HTS FLOW CYTOMETRY, SMALL-MOLECULE DISCOVERY, AND THE NIH MOLECULAR LIBRARIES INITIATIVE - Flow Cytometry in Drug Discovery and Development - page 80

reformatting of the assay to 384-well plates created a challenge with respect to cell
number due to surface to volume ratio of 384-well plates as compared to the 96-well
format in which the assay was submitted. We received approval to perform a virtual
screen to cherry pick from the MLSMR. Since the Center at Emory has previously
screened another ER assay, we used our GPR30 assay to test for relevant molecules
identified in their PubChem uploads as well as other molecules determined by virtual
screening to have similarity with G1. As we had previously reported on a GPR30-
specific antagonist, the screen along with synthetic chemistry has also generated an
antagonist (G15) [44].

Lessons Learned : We recognized early in the initiative that HT flow cytometry was
not well suited to targets that depended on adherent cells and cell permeabilization
when screening a large library was required. To mediate this difficulty, a virtual
screening approach was taken to search for estradiol analogues in the library followed
by screening a small library subset.

4.4.5 Assay for Allosteric Ligands for the VLA-4 Integrin:
X01MH077638-01 (Suspension Cell Singleplex)

Allosteric regulators have proven to be important tools in the regulation of integrin
function. This assay was intended to detect direct binding inhibitors as well as
allosteric regulators. The assay was based on the binding of a fluorescent peptide to
VLA-4 in its resting state or modulated to high affinity by the divalent cation Mn 2 þ .
The signal to background of the assay was 2/1 for the resting state and 4/1 for the high-
affinity state. The screen detected only direct inhibitors with scaffolds similar to
families of already known inhibitors [45, 46]. A novel secondary assay was developed
to help classify the activity of the competitor molecules. This assay uses an antibody
HUTS-241 that is sensitive to VLA-4 conformation. The novel ligand-induced
binding site (LIBS) mAb assay is a result of the interplay between R01HL081062
that has taken advantage of compounds identified through the X01MH077638 that
screened a portion of the MLSMR. A Fast Track proposal for screening the entire
MLSMR is using the improved assay developed to search for novel integrin ligands
that block the binding of traditional ligands and do not in themselves induce the LIBS
mAb binding site.

Lessons Learned : In this assay, we attempted to read each plate twice, once before
and once after the addition of divalent cation. This turned out to be difficult and we
wound up preparing each plate separately.

4.4.6 Small-Molecule Inhibition of Staphylococcus aureus Virulence:

1X01MH078952-01 (Suspension Cell Singleplex)

The bacterial virulence assay contributed by P. Gresham is a cell-based assay that
measures the ability of test compounds to inactivate the quorum sensing pathway of S.
aureus, a pathway responsible for the production of bacterial virulence-associated