Drug transporter assays

Drug transporter proteins can be a very important consideration faced by drug discovery scientists. They are transmembrane proteins which facilitate transport of molecules both in and out of the cell. Therapeutic molecules can be inhibitors or substrates for these transporters. This can have a huge impact on the ADME (absorption, distribution, metabolism and excretion) properties of a therapeutic compound. The most widely studied drug transporter is P-gp (P-glycoprotein), but there are a variety of others expressed within the body.
Driven by the requirement to identify inhibitors of transporter proteins at an earlier stage, a group at Bristol –Myers Squibb have published a 384 well based assay panel in this article J Biomolecular Screening 2013, 19 (9), 1072-1083 http://dx.doi.org/10.1177/1087057113494807 The authors outline a range of assay formats they developed for a variety of different drug transporters using both fluorescence and radioactive methods in 384 well format, for speed and to support higher compound numbers. An example of one assay format developed was with the use of calcein-AM as a fluorescent reporter to measure P-gp inhibition. Calcein-AM can freely pass through the cell membrane and is converted via intracellular esterases into calcein (which does not pass freely). Uninhibited P-gp transporter can efflux the calcein-AM to the external environment before this esterase activity can occur. If the P-gp transporter is inhibited the calcein-AM will remain in the cell to be converted, and can be monitored using cellular imaging techniques. The authors made a comparison of the values obtained in the new 384 format against the current standard 96 well format (3H digoxin) and the correlation between the two methods was acceptable. This 384 well method offers a much faster response to generate data than the earlier 96 well methodology.
Using these assays the authors were able to detect inhibitors of P-gp and other transporters, however currently they are not able to determine if a compound is a substrate for these transporters. If a method could be produced in the same manner and plate format that accomplished this, it would be another step forward for drug transporter assays.

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