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Cuomarin‐6‐sulfonamides May be Useful Photoaffinity‐labeling Reagents for Analyzing the Kinetics of Drug Transport and ATP Hydrolysis in ABC Multidrug Transporters
Author(s) -
Spierenberg Sarah,
Basanagouda Mahantesha,
Kulkarni Manohar V.,
Golin John
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.932.7
Subject(s) - photoaffinity labeling , transporter , atp binding cassette transporter , affinity label , atpase , reagent , chemistry , biochemistry , drug , binding site , enzyme , biology , pharmacology , gene
The photoaffinity reagent [ 125 ]‐iodoarylazidoprazosin (IAAP) is used to study both the ATPase catalytic cycle and the behavior of drug‐binding sites in a number of ABC multidrug transporter proteins including those found in fungi and mammalian cells. This reagent has several limitations, however. It is synthesized with a radioactive label and the lack of a cold counterpart makes measuring the K d at a drug binding site onerous. Furthermore, multidrug transporters have multiple drug binding sites and IAAP labels at best only a minority of these. Both of these limitations are potentially addressed with a set of cuomarin‐6‐sulfonamides that contain a free C4‐azidomethyl group that should be suitable for photoaffinity labeling. We have begun to test some of these reagents using the yeast multidrug transporter Pdr5. We demonstrate that these derivatives are strong Pdr5 drug substrates that don't interfere with the protein's ATPase activity. Using whole cell transport assays, we show that several of these compounds are transported from at least one site that is distinct from the one photoaffinity labeled with IAAP.