Cellular target engagement and selectivity profiling of small molecules for mechanism-of-action studies

A high-quality chemical probe should possess the qualities of being highly specific, potent, and selective against off-target interactions, in order to enable target validation. By contrast, low-quality compounds, which display properties of promiscuity, reactivity and instability can in turn be misleading: such compounds, referred to as Pan-Assay Interference Compounds (PAINS) need to be removed from screening libraries. Target engagement and proteomic studies can often detect undesirable physicochemical properties or polypharmacology within small molecules, as well as help annotate and clean screening libraries and prevent compounds entering biology projects without precautions. Herein are presented a number of target engagement techniques used to annotate compounds. The introduction summarises currently available techniques and provides a literature review pertaining to the Cellular Thermal Shift Assay (CETSA). The second chapter describes how the use of chemoproteomic techniques, including CETSA, has led to the discovery of a number of undesirable characteristics of a literature compound SJ-172550. The third chapter delves into mass spectrometry (MS)-based CETSA techniques, and describes the development of an algorithm to analyse MS-CETSA output data for target validation, using both open-access datasets as well as the dataset obtained from the analysis of a known protein arginine methyltransferase 5 (PRMT5) inhibitor. The fourth chapter describes the use of Immunoblot-CETSA to investigate cellular target engagement of peroxisome proliferator-activated receptor γ (PPARγ)-targeting compounds in the context of downstream induction of the uncoupling protein, UCP1. Subsequently, the fifth chapter concerns the development of an AlphaScreen-based target engagement assay with a high-throughput capacity, and the necessary optimisation steps, and provides the link between AlphaScreen-CETSA and MS-CETSA. The sixth chapter pertains to the proteomic analysis of PPARγ-expressing cells, including an MS-CETSA experiment involving a known PPARγ agonist, rosiglitazone, to investigate its off-target interactions. Chapters 5 and 6 together point to the combined use of AlphaScreen-CETSA for hit detection and MS-CETSA for hit follow-up.