Rapid PROTAC Discovery Platform: Nanomole-Scale Array Synthesis and Direct Screening of Reaction Mixtures

Abstract

Precise length, shape, and linker attachment points are all integral components to designing efficacious proteolysis targeting chimeras (PROTACs). Due to the synthetic complexity of these heterobifunctional degraders and the difficulty of computational modeling to aid PROTAC design, the exploration of structure–activity relationships remains mostly empirical, which requires a significant investment of time and resources. To facilitate rapid hit finding, we developed capabilities for PROTAC parallel synthesis and purification by harnessing an array of preformed E3-ligand-linker intermediates. In the next iteration of this approach, we developed a rapid, nanomole-scale PROTAC synthesis methodology using amide coupling that enables direct screening of nonpurified reaction mixtures in cell-based degradation assays, as well as logD and EPSA measurements. This approach greatly expands and accelerates PROTAC SAR exploration (5 days instead of several weeks) as well as avoids laborious and solvent-demanding purification of the reaction mixtures, thus making it an economical and more sustainable methodology for PROTAC hit finding.

Innovative, combinatorial and high-throughput approaches to degrader synthesis

“…In 2023, AstraZeneca reported their D2B platform, 48 performing PROTAC synthesis in 384-well plates on 120 nmol scale, again using JQ1 as a scaffold to make BRD4-targeting PROTACs as a proof-of-concept. A three-tiered approach to PROTAC optimisation was taken for a given POI, starting with a systematic linker length exploration covering multiple E3 ligases, progressing to a more focused E3 ligase coverage, and finally a collection for bespoke singleton synthesis.…”

Breaking Bad Proteins—Discovery Approaches and the Road to Clinic for Degraders

“…This approach allows miniaturization and more rapid cycles of compound testing; however, care must be taken to assess non-specific impact of crude reagent mixtures on cell viability. Using a D2B approach, SAR around linkers, POI ligands, exit vectors and ligase recruiters can be rapidly explored to identify lead compounds for further refinement, and this strategy has been employed by Janssen, GSK and AstraZeneca in recent reports [148][149][150].…”