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When chimeras meet science: promises from targeted protein degradation

PROteolysis TArgeting Chimeras (PROTACs) are small two-headed (heterobifunctional) molecules that hijack the cell’s protein degradation machinery to irreversibly destroy the target. Each PROTAC consists of a linker with a ligand on one end to confer target specificity, and a different ligand on the other end to engage the cell’s ubiquitin system. The resulting polyubiquitin chain flags the target protein for degradation by the proteasome.

PROTAC technology is emerging as a new therapeutic method to treat diseases such as cancer or neurodegenerative disorders caused by the aberrant expression of a pathogenic protein. While traditional drugs can target only around 20% of the proteome, this new technology could reach the other 80% which is currently undruggable in terms of conventional tools, such as inhibitors and agonist/antagonists.

PROTAC mechanism. Each heterobifunctional PROTAC molecule targets a protein of interest (POI), serving as a bridge to bring it into close proximity with an E3 ligase. The resulting ternary complex catalyzes polyubiquitination of the target protein, tagging it for degradation by the proteasome.

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Targeted Protein Degradation, the next disruption in drug discovery

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Blog post
Blog post

Unlock new parts of the proteome with PROTAC, a promising emerging technology

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PerkinElmer has made available a range of documents with educational content (Webinar, Guide, Literature review, Application Note, and Blog post), and reagents dedicated to PROTAC to help you in your research.

Does PROTAC bind to Targeted Protein ?

To induce protein degradation in cells, a PROTAC must pass through the degradation pathway. To do so, PROTACs assembled by linking two small-molecule ligands via a linker unit must overcome the first three barriers, which are chemical stability, solubility, and membrane permeability, in order to enter cells. Several structural modification strategies have been applied to improve the physicochemical properties of PROTACs. It has been shown that basic nitrogen-containing groups, such as a pyridinyl or piperazinyl group, were empirically introduced into the linker to increase the solubility of the PROTACs. At the same time, an amide bond was avoided in an attempt to improve permeability.
 
Once inside the cell, the degrader must be able to engage either with the target protein to form the corresponding binary complex or with the specific E3 ligase, followed by the involvement of the third binding partner to form the ternary complex. The poly-ubiquitinated target protein would then be recognized and recruited by proteasomes, resulting in the degradation of the target protein.
 
Many target proteins have been successfully degraded by PROTAC, including nuclear receptors, protein kinases, epigenetic regulators, proteins related to neurodegenerative diseases, regulatory proteins, anti-apoptotic proteins, virus-related proteins, and transcription factors.
 
The discovery of PROTAC remains a very empirical process. Therefore, it is essential to quickly and efficiently characterize and classify these difficult-to-obtain compounds and provide accurate, valuable information to guide researchers in compound optimization.

PROTAC pathways to degrade target proteins: from cell entry to target degradation. PROTAC: existing compound amenable to PROTAC derivation (Nature, length, solubility, selectivity) ; POI: Proteins of Interest (kinase, receptor, transcription factors, etc.) ; E3 ligase: Cereblon, VHL, MDM2

Principles of Targeted Protein detection

If the targeted protein is a kinase, assess the binding properties through PerkinElmer’s biochemical offer: Kinase-6HIS Binding Discovery Kit, Kinase-GST Binding Discovery Kit, Kinase-Biotin Binding Discovery Kit.

Find more information and data on the dedicated Guide – Kinase Binding Assay

Assessment of PROTAC binding toward kinase with HTRF Kinase Binding Assay – Screening and pharmacological characterization of PROTAC compounds

On-demand webinar
Webinar

Targeting Proteins and Proteolysis to counter Cancer.

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Application Note
Application Note

Improve PROTAC Targeting Kinase Discovery

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Alpha CETSA® (Cellular Thermal Shift assays, from Pelago Bioscience) kits provide simple & rapid solutions to assess PROTAC compound permeability and bind to the targeted endogenous protein in various cellular models.

Alpha CETSA® assays enable you to monitor the binding of your compounds of interest in the natural living cellular environment, confirming whether your compounds interact with your target of interest or not. The ability to work in an unbiased and more physiologically relevant way means no molecular engineering is required, allows you to choose disease-relevant cell models, and overcomes the limitations of biochemical assays.  

CETSA® gives a quantitative measurement of target engagement by assessing the binding of the compounds to the target, which causes thermal stability upon heating. A thermal melting curve is prepared using known positive and negative controls which identify a certain temperature set point where a compound-stabilized protein target stays in the supernatant, and non-compound binding leads to protein aggregation/precipitation. The compound-stabilized protein in solution is then detected by the well-established Alpha technology.

Alpha CETSA®️ Assay Principle – Principle of the CETSA®️ melting curve and thermal stabilization by compound binding used to assess target engagement early in the drug discovery process, and Alpha assay use for detection

Does PROTAC bind to E3 Ligase ?

The Ubiquitin Proteasome System (UPS) is central to most biological processes, such as apoptosis, mitophagy, inflammatory pathways, cell cycle, etc. In particular, because of their crucial role in the controlled degradation of the regulatory proteins in these processes, essential for proper cell functioning, Ubiquitin E3 ligases are the most diverse proteins. They are the main component of the UPS, which is the last component of an enzymatic cascade that includes a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2). E3 ligases selectively modify proteins by covalently attaching ubiquitin to the lysine, serine, threonine, or cysteine residues of the corresponding E3 substrate. The consequence of protein ubiquitination is ATP-dependent degradation of polyubiquitinated proteins via the 26S proteasome, a very large protease complex that breaks down ubiquitinated proteins into small peptides.

The role of Ubiquitin E3 ligases such as Cereblon, VHL, MDM2, or cIAP in the degradation of a protein of interest.

Principles of the E3 ligase detection

PerkinElmer offers a range of AlphaLISA®️ protein-protein interaction reagents and HTRF®️ protein-protein interaction reagents, as well as AlphaLISA® E3 ligase binding kits and HTRF® E3 ligase binding kits, ensuring a match for your research needs whose operating principles are described below.

Assessment of MT802 binding toward Cereblon with AlphaLISA®️ and HTRF®️ Cereblon binding assay – (PROTAC: MT802; E3 ligand : Pomalidomide)

Is a Ternary complex formed ?

Inside the cell, a PROTAC engages with either the specific E3 ligase or the target protein to form the corresponding binary complex, followed by the involvement of the third binding partner to form the ternary complex. Several properties of the ternary complex determine whether the ubiquitins can be covalently connected to one or more lysine, serine, threonine, or cysteine residue(s) of the target protein. In particular, a stable ternary complex between E3 and a potential substrate is required for degradation.
 
There is increasing evidence that the formation of ternary complexes, the stability of these complexes, and the cooperativity of protein-protein interactions (PPIs) are more predictive of the degradation activity of a PROTAC than its binary interactions.
 
Several different assays can characterize a ternary complex to profile the formation, population, stability, binding affinities, cooperativity, or kinetics of ternary complexes.

To characterize ternary complexes formed between a target protein, a PROTAC degrader, and an E3 ligase, the detection of ternary complex formation is one of the most practical applications of ALPHA and HTRF for PROTAC degraders. By titrating a PROTAC degrader with its target protein and E3 ligase, a bell-shaped curve can be produced by plotting ALPHA and HTRF signals against PROTAC concentrations. The height of the bell-shaped curve reflects the relative population of the ternary complex, enabling scientists to classify PROTAC degraders based on their ability to form ternary complexes. In addition to the ternary complex formation, ALPHA and HTRF can also be repurposed to determine the affinity and cooperativity of these complexes.

Principles of Ternary Complex detection

PerkinElmer offers a range of AlphaLISA®️ protein-protein interaction reagents and HTRF®️ protein-protein interaction reagents,

AlphaLISA and HTRF assays showing increased ternary complex formation with the height of the bell-shaped curve

Literature Review
Lit Review

Novel drugs for cancer therapy development with PROTAC®

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What does cooperativity effect?

In the context of a PROTAC (A-B-C) ternary complex, the binding affinity of PROTAC “B” to a protein partner “C” (binary binding) can be enhanced or reduced by the presence of the second protein partner “A” (ternary binding). This effect can be quantified in terms of the “cooperativity” factor (α), defined as the ratio of the binary and ternary dissociation constants for PROTAC binding to C (α = KDbinary/KDternary). Cooperativity can be described as “positive” (α > 1, increased ternary binding affinity relative to the binary, further stabilizing the complex), “negative” (α < 1, reduced ternary binding affinity relative to the binary, destabilizing the complex), or "noncooperative" (α = 1, no change in binding affinity for C due to the presence of A). Although functional degraders can be generated in the absence of positive cooperativity, numerous studies suggest that improving the cooperativity and stability of ternary complexes may be an effective strategy in PROTAC design.

Define the Ki of PROTAC ligand with increasing concentrations of BTK

Does PROTAC degrade the targeted protein ?

Protein degradation, also known as proteolysis, is a process that results in the hydrolysis of one or more peptide bonds in a protein. The ubiquitin-proteasome pathway (UPP) is one of the significant pathways that mediate the degradation of intracellular proteins.
The poly-ubiquitinated target protein would then be recognized and recruited by proteasomes, resulting in the degradation of the target protein. When the degradation of a target protein is faster than its expression, the net intracellular level of the target protein decreases, resulting in a downstream pharmacological effect.
 
Given the unpredictable nature of PROTAC degraders in inducing target protein degradation, it is crucial to introduce reliable degradation assays to ultimately assess whether the designed PROTAC is well bound to the target and whether it is a good target protein degrader.

Detection principles of Protein degradation

Unlike traditional technologies, PerkinElmer offers a wide range with the AlphaLISA SureFire® Ultra™️ protein kits, Alpha CETSA® kits, HTRF® total protein kits, and HTRF® total cereblon kit, ensuring selectivity, sensitivity, and reproducibility for your research; whose operating principles are described below.

Assessment of MT802 induced-BTK degradation in cells using HTRF or AlphaLISA SureFire total BTK assays

Guide
Guide

Insight into ubiquitin role and protein degradation

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On-demand webinar
Webinar

Targeting Proteins and Proteolysis to counter Cancer.

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What does the future hold for Targeted Protein Degradation ?

Given the unpredictable nature of PROTAC degraders in inducing target protein degradation, it is crucial to introduce reliable degradation assays to ultimately assess whether the designed PROTAC is well bound to the target and whether it is a good target protein degrader.
Targeted protein degradation is a promising new therapeutic modality in drug discovery. By coopting protein degradation pathways, targeted protein degradation facilitates the complete removal of protein molecules inside or outside the cell. While PROTAC technology hijacks the ubiquitin-proteasome system, proteasomes alone are ineffective at degrading specific large proteins or aggregates; newer modalities coopt autophagy or the endo-lysosomal pathway. With these mechanisms, targeted protein degradation is expected to expand the therapeutic space beyond small molecule inhibitors.

ATTEC

ATTEC is a binding compound capable of interacting with both the pathogen protein and the LC3 phagophore protein. It can aim the former at phagophores for autophagic degradation, based on the fact that autophagic protein substrates are engulfed in the double-membrane phagophores where lipidated LC3 proteins are associated.

AUTAC

The autophagy-targeted chimera (AUTAC) is the first degrader using the autophagy mechanism. A typical AUTAC consists of a small unit that binds to a substrate and a guanine derivative (degradation tag) connected by a flexible linker. AUTACs can penetrate cell membranes, and have been successfully used to degrade several cytosolic proteins. Furthermore, the mitochondria-targeting AUTAC promotes mitophagy of small fragmented mitochondria. Induction of mitophagy by AUTAC is mediated by ubiquitin but does not require Parkin or PINK1. Currently, the mode of action of AUTAC is distinct from that of PROTACs, inducing proximity between a ubiquitin ligase and the substrate. The mechanism of recognition of the AUTAC degradation tag is not yet known.

LYTAC

To target extracellular proteins, lysosomal-targeted chimeras (LYTACs) have been developed that allow coopting of the endo-lysosomal pathway.
In a proof-of-concept study, a cation-independent mannose-6-phosphate receptor (CI-M6PR), an essential receptor in the trans-Golgi network, was used to coopt the endocytic pathway. IC-M6PR binds mannose 6-phosphate (M6P)-bearing proteins and insulin-like growth factor 1 (IGF-I) from the extracellular space and targets them to the endosome and lysosome after internalization. The acidic pH of the lysosome triggers the release of the glycosylated cargo, which is degraded by lysosomal enzymes and acid hydrolases. The receptor is then returned to the membrane to repeat the cycle. Thus, the LYTAC technique is suitable for extracellular and membrane-bound proteins.

LYTAC, AUTAC, and ATTEC mechanisms.

TRAFTAC

The development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) can be considered as a generalizable strategy for the targeted degradation of transcription factors. TRAFTACs, consisting of a chimeric oligonucleotide that binds simultaneously to the transcription factor of interest (TOI) and the HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. The delivery of TRAFTACs to two oncogenic TOIs, NF-kB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins with minor changes to the chimeric oligonucleotide.

TRAFTAC mechanisms.

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Targeted Protein Degradation, the next disruption in drug discovery

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