, 1-aminobenzotriazole (<b>ABT</b>). The most potent analogue, 8-[(1<i>H</i>-benzotriazol-1-yl)amino]octanoic acid (<b>7</b>), showed a 60-fold lower shifted-half-maximal inhibitory concentration (IC₅₀) for CYP4Z1 compared to <b>ABT</b>, efficient mechanism-based inactivation of the enzyme evidenced by a <i>K</i>_I = 2.2 μM and a <i>k</i>_inact = 0.15 min^-1, and a partition ratio of 14. Furthermore, <b>7</b> exhibited low off-target inhibition of other CYP isozymes. Finally, low micromolar concentrations of <b>7</b> inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation, selective mechanism-based inhibitor (MBI) will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its association with breast cancer.