Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras

Abstract

Selective degradation of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC molecules incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional molecules require significant linker optimization and possess high molecular weight, which can limit cellular permeation, solubility, and other drug-like properties. We show here that the hetero-bifunctional molecule can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide derivative which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncology targets, BRD4 and ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degradation.

Targeted Protein Degradation: from Chemical Biology to Drug Discovery

“…Its composition impacts cell permeability and an optimized linker length is essential for efficient target ubiquitination. Intracellular PROTAC assembly might offer some advantages in this respect, but a more drug-like character of the components rand higher activities are needed to pursue this idea further (Lebraud et al, 2016). …”

Chemically induced degradation of CDK9 by a proteolysis targeting chimera (PROTAC)

“…23–28 This broad based interest is the direct result of the seminal work by the Bradner lab on the development of bromodomain and extra-terminal domain (BET) degrader. 23 PROTACs are heterofunctional bispecific molecules connected via a linker wherein one fragment interacts with the protein of interest and the other binds to a component of a ubiquitin ligase.…”

“…This results in the poly-ubiquitination of that target protein, which is then proteosomally degraded (Figure 1). 22–24, 26, 27, 29–37 FKBP12, 23 BET, 23, 26, 28 BCR-ABL, 34 and Sirt2 37 proteins have been successfully targeted for degradation using small molecule bifunctional degraders that bind cereblon/Cullin4A E3 ubiquitin ligase. Degradation of the target protein makes this a catalytic process.…”

PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery

“…Recently, Tom D. Heightman's group developed an advanced PROTAC technology named CLIPTAC, in‐cell click‐formed proteolysis‐targeting chimeras, which are actually CRBN‐based PROTACs formed by rapid reaction of a tetrazine‐tagged thalidomide derivative (Tz‐thalidomide) with a trans‐cyclo‐octene (TCO)‐tagged ligand in cells. This CLIPTAC can simultaneously recruit target proteins and the E3 ligase CRBN and promote the proteasomal degradation of the target protein (Figure ) . Notably, the two individual small precursor molecules, which are able to self‐assemble to form a functional PROTAC in cells, have lower molecular weights and better penetrability than previous PROTACs.…”