Role of a cellular thermal shift assay on evaluation of drug‐target engagement for compound selection

Abstract

AbstractBackgroundIdentification of small molecule‐target engagement is important for target‐based drug discovery. To support drug discovery efforts for Alzheimer’s Disease, we developed and implemented cellular thermal shift assays (CETSA) based on HiBiT/LgBiT reconstituted NanoLuc method (Promega). Advantages of the CETSA are testing target engagement in an intact cellular context, simple mix‐and‐read procedures, and high capacity to meet drug discovery project needs.MethodStable cell lines (HEK293t or HMC3) expressing HiBit (11 amino acids; Promega) tagged target were established. The CETSA uses Nano‐Glo HiBiT Lytic Detection kit (Promega) to detect abundance of undenatured HiBit‐tagged target protein in cells, while heat caused protein aggregates to be mostly undetectable. The CETSA procedures are 1) plating cells into 96‐well PCR plates; 2) treating cells with compounds for 60 min in 37 oC incubator; 3) heating cell plates for 3 min in a PCR machine; 4) processing with Nano‐Glo HiBiT Lytic Detection kit (10 min) and reading plates for luminescence. Before testing compounds, an Tagg (temperature at 50% maximal signal during aggregation) for each target was determined and used as baseline. When a compound engages the target, it caused Tagg shifts that can be quantified by the assay.Result1) Tagg is target specific, with INPP5D Tagg = 44.2 °C and PLCG2 Tagg = 45.9 °C. 2) Screening compounds at 100 µM on cells and heated at Tagg, the luminescent signal changes (compared to untreated control) suggesting compound‐target engagement. 3) The CETSA with compound dosing and heating at Tagg was used to confirm the compound engagement. 4) The HiBit tag control CETSA with compound dosing and Tagg heating was used to find non‐specific binders. The assay platform has been implemented for drug discovery efforts targeting INPP5D and PLCG2 respectively; example data will be shown.ConclusionThe CETSA platform is efficient, reproducible and has high capacity, making it well‐suited to target based drug discovery research. Application of the CETSA on projects targeting INPP5D, PLCG2 of microglia has facilitated compound selection based on confirming molecule target engagement, which allowed our project team to quickly focus on selected compounds for further studies.