Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge.

High-Throughput Detection of Ligand-Protein Binding Using a SplitLuc Cellular Thermal Shift Assay.

The confirmation of a small molecule binding to a protein target can be challenging when switching from biochemical assays to physiologically relevant cellular models. The cellular thermal shift assay (CETSA) is an approach to validate ligand-protein binding in a cellular environment by examining a protein's melting profile which can shift to a higher or lower temperature when bound by a small molecule. Traditional CETSA uses SDS-PAGE and Western blotting to quantify protein levels, a process that is both time consuming and low-throughput when screening multiple compounds and concentrations.

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

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA).

Acquired platelet dysfunction may be an aetiologic factor in Heyde's syndrome--normalization of bleeding time after aortic valve replacement.

Background: There is a well-documented relationship, with unknown aetiology, between aortic valve stenosis and occult gastrointestinal bleeding in elderly patients. Despite several studies attempting to determine the prevalence and to discuss the aetiology, there are still many unanswered questions.

Methods: A total of 288 consecutive patients with valvular aortic stenosis--mean age 73 +/- 9 years aortic stenosis group (ASG)--were compared with 129 pacemaker-treated patients, mean age 73 +/- 9 years control group (CG).