Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

Abstract

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

HCV Antiviral Drugs Have the Potential to Adversely Perturb the Fetal-Maternal Communication Axis through Inhibition of CYP3A7 DHEA-S Oxidation

“…We began with an initial screen of these antivirals in order to determine their propensity for inhibition of CYP3A7 metabolism of dibenzylfluorescein (DBF). Although this reaction is not unique or specific to CYP3A7, previous findings determined that CYP3A7 demonstrated maximal activity with this fluorescent substrate, producing the lowest K m and highest signal-tonoise ratio (Work, Kandel, and Lampe 2021). Additionally, these assays utilize only recombinant CYP3A7 and NADPH reductase supplemented with cytochrome b 5 , without interference from other CYP enzymes or alternative clearance pathways.…”

“…In vitro screening assay of recombinant CYP3A7 inhibition. Assays were prepared as previously described (Work, Kandel, and Lampe 2021). In brief, Assays were conducted in triplicate in 96-well black polystyrene microtiter plates (Costar®, catalog # 266) in a 100 µL volume and prepared on ice.…”

“…Herein, we investigated the inhibitory effects of 13 FDA-approved HCV antiviral drugs on CYP3A7 activity. This was initially assessed with a high-throughput fluorescent assay previously developed by our lab that utilizes the probe dibenzylfluorescein (DBF) (Work, Kandel, and Lampe 2021). Drugs that reduced CYP3A7 activity by 50% or more in this assay were further characterized by determining their IC 50 values using an LC-MS/MS-based method and measuring 16α-hydroxy-DHEA-S formation.…”

Fluorescence-Based High-Throughput Assays for Investigating Cytochrome P450 Enzyme-Mediated Drug–Drug Interactions

“…Although the specificity of most reported fluorogenic substrates for human CYPs is not very satisfactory, these substrates could still be applied for HTS of CYP inhibitors when using recombinant CYPs or specific cell preparations as enzyme sources. For example, DBF, a multi-enzyme metabolizing substrate, was identified as a useful tool in HTS assay for CYP3A7 to realize the DDI/HDIs for neonatal population (Work et al, 2021). It is noteworthy that part of the fluorogenic (Li et al, 1997;Renwick et al, 2001;Dai et al, 2017;Ning et al, 2018b;Ning et al, 2019a;Ning et al, 2019c;Jin et al, 2020;Dai et al, 2021;Feng et al, 2022;Tian et al, 2022;He et al, 2023).…”

Repurposing Probenecid to Inhibit SARS-CoV-2, Influenza Virus, and Respiratory Syncytial Virus (RSV) Replication

“…Recent drug screening methods have transitioned away from the more laborious virus plaque assay method traditionally used to quantitate viral replication, shifting to measuring inhibition using automated high-throughput assays that allow for screening of hundreds of drugs. These screening methods are based on random screening of potential antiviral drugs using new assay methods to enable the homogeneous measurement of endpoints such as cytopathic effect, viral protein, or reporter gene expression, and assume that they can serve as markers of viral replication [ 1 , 2 , 3 , 4 ]; however, they typically do not directly measure or quantitate drug inhibition of viral replication. Classical methods to determine drug efficacy assess a reduction in viral replication using a virus plaque assay, and may measure a decrease in viral RNA transcription as determined by real-time polymerase chain reaction (RT-PCR).…”