Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective

Abstract

High-throughput screening (HTS) is a proven method for discovering new lead matter for drug discovery and chemical biology. To maximize the likelihood of identifying genuine binders to a molecular target, and avoid wasting resources following up compounds with unproductive/nonspecific mechanisms of action, it is important to employ a range of assays during an HTS campaign that build confidence of target engagement for hit compounds. Biophysical methods that measure direct target/compound engagement have established themselves as key techniques in generating this confidence, and they are now integral to the latter stages of HTS triage at the European Lead Factory (ELF). One relatively new technique that the ELF is using is microscale thermophoresis (MST), which measures the differences in rate of movement through a temperature gradient that are caused when single molecular species form complexes. Here we provide an overview of the MST assay development workflow that the ELF employs and a perspective of our experience to date of using MST to triage the output of HTS campaigns and how it compares and contrasts with the use of other biophysical techniques.

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

“…Only three compounds returned a pEC 50 > 5 with an acceptable Hill slope. As such all compounds that demonstrated > 20% effect were selected for testing using microscale thermophoresis (MST; as described in Section 2) as an orthogonal biophysical assay to confirm target engagement [26,27,28]. The three compounds that returned a measurable pEC 50 through the competition-binding assay, plus compounds which appeared to bind in the MST experiment and their structural analogues, were combined to give a preliminary hit list of 31 compounds.…”

Mechanism of Action of VP1-001 in cryAB(R120G)-Associated and Age-Related Cataracts

“…Next, we used an orthogonal method, MST, to verify this result using cryAB oligomers. MST relies on tracking the movement of a fluorescently tagged protein through a thermal gradient 39,40. We engineered a unique cysteine residue (E87C) into cryAB ACD and selectively labeled it with a cysteine-reactive Alexa Fluor dye to generate AF488-cryAB(E87C) ACD.…”

Creating and screening natural product libraries

“…165,166 MST is an emerging technology that measures the transit of a uorescent analyte across a temperature gradient. 153,154,167 The interaction of a ligand with the analyte will slow the migration time across this gradient, generating a signal indicative of a binding event. Currently a 96 well MST instrument is commercially available which uses capillaries to sample all wells across the plate.…”

“…Unfortunately, measuring a wellcontrolled gradient requires approximately 20 seconds per sample, limiting its utility. 167 An interesting hybrid assay format is micro/nanocapillary electrophoresis (CE), which continues to be an HTS format that is frequently employed for drug discovery. 168 Improvements in the optics of both excitation and emission using LIF has allowed increasingly small reaction volumes to be deployed in the context of an HTS screening campaign.…”