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Structure‐Activity Analysis of Efavirenz‐Mediated Activation of Pregnane X Receptor

Efavirenz (EFV), a non‐nucleoside reverse transcriptase inhibitor, is the most prescribed antiretroviral worldwide used to treat HIV. EFV increases the expression of drug metabolizing enzymes via activation of both murine and human pregnane X receptor (PXR), and we aimed to identify the structural moieties of EFV that contribute to this. Hepatocytes isolated from wild‐type (WT) male C57BL/6 mice were treated with either 10 μM EFV or with one of 15 EFV analogs and mRNA levels of the murine PXR target gene cytochrome P450 3a11 (Cyp3a11) were measured using qPCR. EFV increased Cyp3a11 mRNA 1.6 ± 0.3‐, 14.4 ± 6.5‐ and 78.7 ± 12.9‐fold compared to control at 24 h, 48 h and 72 h, respectively, and this effect was abrogated in hepatocytes isolated from male mice genetically deficient of PXR. When these experiments were performed using a panel of EFV analogs, the smallest (223 Da) and only analog to lack an intact 6‐membered heterocyclic ring failed to increase Cyp3a11 mRNA even following 72 h of treatment, while several analogs ranging 290 Da to 424 Da in size increased Cyp3a11 mRNA over a similar time course observed with EFV (316 Da) treatment. Further, this effect was not species specific as hepatocytes isolated from humanized PXR male mice exhibited no modulation of Cyp3a11 mRNA expression when treated with the 223 Da analog. In line with these data, luciferase reporter assays performed in HepG2 cells further confirmed the 223 Da analog was unable to directly activate either murine or human PXR. Interestingly, a species specific difference was observed for an analog (317 Da) in which the nitrogen, a hydrogen bond donor, of the heterocylic ring of EFV was substituted with oxygen, a hydrogen bond acceptor. This analog, with the molecular weight difference of one Da from EFV, directly activated human PXR as observed in the luciferase reporter assays but failed to activate murine PXR in either the luciferase reporter assay or in hepatocytes isolated from WT male mice as no modulation of Cyp3a11 mRNA was observed. In addition to these analog studies, we observed the primary oxidative metabolite of EFV, 8‐hydroxy‐EFV (332 Da), with the molecular weight difference of a hydroxyl group compared to the parent drug, failed to activate either murine or human PXR in the luciferase reporter assays nor did it increase Cyp3a11 mRNA expression in hepatocytes isolated from WT or humanized PXR mice, suggesting key ligand‐receptor electrostatic interactions may contribute to ligand stabilization and affinity. In conclusion, a molecular weight dependence for PXR activation has been previously proposed in the literature but has yet to be tested using a panel of structurally similar analogs. Upon testing this, we observed that while the smallest EFV analog (223 Da) indeed did not activate murine or human PXR neither did 8‐hydroxy‐EFV (332 Da) even though it is within the molecular weight range of the analogs that were able to directly activate PXR. Furthermore, these data indicate that molecular structure may be a more important regulator of PXR activation than molecular weight.