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

01 September 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

Direct-to-biology
High-throughput chemistry
Nanoscale
Cell assay
Targeted-Protein Degraders
PROTACs
Automation
Library synthesis
Parallel Chemistry

Supplementary materials

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Supporting Information
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Supporting information containing materials and methods, general procedures, characterisation, proteomic studies and supporting figures.
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