Abstract

Protein denaturation is a key
process controlling protein activity. Certain mutations can de-stabilise
proteins making them more susceptible to denature due to factors such as an
increase in temperature, leading to an abnormal or non-functional protein.
Therefore small molecule correctors that stabilize the native structure of the
protein at high temperatures are of interest. Thermal shift assays are widely
used in hit-to-lead projects where the temperature at which proteins “melt” in
the presence and absence of test compounds can be determined but this format is
low throughput and not amenable to high throughput screening (HTS).  In
this project we have utilized Time-Resolved Fluorescence Energy Transfer
(TR-FRET) to monitor a temperature-induced conformational change in the protein
to enable a HTS to identify small molecule correctors of the target protein.
Interestingly when exploiting thermal stabilisation assay for the purpose of
HTS many challenges were encountered, particularly as the assay was scaled-up,
and needed to be overcome if throughput was to be achieved.

Here
we highlight Charles River’s approach to successfully execute a challenging
155,000 compound HTS campaign using a thermal stabilization as an assay readout
resulting in the identification of novel chemical matter for the target class
under investigation by our Partner