High-throughput profiling of PROTAC-induced protein degradation using mePROD quantitative proteomics

Targeted protein degradation (TPD) is an effective and potent strategy in drug discovery that involves the elimination of proteins of interest (POIs) using small-molecules as degraders. Proteolysis targeting chimeras (PROTACs) are a major class of such compounds that catalytically mediate the transfer of ubiquitin units from an E3 ligase complex to a protein of interest thereby marking the POI for subsequent proteasomal degradation. Since the major perturbation induced by the degraders is at the protein level, mass spectrometry-based quantitative proteomics serve as a one of the most direct methods of assessing their efficacy and selectivity. These methods are essential to illustrate the downstream effects of PROTACs, especially as they do not require prior knowledge of the specific target of the selected molecule. The disadvantage of these routine proteomics approaches, such as Label Free, Tandem Mass Tag or even SILAC, is that they provide insights into global proteome changes induced by degraders, without clear discrimination between primary and secondary targets. Moreover, such analyses are normally applied to a small subset of molecules, often when the final drug candidate has already been selected and partially optimized. In addition, the high dynamic rate of the changes at the proteome level, is often investigated to obtain details into the activity of PROTAC candidates but these approaches require long labelling times, which can be counterproductive in this context of understanding PROTACs impact on protein translation, modification and degradation. To address these limitations, we present the use of our multiplexed enhanced protein dynamics (mePROD) proteomics approach to obtain information on protein degradation and translation upon treatment with different PROTACs molecules. As a higher throughput approach would be beneficial to discriminate and confirm putative targets and to investigate the specificity of newly synthesized molecules, we have developed a pipeline to carry out the entire procedure in a 96-well format, limiting input and handling times to allow simultaneous proteome, degradome and translatome testing of over 20 PROTACs in replicates in a single plate. This comprehensive strategy promises to increase the throughput of analyzed small molecule degraders while providing a standardized method to generate large datasets for a more rigorous analysis of on- and off- targets effects of multiple known and unknown degraders, improving compound evaluation and optimization. By accelerating the discovery of therapeutic targets and deepening our understanding of protein dynamics in this cellular context, this approach is expected to facilitate the advancement of drug discovery and development of new PROTAC compounds.