HTS FLOW CYTOMETRY, SMALL-MOLECULE DISCOVERY, AND THE NIH MOLECULAR LIBRARIES INITIATIVE - Flow Cytometry in Drug Discovery and Development - page 83

high-throughput multiplexed flow cytometry assay based on RGS binding to fluor-
escently labeled G a 0 ) is incubated with

purified biotinylated RGS proteins that are immobilized onto streptavidin micro-
spheres. Five RGS family members were studied: RGS4, RGS7, RGS8, RGS16, and
RGS19. As a small number of inhibitors identified have the potential of covalently
modifying the cysteine residues, the current goal of this project is to identify
reversible small-molecule inhibitors of RGS.

a . AlexaFluor 488-conjugated G a 0 (AF-G

Lessons Learned : Multiplexing was extended to biotinylated proteins and
streptavidin beads.

4.4.11 Multiplex Screening for ABC Transporter Inhibitors:
1R03MH078946-01

Upregulation of ATP binding cassette (ABC) transporter expression results in the
selection of resistant cancer cells and cancer multidrug resistance to diverse trans-
porter substrates. The assay submitted by R. Larson was designed to identify
inhibitors of three human ABC transporters, ABCB1, ABCC1, and ABCG2 that
have been shown to contribute to cancer multidrug resistance. The proposed
screen involved two separate duplex assays: one in which ABCB1 and ABCG2
transporters are evaluated in parallel using fluorescent JC-1 as substrate and the
other in which ABCB1 and ABCC1 transporters are evaluated in parallel using
fluorescent calcein acetoxymethyl ester (CaAM) as substrate [54, 55]. An active test
compound is one that prevents test cells from transporting fluorescent substrate out
of the cytoplasm and is detected by an increase in cell fluorescence intensity.
Cells expressing different transporters are color coded to distinguish which trans-
porter is affected by a compound. Although we hoped that it would be possible to
formulate a cell-based triplex for three pumps (ABCB1, ABCC1, and ABCG2), the
target assay involved duplexes, involving two dyes, that each recognized two of three
transporters. As a screen for ABCC1 inhibitors was previously performed elsewhere,
we performed HTS on the ABCB1/ABCG2 duplex assay. For the primary screening
protocol, a duplex assay was constructed in which ABCB1 and ABCG2 transporters
were evaluated in parallel using the fluorescent J-aggregate-forming lipophilic cation
5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide (JC-1) as
substrate. ABCB1-expressing cells (CCRF-Adr) were color coded with FarRed
DDAO CellTrace SE (Invitrogen) to distinguish them from ABCG2-expressing cells
(Ig-MXP3) via a red fluorescence emission wavelength of 665

10 nm (635 nm
excitation). The output of the primary screen included compounds that inhibited
pumps and were themselves nontoxic. The compound family is now being evaluated
for selectivity in anticipation of a probe report.

þ /

Lessons Learned : HTS flow cytometry is well suited to ABC transporter assays
involving suspension cells. Although the approach can be multiplexed, the substrate
selectivity of the transporters is such that there is no guarantee that a single fluorescent
substrate will be applicable to the collection of transporters assembled in a multiplex.