Evaluation of Fluorescence- and Mass Spectrometry–Based CYP Inhibition Assays for Use in Drug Discovery

Bell, Leslie; Bickford, Shari; Nguyen, Phong Hung; Wang, Jianling; He, Timothy; Zhang, Bailin; Friche, Yannick; Zimmerlin, Alfred; Urbán, László; Bojanic, Dejan

doi:10.1177/1087057108317480

Cited by 40 publications

(27 citation statements)

References 22 publications

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“…Bell et al found that nearly 75% of marketed drugs yielded acceptable correlations between fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9, which supports the use of fluorogenic assays for rapid early risk binning based on IC 50 values [26] . In the present study, fluorescent probes were used to study the effects of three pungent constituents of ginger on P450-mediated drug metabolism.…”

Section: Discussionmentioning

confidence: 99%

Pungent ginger components modulates human cytochrome P450 enzymes in vitro

Chen

Yue

et al. 2013

Acta Pharmacol Sin

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Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC 50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8-and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes.

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Section: Discussionmentioning

confidence: 99%

Pungent ginger components modulates human cytochrome P450 enzymes in vitro

Chen

Yue

et al. 2013

Acta Pharmacol Sin

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“…In addition, several CYP isoforms may contribute to the formation of the ultimate CYP inhibitor. Because the substrates for fluorescent assays are not CYP selective and necessitate the use of recombinant single enzyme systems, the inhibitory effect of metabolites generated by one CYP on other CYPs cannot be tested (Bell et al, 2008, Cohen et al, 2003. However, despite these problems, time-dependent fluorimetric assays are used to study compounds with respect to potency of MBI (Atkinson et al, 2005;Ghanbari et al, 2006, Riley et al, 2007.…”

Section: Discussionmentioning

confidence: 99%

Mechanism-Based Inhibition: Deriving KI and kinact Directly from Time-Dependent IC50 Values

Krippendorff¹,

Neuhaus²,

Lienau³

et al. 2009

SLAS Discovery

128

148

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The potential of enzyme inhibition of a drug is frequently quantified in terms of IC50 values. While this is a suitable quantity for reversible inhibitors, concerns arise when dealing with irreversible, or mechanism-based inhibitors (MBI). IC50 values of MBI are time-dependent, causing serious problems when aiming at ranking different compounds with respect to their inhibitory potential. As a consequence, most studies and ranking schemes related to MBI rely on the inhibition constant (K I ) and the rate of enzyme inactivation (k inact ) rather than on IC50 values. In this article we derive a novel relation between potentially time-dependent IC50 values and K I , k inact parameters for different types of inhibition. This allows for direct estimation of K I and k inact values from time-dependent IC50 values, even without the need of additional pre-incubation experiments. The application of this approach is illustrated using a fluorimetric assay to access the drug-drug interaction potential associated with new chemical entities. The approach can easily be implemented using standard software tools (e.g., XLfit) and may also be suitable for applications where mechanism-based inhibition is a desired mode of actions, e.g., at particular pharmacological drug targets.

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“…Probes based on known drugs are traditionally detected by radiometric assays (Stresser et al, 1996;Moody et al, 1999) and mass spectrometry, yet these assays are disadvantaged by concerns over the safety and disposal of radioactive isotopes or by low throughput, respectively. Chromogenic, fluorogenic (Friden et al, 2006), and bioluminogenic probes (Cali et al, 2006) are rapid and sensitive, yet concerns regarding their analogy to traditional drug probes (Bell et al, 2008) must be addressed by empirical correlation to traditional probes. A complexity in interpreting correlative studies is associated in particular with CYP3A4, which demonstrates substrate-dependent responses to certain analytes (Wang et al, 2000).…”

Section: Introductionmentioning

confidence: 99%