An Integrated Direct-to-Biology Platform for the Nanoscale Synthesis and Biological Evaluation of PROTACs

ABSTRACT: Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co opt the cell’s natural proteasomal degradation mechanisms to selectively tag and degrade undesired proteins. However, a challenge associated with PROTACs is the difficult optimisation required to identify new degraders, thus the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This ‘Direct-to-Biology’ (D2B) approach was validated, then exem-plified in a medicinal chemistry campaign to identify novel BRD4 PROTACs from a BRD4-binding scaffold previously unexplored for targeted protein degradation. Using the D2B platform, the synthesis of over 600 PROTACs was carried out in a 1536-well plate and subsequent biological evaluation of these candidates was performed by a single scientist in less than one month, to identify a set of picomolar BRD4 degraders. Due to its ability to hugely accelerate the optimisation of new degraders, we anticipate our platform to transform the synthesis and testing of PROTACs.