Cytochrome P450 2B6 (CYP2B6) is primarily responsible for the metabolism of the anti-HIV drug efavirenz (EFV). We set out to explore the molecular basis for CYP2B6 activity toward EFV by examining the metabolism of eight EFV analogues. cDNA-expressed CYP2B6 formed monooxygenated metabolites from EFV analogues containing an intact oxazinone or oxazine ring, but not from analogues with a disrupted ring, suggesting this ring is important for metabolism of EFV by CYP2B6. Subsequent substrate depletion analysis of EFV and EFV analogues found to be CYP2B6 substrates revealed further differences between these CYP2B6 substrates. Compounds that were not found to be CYP2B6 substrates were still able to inhibit CYP2B6 activity toward a known substrate, bupropion, suggesting they do gain access to the CYP2B6 active site. Taken together, these data reveal structural characteristics of EFV, namely, the oxazinone ring, that are critical for CYP2B6 metabolism of compounds with the EFV chemical scaffold.

Previously, we observed that the oxazinone ring is important for CYP2B6 activity toward efavirenz ((4S)-6-Chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one), a CYP2B6 substrate used to treat HIV. Here, to further understand the structural characteristics of efavirenz that render it a CYP2B6 substrate, we test the importance of each heteroatom of the oxazinone ring. We assembled a panel of five analogues: 6-Chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-2-methyl-4-(trifluoromethyl)-2H-3,1-benzoxazine (1), (4S)-6-Chloro-4-[(1E)-2-cyclopropylethenyl]-3,4-dihydro-4-(trifluoromethyl)-,2(1H)-quinazolinone (2), (4S)-6-Chloro-4-(2-cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (3), 6-Chloro-4-(cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinolinone (4), and 6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-4H-benzo[d][1,3]dioxin-2-one (5). Metabolism of 1–5 was investigated using human liver microsomes, individual P450s, and mass spectrometry or UV absorbance detection. Steady-state analysis of CYP2B6 metabolism of 1–5 showed KM values ranging from 0.3 to 3.9 fold different than observed for efavirenz (KM of 3.6 ± 1.7 μM). The lowest KM values approximating 1 μM, were observed for metabolism of 1, while the largest KM, 14 ± 6.4 μM, was found for 4. Our work reveals that analogues with heteroatom changes in the oxazinone ring are still CYP2B6 substrates, though the changes KM suggest altered substrate binding.

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