Abstract

In collaboration with our Client we have developed a
Fluorescence Polarisation (FP) assay using BellBrook Labs Transcreener® UDP²
Assay kit in both endpoint and kinetic read formats for high throughput screen
(“HTS”) purposes to identify small molecule inhibitors of our Target. An
orthogonal biochemical colorimetric assay was also developed by our Client for
screening purposes. We assessed both formats in a pilot screen and the FP
format was selected based on robustness for HTS purposes, with the other format
being used as an orthogonal assay in follow up potency studies. The FP assay
was used to screen approximately 300,000 Discovery UK library compounds
comprising of the entire Discovery UK Lead-Like Library of approximately
155,500 compounds together with 150,000 compounds selected by the Client using
a plate-based selection from the Discovery UK Diversity Library of 625,000
compounds.

The Biocel fully automated platform was employed to enable
the HTS assays to be run in kinetic mode. Performing the assays in kinetic mode
enabled analysis of the compound effect by both calculation of slope and
endpoint readings. A kinetic read has a significant advantage over an endpoint
reading, as is common in HTS programs, in terms of identifying false positives
and interfering compounds. A comparison of kinetic slope analysis and endpoint
readings will be discussed highlighting common pitfalls in assessing the
activity of compounds.