Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase

Abstract

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.

Proteolysis‐targeting chimeras in drug development: A safety perspective

“…The hook effect is an intrinsic property of any PROTAC, and the underlying mechanisms in terms of ternary complex formation is generally understood (Douglass, Miller, Sparer, Shapiro, & Spiegel, ). However, while ternary complex formation is an essential step for PROTAC‐mediated protein degradation, this is not sufficient to induce degradation (Smith et al, ). Consequently, the onset of the hook effect observed from binding experiments may be very different from what is observed in cell‐based degradation assays.…”

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

“…VHL ligands have several known functionalization sites based on their prior incorporation into bivalent degraders providing multiple possible exit vectors. Importantly, the exit vector chosen can have a large impact on ternary complex formation (Cromm and Crews, 2017;Chan et al, 2018;Smith et al, 2019).…”

PRosettaC: Rosetta based modeling of PROTAC mediated ternary complexes

“…PROTACs offer several advantages over traditional small molecule inhibitors, including: complete inhibition of function, not limited to enzymatic function, or a specific site on the target, long duration-of-action that is proportional to the protein turn-over rate, enhanced selectivity [4][5][6][7] and the ability to work at sub-stoichiometric concentration 8 , since a single PROTAC molecule can degrade multiple copies of the target. These advantages propelled wide interest in their development as chemical tools and potential drugs.…”