High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

TLDR

Ligand binding stabilizes proteins through energetic coupling of ligand‑binding and protein‑melting reactions, a well‑understood phenomenon. The authors aimed to create a universally applicable, fully automated, miniaturized fluorescence‑based thermal shift assay for high‑throughput drug screening. They implemented automated microplate instrumentation and validated it on over 100 protein targets by measuring ligand‑induced thermal shifts. The assay proved broadly applicable, eliminating the need for target‑specific assay design and enabling quantitative screening of genomics‑derived targets without prior functional knowledge.

Abstract

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.