Abstract

Developing small molecule
inhibitors requires a robust screening cascade which can efficiently identify compounds
which engage the target protein, determine selectivity over undesirable related
proteins and demonstrate activity in downstream functional assays.

Here we present data on the
screening cascade we have successfully developed for a client project to
identify small molecule inhibitors of a DNA damage response protein. Utilising
the Promega NanoBRET™ Target Engagement Assay has enabled us to distinguish
molecules which bind to the target protein in a cellular context using full
length protein constructs whilst also showcasing differences in affinity to a
related but off-target protein. CellTiter-Glo® has been effectively used to
identify compound-induced toxicity and establish effective non-toxic
concentration ranges for downstream functional assays. To demonstrate on-target
activity we developed two plate-based flow cytometry assays to interrogate the
mechanism of action of the protein of interest. A TUNEL assay examines the
ability of test compounds to inhibit DNA single strand break formation whilst
hits are confirmed in an orthogonal cell cycle assay using Bromodeoxyuridine (BrdU)
and 7-Aminoactinomycin D (7-AAD) staining to categorise the position of cells
within the cell cycle. Overall, this has resulted in a robust screening cascade
which has been effectively utilised to screen test compounds and identify
inhibitors of the protein target.