NADPH-Independent Inactivation of CYP2B6 and NADPH-dependent Inactivation of CYP3A4/5 by Pyrrolobenzodiazepine Dimer (PBD) and Potential Implication for Assessing Covalent Modulators for Time-dependent Inhibition.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) is a DNA-minor groove alkylating agent with broad antitumor properties currently under development as a highly potent cytotoxic antibody-drug conjugate warhead against a variety of oncology targets. During a routine assessment of reversible CYP inhibition, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 (IC50 >10 µM). In contrast, PBD is a classic time-dependent inhibitor (TDI) of CYP3A4, with >30-fold shift in IC50 following a 30 min pre-incubation in the presence of NADPH. All other CYP isoforms tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed following a 30-minute pre-incubation both in the absence and presence of NADPH, an unexpected observation given the fact that no CYP2B6 inhibition was observed in the direct reversible inhibition assay up to 10 µM of PBD. No other CYP isoforms were susceptible to this apparent non-NADPH dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the HLM pellet after 45 min incubation with PBD did not fully recover the CYP2B6 activity, indicating that PBD is covalently binding to CYP2B6 leading to inactivation of the enzyme. To further investigate the mechanism of NADPH independent inhibition, the IC50-shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent CYP2B6 inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition both in the presence and absence of cofactor (NADPH) to adequately characterize the in vitro CYP inhibitory properties of various therapeutic agents with reactive moieties such as covalent modulators. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of PBD as a potent reversible CYP2C8 inhibitor, a NADPH dependent CYP3A TDI inhibitor and a NADPH independent CYP2B6 TDI inhibitor, and inhibition can occur through distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized. The American Society for Pharmacology and Experimental Therapeutics.