Biomedical Engineering Reference
In-Depth Information
It is also worth noting that because ABC transporters have multiple binding sites,
different fluorescent substrates may identify different sets of inhibitors. We have
profiled more than 100 fluorescent substrates against
this trio of transporters
(publication in preparation).
4.4.12 TR-FRETHTS Assay for Inhibitors ofMEKK2-MEK5 PB1 Domain
Interactions: 1R03MH084830-01
This project developed as a singleplex TR-FRETassay [56] by K. Nakamura aimed to
identify small-molecule modulators of PB1 domain interactions between MEK5 and
MEKK2. It was reformulated at a triplex bead-based assay, which also included
MEKK3, and a MEKK2 mutant with a lysine to alanine mutation in the PB1 domain
that results in altered binding to MEK5 and a loss of downstream activation of ERK5.
For each target, the components of the multiplex consist of a streptavidin-functio-
nalized polystyrene bead, a biotinylated MEKK-fusion protein target, and a fluor-
escent peptide probe (AlexaFluor 488-GST-MEK5 WT). The similarly sized bead
sets (
5
m
m) are distinguished by emission at 750 nm with excitation at 635 nm.
Lessons Learned : Although the conversion of TR-FRETassay to a bead-based assay
was straightforward, the screen was plagued by a large number of compounds that
appeared to represent high background fluorescence. No unequivocally active
molecules have yet been identified.
4.4.13 HTS for DevelopingTCell ImmuneModulators (1X01MH085707-01)
(Pimary Cell-Based Assay)
The LFA-1 integrin plays important roles in T cell immunity. Along with its ligand
partner ICAM-1, this integrin is involved in the immune synapse that stabilizes the
interaction between na ı
ve T cells and antigen presenting cells (APCs), promoting T
cell proliferation and the generation of effector functions. The complex nature of
T/APC interaction (due to involvement of multiple receptor/ligand pairs), however,
makes it difficult to use APCs to study the role of individual receptor/ligand
interactions in T cell activation. The screening campaign currently underway is to
find small chemical compounds that modulate the LFA-1/ICAM-1 interaction. The
assay developed by I. Hwang [57] and coworkers detects the interaction of exosome-
like membrane vesicles (eMVs) that express murine ICAM-1, B7.1, and L d (trans-
genically engineered surrogate APC prepared from S2 drosophila cells) with murine
CD8 þ T lymphocytes isolated from 2C TCR transgenic mice. 2C TCR Tg T cells
actively absorb eMVs that carry the cognate QL9 antigen. The binding of eMVs to T
cells is detected by dual staining using anti-CD8 and anti-B7.1 monoclonal anti-
bodies. Small molecules of interest could act as inhibitors or potentiators of the
extracellular molecular interactions or intracellular pathways.
Lessons Learned : This assay is an example of the potential to use flow cytometry for
screening primary cells from transgenic animals. Considerations involve breeding
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