Current strategies for the design of PROTAC linkers: a critical review.

Robert I Troup,Matthias G J Baud,Charlene Fallan

doi:10.37349/etat.2020.00018

Abstract

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands; an “anchor” to bind to an E3 ubiquitin ligase and a “warhead” to bind to a protein of interest, connected by a chemical linker. Targeted protein degradation by PROTACs has emerged as a new modality for the knock down of a range of proteins, with the first agents now reaching clinical evaluation. It has become increasingly clear that the length and composition of the linker play critical roles on the physicochemical properties and bioactivity of PROTACs. While linker design has historically received limited attention, the PROTAC field is evolving rapidly and currently undergoing an important shift from synthetically tractable alkyl and polyethylene glycol to more sophisticated functional linkers. This promises to unlock a wealth of novel PROTAC agents with enhanced bioactivity for therapeutic intervention. Here, the authors provide a timely overview of the diverse linker classes in the published literature, along with their underlying design principles and overall influence on the properties and bioactivity of the associated PROTACs. Finally, the authors provide a critical analysis of current strategies for PROTAC assembly. The authors highlight important limitations associated with the traditional “trial and error” approach around linker design and selection, and suggest potential future avenues to further inform rational linker design and accelerate the identification of optimised PROTACs. In particular, the authors believe that advances in computational and structural methods will play an essential role to gain a better understanding of the structure and dynamics of PROTAC ternary complexes, and will be essential to address the current gaps in knowledge associated with PROTAC design.

Highlights

General considerationsProteolysis targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands connected by a linker [1,2,3,4,5]
The authors believe that advances in computational and structural methods will play an essential role to gain a better understanding of the structure and dynamics of proteolysis targeting chimeras photostationary states (PSS) (PROTAC) ternary complexes, and will be essential to address the current gaps in knowledge associated with PROTAC design
Docking of analogous and equipotent (DC50 = 3.8 nM in MM.1S cells) PROTAC NP8 (100) [145], which possessed an alternate linker conjugation site, highlighted a different set of interactions stabilising the HDAC6-NP8-CRBN ternary complex (TC) (Figure 21): a H-bond interaction between the triazole moiety of 100 and Lys157 of CRBN; a H-bond between the urea group in 100 and Ser531 in HDAC6; and a different assortment of protein-protein interactions (PPIs). These results demonstrated that PROTACs with different linker conjugation sites could still form productive TCs stabilised by different interactions, and achieve equivalent degradation potency

Summary

Introduction

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands connected by a linker [1,2,3,4,5]. CRBN PROTACs (60) containing intermediate length linkers (1-2 PEG units) showed reduced BRD4 degradation potency (DC50 >5 μM) in H661 cancer cells compared to those with shorter and longer linkers (0, 4-5 PEG units, < 0.5 μM) This unexpected pattern was not replicated in the VHL series (61), in which potency decreased as linker length increased, and further highlights the crucial requirement to optimise linker length for each ligand pair when designing PROTACs. Triazole click chemistry has been used for the combinatorial PROTAC synthesis and rapid identification of anchorlinker-warhead combinations displaying optimal degradation efficiency. Zhao et al [88], generated a series of potential PARP1 degraders by conjugating the same acid and azide functionalised linker intermediate (62) to either a niraparib (63) or olaparib (64) derived warhead and a ligand for VHL, CRBN or MDM2 Their linker contained an amine linked to the anchor through amide bond formation or SNAr, and an azide that could be coupled to the alkyne moieties in 63 or 64 through click chemistry (Figure 11). By using crystal structures of their PROTACs in TC formation, the authors were able to develop a potent SMARCA2/4 degrader in only three design iterations

Computational methods

Findings

Conclusion and outlook