Biomedical Engineering Reference
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proteins. The binding of fluorochrome-conjugated BH3 peptides (including Bim) to
Bcl-2 family members thus provides the basis for construction of fluorescence-based
assays amenable to flow cytometry high-throughput screening for small-molecule
regulators of these interactions. The high-throughput screen was a multiplexed assay
to identify small-molecule regulators of protein interactions between the BH3 peptide
of Bim and the following six Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1,
Mcl-1, and Bcl-2 (the eponymous founding member of the Bcl-2 family). HTS has
been completed and reported in PubChem and we continue to characterize the
activities of the molecules, some of which may be chemically reactive, in secondary
assays.
Lessons Learned : The setup for this screen depended on the unexpected observation
thatwhenGSTis arrayedat highdensities onmicrospheres, the attachment is stabledue
to rebinding [49]. Even though the monovalent affinity of GSH to GST is
10
m
M,
individual proteins can be attached to separately color-coded beads.
4.4.9 Low Molecular Weight G-Protein Multiplex Assay:
1R03MH081231-01 (Bead-Based Multiplexes)
The lowmolecular weight G-protein assaywas submitted byA.Wandinger-Ness [50].
A multiplex assay screened the binding of fluorescent GTP to G-protein-GST fusion
proteins onGSHbeads. A set of sixG-proteins (Rac1wt, Rab7wt, Rac1 activated, Ras
wt, Rab2 wt, and Cdc42 wt) were arrayed. The dose-responses have also included
activated mutants of Ras and Cdc. Dose-response curves have been performed on a
total of nine proteins. The screens have identified a pan-activator series (probe report,
publication in preparation), a pan-inhibitor series (undergoing optimization), a rho
family selective series [51], and a Cdc42 selective inhibitor (probe report and
publication in preparation).
Lessons Learned : A successful multiplex screening campaign has the potential of
revealing molecules active against one or more of the targets.
4.4.10 HTS to Identify Small-Molecule Regulators of RGS Family
Protein Interactions: NIH R21NS057014 (Bead-Based Multiplex)
The RGS assay [52, 53] submitted by R. Neubig and coworkers involves the assembly
of five biotinylated RGS proteins on streptavidin beads. Regulators of G-protein
signaling (RGS) proteins are a diverse set of intracellular proteins that modulate G-
protein-coupled receptor (GPCR) signaling. As a subfamily of GTPase Accelerating
Proteins (GAPs), RGS proteins bind directly to G
-GTP, accelerate the rate of GTP
hydrolysis, and shorten the lifetime of activeG-proteins up to several 1000-fold. Thus,
RGS-G
a
interactions are central to the downstream regulation of GPCR signaling
events. Since GPCRs control numerous physiological processes in diverse tissues,
including brain, heart, liver, and lung, modulation of the RGS/G-protein interaction
has become an attractive target for drug discovery. The primary screen was a
a
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