Vesicle Assay Design

Transporter-Expressing Vesicles

The efflux transporter-expressing vesicle is a useful tool to evaluate if a drug is a substrate or inhibitor of an efflux transporter. Inside out membrane vesicles are prepared from insect cells transiently transfected with a single efflux transporter or from mammalian cells chemically selected for the expression of a transporter. Because the vesicles are in an inside out orientation, the efflux transporter pumps substrates into the vesicles. To determine if a compound is an inhibitor of an efflux transporter, the effect on the accumulation of a probe substrate is measured. Because the transport is ATP ependent, AMP samples serve as a control. The accumulation of test compounds in transporter expressing vesicles and control vesicles can be measured to determine if the compound is a substrate of the transporter. However, hydrophobic drugs will either saturate the membrane vesicles of the transporter expressing and control vesicles masking any transporter mediated uptake or will permeate out of the vesicle before it can be quantified. The inhibiiton assay is a quick, reproducible method to determine if a compound is an inhibitor of a particular transporter. The vesicle inhibition assay is noted by the ITC as being an acceptable alternative to bi-directional transporter on monolayers in certain circumstances.

Identification of a Substrate

Comparison of the uptake activity in the transport-expressing vesicles with that of the control vesicle is used to calculate if the candidate is a substrate of the efflux transporter. Km and Vmax are calculated from the relationship between substrate concentration and uptake activity.

Identification of an Inhibitor

The inhibitory effect of the candidate is evaluated by IC50 determination (concentration causing 50% inhibition of transporter-mediated uptake of the typical substrate).

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