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Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes

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Abstract

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.

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Abbreviations

ABT:

1-Aminobenzotriazole

ADME:

Absorption, distribution, metabolism, and excretion

AO:

Aldehyde oxidase

CBR:

Carbonyl reductase

CE:

Collision energy

CES:

Carboxylesterase

CO2 :

Carbon dioxide

CXP:

Collision cell exit potential

CYP:

Cytochrome P450

DDI:

Drug-drug interaction

DMSO:

Dimethyl sulfoxide

DP:

Declustering potential

EPHX:

Epoxide hydrolase

f m :

Fraction metabolized

FMO:

Flavin-containing monooxygenase

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HHEP:

Human hepatocytes

HLM:

Human liver microsomes

HPLC:

High-performance liquid chromatography

HTS:

High throughput screening

IC50 :

Half-maximal inhibitory concentration

IVIVE:

In vitro-to-in vivo extrapolation

K I :

Inactivation rate constant

k inact :

The maximum rate of enzyme inactivation

K i,u :

Unbound inhibition constant

K m :

Michaelis constant

LC-MS/MS:

Liquid chromatography with tandem mass spectrometry

MAO:

Monoamine oxidase

MRM:

Multiple-reaction monitoring

4-MU:

4-Methylumbelliferone

M/Z:

Mass-to-charge ratio

Na2CO3 :

Sodium carbonate

NADPH:

Nicotinamide adenine dinucleotide phosphate, reduced form

NAT:

N-acetyltransferase

PK:

Pharmacokinetics

Q1:

First quadrupole mass filter

Q3:

Second quadrupole mass filter

TDI:

Time-dependent inhibition

UGT:

Uridine 5′-diphospho-glucuronosyltransferase

WEM:

Williams E medium

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Acknowledgements

Authors greatly appreciate the help of Sophia M. Shi in editing the manuscript and many colleagues for their helpful discussion.

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Author notes

  1. Li Di

    Present address: Recursion Pharmaceuticals, Salt Lake City, UT, USA

Authors and Affiliations

  1. Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Groton, Connecticut, USA

    Jianhua Liu, Daria Vernikovskaya, Gary Bora, Anthony Carlo, Samantha Jordan, Lloyd Wei Tat Tang, George Chang, Matthew D. Troutman & Li Di

  2. Global Biometrics and Data Management, Pfizer Worldwide Research and Development, Groton, Connecticut, USA

    Woodrow Burchett

  3. Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, Massachusetts, USA

    Joy Yang

  4. Discovery Sciences, Pfizer Worldwide Research and Development, Groton, Connecticut, USA

    Ye Che

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Contributions

Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: JL, DV, GB, AC, WB, SJ, LT, JY, YC, GC, MT, and LD. Drafting the work or revising it critically for important intellectual content: JL, DV, WB, SJ, LT, and LD. Final approval of the version to be published: JL, DV, GB, AC, WB, SJ, LT, JY, YC, GC, MT, and LD. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: JL, DV, GB, AC, WB, SJ, LT, JY, YC, GC, MT, and LD.

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Correspondence to Li Di.

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Authors are employees of Pfizer Inc., New York, NY, USA, and may hold Pfizer stocks.

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Liu, J., Vernikovskaya, D., Bora, G. et al. Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes. AAPS J 26, 36 (2024). https://doi.org/10.1208/s12248-024-00908-8

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