Quick Definition
What is an ATPase Assay for ABC Transporters?: An ATPase assay measures ATP hydrolysis by an ABC transporter as a surrogate for transport activity. Transported substrates stimulate the basal ATPase activity of P-gp, BCRP and other ABC transporters; inhibitors reduce this stimulated activity. Inorganic phosphate (Pi) released during hydrolysis is quantified colorimetrically, giving a rapid readout of transporter interaction.
When to use ATPase vs other methods
ATPase is most valuable for: early discovery screening of large compound series (50–500 compounds) needing rapid rank-ordering of P-gp and BCRP interaction potential; preliminary hit identification before vesicular transport assays; and as supplementary data in regulatory submissions.
Do NOT use as standalone: FDA and EMA do not accept ATPase data as sole DDI IC50 evidence; not reliable for BSEP, MRP2, MRP3, MRP4 where substrate stimulation is variable; not for clinical DDI prediction without vesicular transport confirmation.
Key Facts
- Principle: ABC transporters hydrolyse ATP → Pi released → measured by molybdate/malachite green colorimetric assay
- Vanadate-sensitive ATPase: sodium orthovanadate specifically inhibits ABC transporter ATPase — vanadate-sensitive Pi = specific transporter activity
- Throughput: very high — colorimetric, 96/384-well plate, same-day results, no specialised equipment beyond plate reader
- Substrate ID: stimulation of basal ATPase above background = potential substrate
- Inhibitor ID: reduction of stimulated ATPase = potential inhibitor
- Limitation: non-linear relationship between ATPase activity and transport rate — not standalone for regulatory DDI submissions
- Best use: early-phase high-throughput screening to rank-order compound series before vesicular transport assay confirmation
Further Reading
- What is a Vesicular Transport Assay?
- Vesicular vs Cell-Based vs ATPase Assay
- What is ABC Transporter?
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