SOLVO® Vesicular Transporter Assays

Membrane preparations for vesicular transport assays are suitable for general drug-efflux transporter interaction studies. Both substrate and inhibitor interactions can be assessed using membrane vesicles prepared from mammalian cell lines over-expressing key human transporters.

Human efflux transporters can be stably expressed in mammalian cells. Membrane preparations from these cells contain closed membrane vesicles, a significant percentage of which are oriented “inside-out.” Due to the reverse orientation of the efflux transporter on these membranes, transported substrates accumulate inside the vesicles. Rapid filtration of the membrane suspension through a filter allows separation of membrane vesicles and quantification of transported molecules by LC-MS/MS.

The rate of transport in these vesicle preparations is both temperature and ATP dependent. These assays are best suited for test compounds with low to moderate passive permeability.

We utilize SOLVO Biotechnology’s vesicular transport and ATPase models which provide superior transporter activity, higher dynamic range and more physiologically-relevant lipid composition allowing optimal investigations of drug-transporter interactions.

Substrate Assessment
Identification of a test compound as a substrate for a given efflux transporter can be accomplished by measuring the ATP-dependent uptake (accumulation) of the compound into membrane vesicles. This can also be confirmed by the use of a known small molecule inhibitor of the transporter. Negative control membranes (prepared from a non-transfected cell line) and AMP are typically used as negative controls.

Inhibition Assessment
Identification of a test compound as an inhibitor for a given efflux transporter can be addressed by measuring effects on the rate of uptake of a known substrate for the transporter under consideration. Concentration-dependent inhibition of substrate uptake by the test compound can be used to determine an IC50.
 

 Membrane Vesicle Transport Assay Protocol

 
Test System SOLVO membrane vesicles including:
BSEP
P-gp
BCRP
MRP1
MRP2
MRP3
MRP4
MRP5
Test Compound Concentration 0.1 -300 µM in half log increments, or custom
Assay Length 3-15 minutes
Number of Replicates 3
Negative Controls Negative control membrane stock (5 mg/ml)
Adenosine monophosphate (AMP)
Positive Controls Transporter/Substrate/Inhibitor BSEP/Taurocholate/Cyclosporine A
P-gp/N-methyl-quinidine/Verapamil
BCRP/Estrone 3-Sulfate/Ko143
MRP1/Leukotriene C4/MK-571
MRP2/Estradiol 17-(beta-D-glucuronide)/Benzbromarone
MRP3/Estradiol 17-(beta-D-glucuronide)/Sulfasalazine
MRP4/Dehydroepiandrosterone sulfate (DHEAS)/MK-571
MRP5/cGMP/Benzbromarone
Or custom
Compound Requirements 50 µL of 10 mM solution
Analysis Method LC-MS/MS
Data Delivery Assay methods
Rate of uptake (pmol/mg/min)
Km or IC50 as appropriate

Click here to contact an expert today for your vesicular transporter testing needs.