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Using Protein Degradation To Target the Undruggable

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The life of a protein

Proteins are synthesized based on mRNA templates in the process of translation, which takes place in an organelle known as the ribosome (Figure 1). Ribosomes are ribonucleoprotein complexes that are found either in the cytosol or associated with the endoplasmic reticulum (ER). After translation, newly produced proteins fold into their native structures, often with the help of chaperones, and are transported to their target cellular compartment.

The half-life of a protein varies and can range from minutes to even years, as is seen for some components of the extracellular matrix. When proteins do degrade, they are broken down via two main routes:

  1. Secreted proteins and plasma membrane proteins are endocytosed and trafficked to lysosomes, where they are degraded via lysosome proteases. These proteases also play roles in proteolytic degradation during micro- and macro-autophagy.

  2. The other route of protein degradation is ubiquitin-mediated proteasome-dependent degradation.

In this article, we will discuss the mechanism of ubiquitin-mediated proteasome-dependent degradation and discuss how it has been employed to design novel therapeutics that can turn any class of human proteins into a therapeutic target.

Figure 1. The life of a protein (Credit: Proteintech) 
Left: Secreted and plasma membrane proteins are synthesized on ribosomes associated with the ER and degraded predominantly in lysosomes via endocytic route. Right: Intracellular proteins are synthesized on ribosomes in the cytoplasm and follow ubiquitin-mediated degradation in proteasomes.

Ubiquitin and proteasome-dependent degradation

Ubiquitin is a small ~9kDa protein that is present in virtually every human cell, usually in the form of multimers or linked with other proteins. Conjugation of proteins with ubiquitin is the basis of a ubiquitin-mediated proteasome-dependent degradation. This is a highly regulated mechanism that involves three types of enzymes (Figure 2).

The initiator of this reaction is ubiquitin-activating enzyme E1, which produces the active thiolester form of ubiquitin. E2 ubiquitin-conjugating enzyme binds to activated ubiquitin, while E3 ligase recruits the cellular protein targeted for degradation and mediates the transfer of ubiquitin to a lysine residue of the target protein. In most cases, this process is recurrent, leading to extension of the ubiquitin chain because ubiquitin molecules also possess a lysine group. The polyubiquitinated proteins are then recognized by the 26S proteasome, a multimeric complex composed of a 20S core and 19S regulatory particles present at one or both ends. 19S particles recognize polyubiquitinated proteins as substrates, while the 20S core executes protein degradation.

Figure 2. Protein ubiquitination (Credit: Proteintech) 
A cascade of ubiquitin activating enzyme E1, conjugation enzyme E2 and ubiquitin ligase E3 is responsible for the transfer of a ubiquitin molecule onto a target protein.

PROTACs  drugs that use E3-ligase-mediated protein degradation

Proteolysis targeting chimeras (PROTACs) are a new class of therapeutics that employ targeted protein degradation as a mode of action. They comprise of two domains – a binding domain for the target protein and a ligand for E3 ligase recruitment (Figure 3). Association of PROTACs with both the target protein and the E3-ubiquitin ligase complex brings the degradation machinery in close proximity to the target protein, leading to its subsequent polyubiquitination. The ubiquitinated target protein is then recognized by the 26S proteasome domain and proteolytically digested. Throughout this process, PROTACs act as catalyzers and are not degraded; therefore, they are particularly potent, as they remain able to catalyze the subsequent ubiquitination of many molecules of the target protein.

The first generation of PROTACs used peptides derived from natural ligands of E3 ligases for their recruitment6,7
, which provided the desired activity but were often not cell permeable, or required micromolar concentrations for efficient target degradation. A shift to the use of engineered small molecule E3 ligase ligands lowered their molecular weight, improved cell permeability and potency.1,2

Figure 3. Mechanism of action of PROTACs (Credit: Proteintech) 
PROTACs mediate interaction between the target protein and the E2-E3 complex, allowing transfer of ubiquitin onto the target protein. PROTACs protein targets include nuclear receptors (e.g. AR), transcription factors (e.g. ARNT), kinases (e.g. HER2) and scaffolding proteins (e.g. BRD9). Immunofluorescent analysis of 10% Formaldehyde fixed HepG2 cells using 22576-1-AP (androgen receptor antibody) at dilution of 1:50. WB result of ARNT (HIF1B) antibody (14105-1-AP; 1:3000; incubated at room temperature for 1.5 hours) with sh-Control and sh-ARNT (HIF1B) transfected HeLa cells. 18299-1-AP (HER2/ErbB2 antibody) at dilution of 1:600 (under 40x lens) heat mediated antigen retrieved with Tris-EDTA buffer(pH9). HeLa cells were subjected to SDS PAGE followed by western blot with 24785-1-AP (BRD9 Antibody) at dilution of 1:1000 incubated at room temperature for 1.5 hours.

Advantages of PROTACs over other modalities

Generally, the two most common types of therapeutics are small molecules and monoclonal antibodies (mAbs). Small molecules are quite versatile and can act on both extracellular and intracellular targets, unlike mAbs that predominantly target extracellular proteins such as cell receptors (Table 1). Both small molecule and mAbs mostly act via binding to a target molecule in order to affect a given result – for example, prevent its activity, inhibit binding with other partners or activate a certain pathway. MAbs usually provide high specificity with low toxicity, which is often difficult to achieve with small molecule drug candidates. On the other hand, small molecules usually have better tissue penetration due to low molecular weight, with a shorter half-life and shorter pharmacodynamics. Novel modalities, such as antibody-drug conjugates (ADCs) or Fc-fusion proteins try to overcome some of these challenges. However, they do not overcome the main obstacle: they do not considerably expand the class of proteins that are druggable by these modalities.

PROTACs’ mechanism of action is based on selective binding to a target protein to trigger its catalytic degradation. Unlike other modalities, the binding site does not have to be restricted only to certain protein domains, e.g. active sites of enzymes in order to compete with their substrates or protein interaction sites to block the binding.

Theoretically, any site on the protein surface can be targeted by PROTACs as long as it provides specificity for the given molecule. This also offers a great advantage over small molecules for selectively targeting mutated proteins or protein isoforms. An interesting concept is to use target protein binding domains that are functional only in certain cell states – for example phosphoPROTACs are active only in a phosphorylated state
.3 Because the PROTAC mechanism of action involves catalytic degradation of the target protein, PROTACs can target scaffolding proteins, non-enzymatic protein-protein interaction targets or toxic protein aggregates. Even low doses of the drug should be sufficient for its action.

Table 1. Comparison of drug properties of small molecules, antibodies and PROTACs.

FeatureSmall moleculesAntibodiesPROTACs
Extracellular targetsYesYesNo
Intracellular targetsYesNoYes
Scaffolding protein targetsLimitedNoYes
Protein-protein interaction targetsLimitedLimitedYes
Targeting mutant proteins/protein isoformsLimitedLimitedYes
Mechanism of actionBinding to trigger inhibition/activation of the targetBinding to trigger inhibition/activation of the target
Binding and catalytic degradation of the target
SizeSmall (below 1 kDa)Big (ca. 150 kDa)Small-medium (up to 1.5 kDa for small molecule PROTACs)
Half-lifeTypically hoursDays to weeksTBD
PharmacodynamicsShort actingLong actingTBD
Tissue penetrationGoodLimitedGood

*Predicted lower doses compared to small molecules

The future of PROTACs 

PROTACs undoubtedly have the potential to change the drug discovery landscape in the forthcoming years. Proof-of-principle studies have demonstrated their degradation activity towards various protein targets, including nuclear receptors, transcription factors, kinases and scaffolding proteins in cell-based experiments (Figure 3). The first PROTAC molecules are now reaching clinical stages of development. Current efforts focus on discovery and improvement of existing E3 ligase linkers, and also on gaining a better understanding of tertiary complexes between target proteins to decipher what drives target degradation. For example, it has been recently shown that PROTACs designed to target motifs present in many proteins can successfully associate with the vast majority of them, but trigger degradation of only a fraction of them
.4 At present, there are no in silico models that would correctly predict success. Others focus on improving cell permeability of PROTACs by altering ligands and re-designing linkers. An interesting approach is delivery of two PROTAC precursors that would assemble into a functional PROTAC molecule inside the cell using click chemistry (so called CLIPTACs).5


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