Aug 03, 2025
Drug transporter (ABC), P-gp, BCRP, BSEP inhibition using drug substrate assay
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. Drug transporter (ABC), P-gp, BCRP, BSEP inhibition using drug substrate assay. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbkbzngpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2025
Last Modified: August 03, 2025
Protocol Integer ID: 219057
Keywords: ADME, DMPK, drug discovery, drug transporter, pharmacokinetic, transporter assay, vesicular transport assay, drug transporter, drug substrate, crucial for drug absorption, using drug substrate, specific abc transporter, crucial role in drug absorption, drug absorption, probe substrate by the transporter, drug interaction, pharmacokinetic property, atp, bsep inhibition, interaction of drug, inhibitor, atpase, potential for drug, transport, common assay type, drug, transporter, assay, bcrp, family of protein pump, protein pump, inhibition, substrate, molecules across cell membrane, cell membrane
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Abstract
ATP-binding cassette (ABC) transporter assays are used to study the activity and interaction of drugs with ABC transporters P-gp, BCRP, and BSEP, which are crucial for drug absorption, distribution, and excretion.
These assays measure the effect of a test compound on the transport of a known probe substrate by the transporter. By quantifying the amount of substrate transported in the presence and absence of the test compound, researchers can determine the degree of inhibition.
ATP-binding cassette (ABC) transporters are a family of protein pumps that move molecules across cell membranes, playing a crucial role in drug absorption, distribution, and excretion.
These assays help determine if a drug is a substrate or inhibitor of a specific ABC transporter, impacting its pharmacokinetic properties and potential for drug-drug interactions. Common assay types include vesicular transport assays, cell-based uptake assays, and ATPase assays.
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Summary
These assays measure the effect of a test compound on the transport of a known probe substrate by the transporter. By quantifying the amount of substrate transported in the presence and absence of the test compound, researchers can determine the degree of inhibition.
- Principle: These assays utilize a "probe substrate" - a compound known to be transported by the specific ABC transporter.
- Inhibition Assay: The test compound (potential inhibitor) is co-incubated with the probe substrate and the transporter, and the amount of substrate transported is measured. If the test compound inhibits the transporter, the amount of substrate transported will be reduced.
- IC50: The concentration of the test compound required to inhibit the transporter by 50% is measured
Sample Preparation
The membrane vesicles (MV) prepared from HEK293 cells over expressing human or other species specific ABC transporters (from SOLVO) and transporter specific substrates are added into a standard 96 well plate.
Assay
Test compounds, reference inhibitor or vehicle control (1% DMSO) are pre-incubated
with the MV at 37 °C for 15 min.
Subsequently ATP or AMP is added to the plate and incubated at 37 °C for 5 min (BESP) or 1 min (P-gp and BCRP).
The reaction is stopped by adding cold washing buffer.
The membrane vesicles are washed.
The amount of transporter specific substrate accumulated inside the MV is quantified by LC-MS using peak ratio of the peak area of transporter specific substrate vs. the peak area of the internal standard.
| Transporter | Vesicles | Substrate | Reference Inhibitor | |
| P-gp | P-gp-HEK Vesicles | N-methyl-quinidine (1 µM) | verapamil | |
| BCRP | BCRP-HEK Vesicles | Estrone 3-sulfate (1 µM) | Ko143 | |
| BSEP | BSEP-HEK Vesicles | taurocholic acid (0.1 µM) | cyclosporine A |
Data Analysis
The percent of control is calculated using the equation below.
The IC50 value is determined by non-linear regression analysis of the
concentration-response curve using the Hill equation.
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 (%) = (𝐶𝑜𝑚𝑝𝑜𝑢𝑛𝑑 − 𝐵𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑) / (𝑉𝑒ℎ𝑖𝑐𝑙𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐵𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑) × 100
- Compound is the individual peak ratio obtained in the presence of the test compound and ATP.
- Vehicle control is the mean peak ratio obtained in the presence of vehicle control (1% DMSO) and ATP.
- Background is the mean peak ratio obtained in the presence of vehicle control and in the absence of ATP.
Protocol references
Prediction of drug–ABC-transporter interaction — Recent advances and future challenges
Acknowledgements
Grateful to Concept Life Sciences for supplying the original protocol summary