Impact of linker length on the activity of PROTACs

Abstract

Conventional genetic approaches have provided a powerful tool in the study of proteins. However, these techniques often preclude selective manipulation of temporal and spatial protein functions, which is crucial for the investigation of dynamic cellular processes. To overcome these limitations, a small molecule-based novel technology termed “PROteolysis TArgeting ChimeraS (PROTACs)” has been developed, targeting proteins for degradation at the post-translational level. Despite the promising potential of PROTACs to serve as molecular probes of complex signaling pathways, their design has not been generalized for broad application. Here, we present the first generalized approach for PROTAC design by fine-tuning the distance between the two participating partner proteins, the E3 ubiquitin ligase and the target protein. As such, we took a chemical approach to create estrogen receptor (ER)-α targeting PROTACs with varying linker lengths and the loss of the ER in cultured cells was monitored via western blot and fluorometric analyses. We found a significant effect of chain length on PROTAC efficacy, and in this case, the optimum distance between the E3 recognition motif and the ligand was a 16 atom chain length. The information gathered from this experiment may offer a generalizable PROTAC design strategy to further the expansion of the PROTAC toolbox, opening new possibilities for the broad application of the PROTAC strategy in the study of multiple signaling pathways.

Targeted Protein Degradation: from Chemical Biology to Drug Discovery

“…Therefore, it is important to test a variety of warheads (if applicable), linkers and E3s when optimizing a specific PROTAC. In particular, the linker connecting the two functional moieties has a significant influence on PROTAC activity (Cyrus et al, 2011; Buckley et al, 2015). Its composition impacts cell permeability and an optimized linker length is essential for efficient target ubiquitination.…”

Small‐Molecule PROTACS: New Approaches to Protein Degradation

“…[40] While both PROTACS induce degradation of BRD4 within 24 h, MZ1, which possesses as horter PEG linker, exhibited higher efficacy.T his finding demonstrates once again that linker composition and length are crucial variables in determining the potency of PROTACs. [41] Interestingly, only partial degradation of BRD2 and BRD3 was observed after 24 hw ith these compounds, [40] in contrast with the cereblon-based BRD4-targeting PROTACsA RV-825 [34] and dBET1. [36] These conflicting results raise the question of whether recruiting different E3 ligases can lead to different selectivity profiles.However,even though MZ1 (90 %BRD4 reduction at 100 nm)isless potent than ARV-825 in decreasing BRD4 levels,i ti sc learly as potent as dBET1.…”

Degradation of Polycomb Repressive Complex 2 with an EED-targeted Bivalent Chemical Degrader

“…Functionalization off of the terminal amine of VH032 has been extensively used in the design of bivalent degraders so we chose this position for linker appendage (Frost et al, 2016;Chan et al, 2018;Girardini et al, 2019). To connect the two ligands, different length alkyl (UNC6851-UNC6853) and PEG linkers (UNC6845-UNC6847) were incorporated to assess the distance required to induce successful EED degradation upon formation of the EED-degrader-VHL ternary complex (Table 1) (Cyrus et al, 2011). To enable this approach, we synthesized a carboxylic acid functionalized EED ligand via a Suzuki-Miyaura reaction with (4-(methoxycarbonyl)phenyl)boronic acid and 8-bromo-N-(furan-2-ylmethyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine (1) and subsequent basic hydrolysis to yield (2) (Scheme S1).…”