A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

Abstract

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase

“…For instance, potency values are right-shifted when the heat duration is increased, as demonstrated for LDHA. A rightward shift in compound potency was also reported for a set of SMYD3 inhibitors when CETSA and cellular methylation assays were compared 21 . In the context of high-throughput screening, prolonged heating times are expected to reduce the capacity to detect compounds that bind weakly, which highlights the importance of empirically defining the optimal assay conditions for each target, while also taking into consideration the acceptable hit rate for the screening campaign.…”

“…Although we did not test the HiBiT system directly, we anticipate that HiBiT reagents will be well-suited for high-throughput CETSA. A CETSA assay platform based on the DiscoverX β-galactosidase enzyme fragment complementation technology was also recently reported by GlaxoSmithKline 21 . With this approach, target proteins are fused to the DiscoverX 42-amino acid ePL tag at the C-terminus and expressed in cells via BacMam transduction.…”

“…In the initial iterations of this system, compound binding was detected as a protein stabilization event under physiologic conditions, as demonstrated for MEK1 18 , PRMT3 19 , and BRD4 20 . More recently, the approach was adapted to a CETSA format to profile 123 SMYD3 inhibitors for thermal stabilization 21 . To date, a 1,536-well compatible CETSA assay able to support ultra-high-throughput drug screening campaigns has not been reported.…”

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells

“…The CETSA classic method has been demonstrated to work with reporter tagged proteins for example (i) using Enzyme Fragment Complementation (EFC) between two inactive b-galactosidase fragments (InCELL Hunter Target Engagement Assay, DiscoveRx) or (ii) using a Nanoluc-tagged protein (Promega). This approach has recently been utilized to develop a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA) based on BacMam titratable expression of ePL tagged full-length target proteins [ 20 ]. The possibility therefore exists to generate and detect proteins tagged with a fluorescent protein (e.g.…”

Applied Biophysical Methods in Fragment-Based Drug Discovery

“…15 More recently, intrinsic tryptophan fluorescence has made a return to Tm shift buffer screens, 16 and the thermal shift principle has also been adapted to a cell-based readout and the issue of target engagement. [17][18][19][20][21] The following section discusses Tm shift fragment screening monitored by Sypro Orange binding, but similar principles should apply to any fluorescent method that is used to monitor the thermal denaturation process.…”