Editing ABC transporter (section)

== Methods to characterize ABC transporter interactions ==
There are a number of assay types that allow the detection of ABC transporter interactions with endogenous and xenobiotic compounds.<ref>{{cite journal | vauthors = Glavinas H, Krajcsi P, Cserepes J, Sarkadi B | title = The role of ABC transporters in drug resistance, metabolism and toxicity | journal = Current Drug Delivery | volume = 1 | issue = 1 | pages = 27–42 | date = Jan 2004 | pmid = 16305368 | doi=10.2174/1567201043480036}}</ref> The complexity of assay range from relatively simple membrane assays.<ref>{{cite journal | vauthors = Glavinas H, Méhn D, Jani M, Oosterhuis B, Herédi-Szabó K, Krajcsi P | title = Utilization of membrane vesicle preparations to study drug-ABC transporter interactions | journal = Expert Opinion on Drug Metabolism & Toxicology | volume = 4 | issue = 6 | pages = 721–32 | date = Jun 2008 | pmid = 18611113 | doi = 10.1517/17425255.4.6.721 | s2cid = 86198612 }}</ref> like vesicular transport assay, [[ATPase assay]] to more complex cell based assays up to intricate ''in vivo''{{cite journal | vauthors = Jeffrey P, Summerfield SG | title = Challenges for blood-brain barrier (BBB) screening | journal = Xenobiotica | volume = 37 | issue = 10–11 | pages = 1135–51 | year = 2007 | pmid = 17968740 | doi = 10.1080/00498250701570285 | s2cid = 25944548 }} detection methodologies.<ref>This entire volume is dedicated to various methods used: {{Cite book | series = Methods in Enzymology | volume = 292 | pages = 3–853 | year = 1998 |vauthors=Nikaido H, Hall J | title = ABC Transporters: Biochemical, Cellular, and Molecular Aspects |pmid=9711542 | doi = 10.1016/s0076-6879(00)x0188-7 | isbn = 9780121821937 }}</ref>

=== Membrane assays ===
The ''vesicular transport assay'' detects the translocation of molecules by ABC transporters.<ref>{{cite journal | vauthors = Horio M, Gottesman MM, Pastan I | title = ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 85 | issue = 10 | pages = 3580–4 | date = May 1988 | pmid = 3368466 | pmc = 280257 | doi = 10.1073/pnas.85.10.3580 | bibcode = 1988PNAS...85.3580H | doi-access = free }}</ref> Membranes prepared under suitable conditions contain inside-out oriented vesicles with the ATP binding site and substrate binding site of the transporter facing the buffer outside. Substrates of the transporter are taken up into the vesicles in an ATP dependent manner. Rapid filtration using glass fiber filters or nitrocellulose membranes are used to separate the vesicles from the incubation solution and the test compound trapped inside the vesicles is retained on the filter. The quantity of the transported unlabelled molecules is determined by HPLC, LC/MS, LC/MS/MS. Alternatively, the compounds are radiolabeled, fluorescent or have a fluorescent tag so that the radioactivity or fluorescence retained on the filter can be quantified.

Various types of membranes from different sources (e.g. insect cells, transfected  or selected mammalian cell lines) are used in vesicular transport studies. Membranes are commercially available or can be prepared from various cells or even tissues e.g. liver canalicular membranes. This assay type has the advantage of measuring the actual disposition of the substrate across the cell membrane. Its disadvantage is that compounds with medium-to-high passive permeability are not retained inside the vesicles making direct transport measurements with this class of compounds difficult to perform.

The vesicular transport assay can be performed in an "indirect" setting, where interacting test drugs modulate the transport rate of a reporter compound. This assay type is particularly suitable for the detection of possible drug-drug interactions and drug-endogenous substrate interactions. It is not sensitive to the passive permeability of the compounds and therefore detects all interacting compounds. Yet, it does not provide information on whether the compound tested is an inhibitor of the transporter, or a substrate of the transporter inhibiting its function in a competitive fashion.  A typical example of an indirect vesicular transport assay is the detection of the inhibition of taurocholate transport by ABCB11 ([[ABCB11|BSEP]]).

=== Whole cell based assays ===
Efflux transporter-expressing cells actively pump substrates out of the cell, which results in a lower rate of substrate accumulation, lower intracellular concentration at steady state, or a faster rate of substrate elimination from cells loaded with the substrate. Transported radioactive substrates or labeled fluorescent dyes can be directly measured, or in an indirect set up, the modulation of the accumulation of a probe substrate (e.g. fluorescent dyes like rhodamine 123, or calcein) can be determined in the presence of a test drug.<ref name="ReferenceA"/>

Calcein-AM, A highly permeable derivative of [[calcein]] readily penetrates into intact cells, where the endogenous esterases rapidly hydrolyze it to the fluorescent calcein. In contrast to calcein-AM, calcein has low permeability and therefore gets trapped in the cell and accumulates. As calcein-AM is an excellent substrate of the MDR1 and MRP1 efflux transporters, cells expressing MDR1 and/or MRP1 transporters pump the calcein-AM out of the cell before esterases can hydrolyze it. This results in a lower cellular accumulation rate of calcein. The higher the MDR activity is in the cell membrane, the less Calcein is accumulated in the cytoplasm. In MDR-expressing cells, the addition of an MDR inhibitor or an MDR substrate in excess dramatically increases the rate of Calcein accumulation. Activity of multidrug transporter is reflected by the difference between the amounts of dye accumulated in the presence and the absence of inhibitor. Using selective inhibitors, transport activity of MDR1 and MRP1 can be easily distinguished. This assay can be used to screen drugs for transporter interactions, and also to quantify the MDR activity of cells. The calcein assay is the proprietary assay of SOLVO Biotechnology.