NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD : Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition

Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics..

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. 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 human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound 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 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 w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three 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.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:48

Enthalten in:

Drug metabolism and disposition: the biological fate of chemicals - 48(2020), 8 vom: 01. Aug., Seite 655-661

Sprache:

Englisch

Beteiligte Personen:

Kosaka, Mika [VerfasserIn]
Zhang, Donglu [VerfasserIn]
Wong, Simon [VerfasserIn]
Yan, Zhengyin [VerfasserIn]

Links:

Volltext

Themen:

12794-10-4
53-59-8
Antineoplastic Agents
Benzodiazepines
CYP2B6 protein, human
CYP3A4 protein, human
CYP3A5 protein, human
Cytochrome P-450 CYP2B6
Cytochrome P-450 CYP2B6 Inhibitors
Cytochrome P-450 CYP3A
Cytochrome P-450 CYP3A Inhibitors
EC 1.14.14.1
EC 1.14.14.55
Journal Article
NADP
Pyrroles
Pyrrolo(2,1-c)(1,4)benzodiazepine
Recombinant Proteins

Anmerkungen:

Date Completed 13.09.2021

Date Revised 13.09.2021

published: Print-Electronic

Citation Status MEDLINE

doi:

10.1124/dmd.120.090878

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM310631505