Methods to accelerate PROTAC drug discovery

Abstract

Proteolysis-targeting chimeras (PROTACs) represent a novel and promising modality for probing biological systems, elucidating pharmacological mechanisms, and identifying potential therapeutic leads. The field has made significant strides, as demonstrated by the growing number of PROTACs advancing to clinical trials. Despite this progress, the development of PROTACs faces significant challenges, which is partially due to the heterobivalent nature of this class of molecules. PROTACs must simultaneously bind to a protein of interest and an E3 ubiquitin ligase. This means PROTACs are significantly larger and more complex than conventional small molecules. This complexity impacts their design and synthesis, requiring strategic approaches to create libraries of PROTACs with various combinations of target ligands, linkers, and E3 ligase-recruiting elements. To fully realise the potential of this innovative technology, there is a need for novel approaches to accelerate the development of PROTACs. This review focuses on three critical areas to accelerate PROTAC development: appropriate target selection, modular chemical synthesis, and high-throughput biological evaluation. By appropriate selection of target proteins for degradation, optimizing synthesis methods to handle the complexity of PROTAC molecules, and employing robust high-throughput biological assays to evaluate PROTAC activity, researchers in academia and industry have streamlined the development and increased the success rate of PROTAC-based discovery programmes.

Keywords: DNA-encoded library; click chemistry; direct-to-biology; proteolysis-targeting chimeras; solid-phase synthesis.

Figure 1:. Schematic representation of PROTAC mechanism of action.

The PROTAC engages both the E3 ligase and the POI in a ternary complex. This allows an E2 conjugating enzyme to polyubiquitinate the POI, which labels the POI for proteasomal degradation. The PROTAC is released and recycled.

Figure 2:. Simulated characterisation of PROTAC efficacy.

The curve shows POI degradation as a function of PROTAC concentration. DC₅₀ (orange) and D_max (green) values are shown with respective relationship to the corresponding datapoints on the curve. The Hook effect is highlighted in purple, where, at higher PROTAC concentrations, the binary complexes of POI–PROTAC and PROTAC–E3 compete with ternary complex formation.

Figure 3:. Schematic of a tTPD system.

(a) Representative structure of a tTPD. (b) Steps in the mechanism of a tTPD system. The respective proteins are labelled and ubiquitin is shown as orange spheres.

Figure 4:. Chemical structures of E3RE and POI ligands discussed in the text.

Arrows represent vectors where the ligands can be elaborated without loss of binding to their respective protein targets.

Figure 5:. Comparison between click reactions and solid-phase organic synthesis.

(A) Examples of some of the click chemistry reactions that have been used in PROTAC synthesis. (B) Schematic of the steps involved in solid phase organic synthesis. (C) Comparison of the two approaches.

Figure 6:. DEL workflows for PROTAC identification.

(a) & (b) VHL DEL libraries showing position of DNA tag; (c) example connectors from both VHL-based PROTAC DELs; (d) VHL DEL screening cascade; (e) CRBN DEL and control DEL without CRBN recruiting ligand, featuring trifunctional linker with attached DNA tag; (f) CRBN DEL screening cascade.

Figure 7:. Depiction of D2B workflows vs. conventional design, synthesis and test cycles, comparing degrees of throughput.

Figure 8:. Chemical synthesis of D2B libraries.

(a) RAPID-Tac acyl hydrazone synthesis. (b) Second-generation RAPID-Tac phthalimidine synthesis. (c) Telescoped three-step amide synthesis employed by Janssen. (d) One-step amide synthesis employed by GSK and AstraZeneca.