Structure–Activity Studies Reveal the Oxazinone Ring Is a Determinant of Cytochrome P450 2B6 Activity Toward Efavirenz

Abstract

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.

Single Heteroatom Substitutions in the Efavirenz Oxazinone Ring Impact Metabolism by CYP2B6

“…We previously showed that elimination of this position altogether, resulted in a lower K M and higher V max compared to EFV. [13] Our data obtained here demonstrate that addition of a hydrophobic methyl group also results in tighter binding, as is suggested by the lower K M value. Thus, our work here further supports that the presence of a carbonyl oxygen atom seems to decrease the affinity of CYP2B6 for EFV.…”

“…We then incubated CYP2B1 with each of the eight EFV analogues reported previously. [13] We again found that CYP2B1 mirrored CYP2B6 activity towards our entire panel of EFV analogues, including M2 formation from E-dihydroefavirenz, M4 and M5 formation from the pentynyl analogue, and M2 formation from the oxazine analogue. Moreover, with the exception of 2 -M2, 3 -M1, 4 -M2, and 5 -M1; metabolite formation from our panel of EFV analogues differed by less than 3-fold between CYP2B6 and CYP2B1 (Figure 5).…”

“…Previously, we demonstrated an intact oxazinone ring to be important for metabolism of EFV by CYP2B6. [13] Additionally, in those studies, we found that an analogue lacking only the carbonyl oxygen of EFV was a better CYP2B6 substrate than EFV itself. Here we continue this work by examining the impact of each heteroatom position of the EFV oxazinone ring on metabolism by CYP2B6.…”

“…Results from our previous work with other EFV analogues suggested differences in binding affinity, [13] thus we sought to explore whether these analogues also differed from EFV in their affinity for CYP2B6. In order to explore this question, we performed product formation kinetic analysis with 1 – 4 using uHPLC-MS/MS and with 5 using uHPLC-UV detection, and compared the results to the K M associated with 8-OH EFV formation (Table 1).…”

“…Interestingly, CYP2B1 displayed activity towards the both (R) and (S) -5-Chloro-α-(cyclopropylethynyl)-2-amino-α-(trifluoromethyl) benzenemethanol analogues, which we did not observe previously with CYP2B6. [13] We then developed a more sensitive selected reaction monitoring method to detect metabolites from these analogues and found CYP2B6 to also form a monooxygenated metabolite from these enantiomers (Figure S3). Taken together, these data suggest that CYP2B1 and CYP2B6 share a parallel profile of activity towards EFV analogues.…”