Category: Drug Target Strategies

1279-E - Cellular thermal shift assays in high throughput: a 1536-well cellular target engagement assay for drug discovery

Wednesday, February 7, 2018
11:30 AM - 12:30 PM

                Thermal shift assays (TSA) reveal changes in protein structure upon binding to small molecules due to a resultant change in the thermal melting temperature of the protein. In drug discovery, this assay can reveal drug binding to its protein target, or “target engagement”. Experimentally, this change in melt temperature can be measured by exposure of the protein to a temperature gradient, followed by quantification of the protein level or activity at each temperature. Originally, protein thermal shift experiments were performed with purified protein samples, but in the last few years, the TSA was reported in a cellular context, and the cellular thermal shift assay (CETSA) was born. We have combined CETSA with a high throughput protein detection method, to increase the throughput of the assay, since traditional protein detection methods such as western blots are low throughput. To develop high throughput 1536-well CETSA, we used a protein reporter system in a homogeneous (additions only, no washing) assay format. We recently published on this assay in 384-well format, and described its application to small molecule compound dose response curves [McNulty et al, SLAS Discovery 2018, 23(1)34-46)]. Those experiments showed the successful use of a cellular reporter system to generate small molecule compound-target engagement potency values in drug discovery. To further develop this system, we have successfully increased the throughput of the assay, and moved from 384-well plates into 1536-well plates.  Assay parameters tested during optimization of assays included the expression level of the target protein, and the amount of detection reagents added after thermal melting. Flexible and precise control over the expression level of the tagged target in cells was achieved using the BacMam transient expression system. Innovation in equipment and methods used for thermal melting contributed to our ability to successfully translate this technology into an assay format that can be used for high throughput screening. Assay development results, and assay statistics in 1536-well format will be presented.

Lorena A. Kallal

Collegeville, PA

Lorena Kallal, PhD. Manager, and Technologies and Platforms Group Leader, GlaxoSmithKline.

Drug discovery scientist, with 18 years of experience in the pharmaceutical industry. I have a passion for applying biology and phenotypic approaches to drug discovery. I seek new technologies that can accelerate medicine discovery. My leadership style is to focus on the science and create active and engaged teams that accomplish what we thought might not be possible.