Profiling E3 ligase compounds and the Implications in Drug Discovery

By September 8, 2022 No Comments

By: Abdul Haseeb, Ph.D.


Drugging the Undruggable

Tremendous progress in genomics, transcriptomics and proteomics has led to the discovery of thousands of new drivers of disease, but drug discovery is still catching up to these new potential drug targets because most of these new players are intractable to traditional pharmacological targeting. Traditional small molecule drugs require protein pockets in their target molecules to attach whether to inhibit or activate and many recently discovered disease-causing proteins lack these pockets. Development of new ways to target such proteins is ongoing. In this article I discuss the utilization of the cellular ubiquitin proteasome system as one of the approaches to drug undruggable proteins.

The Ubiquitin Proteasome System (UPS) – The Protein Recycling System of the Cell

Our cells have a very elaborate and efficient machinery to recycle the proteins that become unfit. Recycling of those proteins is performed though the ubiquitin proteasome system, which consists of hundreds of enzymes and a protein shredder, the 26S proteasome. Proteins that need to be degraded are ubiquitinated – marked with poly-ubiquitin chain tags for degradation through a series of cascading events carried out by a number of enzymes. The 26S proteasome recognizes these tags and chops the proteins into smaller pieces. These small pieces (amino acids) are then used as building blocks to make new proteins. Addition of ubiquitin tags is carried out sequentially by three classes of enzymes: an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligase. Our cells harbor >60 E2 conjugating enzymes and >600 E3 ligases. This process is tightly regulated by approximately 100 deubiquitinases (DUBs) that remove the tags if they become erroneously attached.

E3 ligases as drug targets

The regular turnover of proteins ensures that cells maintain their homeostasis and its disruption leads to many diseases including multiple types of cancer. Ubiquitination of proteins not only marks them for degradation but also controls their trafficking inside the cells. Since E3 ligases determine the specificity of the ubiquitination machinery, they can be pharmacologically targeted to regulate key disease-causing proteins. A number of E3 ligase inhibitors obtained by high throughput screening and refined based on protein structures are in various stages of drug development (see table 1).


E3 ligases can also be pharmacologically directed towards the proteins of interest to be degraded. This approach has led to the growth of a new drug modality called targeted protein degradation (TPD).
Heterobifunctional molecules called PROTACs (PROteolysis TArgeting Chimeras) bind to an E3 ligase at one end and to a selected target protein at the other end, thereby ubiquitinating the target protein for degradation by the UPS, has opened a new field of chemical biology. PROTAC based approaches for novel therapeutics offer many advantages over traditional therapeutic strategies based on small-molecule occupation of a protein’s binding site. First, using PROTACs the target can be selectively degraded with catalytic specificity. Second, even the weak binders of either protein, E3 ligase or the target, can be converted into selective PROTAC drugs. And third, overexpressed or mutant targets can be degraded. Cereblon and VHL are the only E3 ligases that are largely being used as vehicles to ubiquitinate and degrade multiple target proteins, such as nuclear receptors, kinases, transcription factors, and neuronal proteins including tau and α-synuclein.
Along with their advantages, PROTACs have the disadvantage of being very large molecules limiting their approach to the desired tissue and entry into the cells.

Figure 1: PROTAC technology comparison to molecular gluesUbiquitination of the target proteins by molecular glues and PROTACs

Molecular glues

A new class of molecules, molecular glues, address the size problem of PROTACs. Molecular glues (MGs) reshape a protein of interest (POI) to generate a new molecular surface to help it bind with another protein. MGs are being developed as agents that can facilitate the binding of E3 ligases with their targets to induce ubiquitination of targets followed by their degradation.
Despite the attraction of MGs as small protein degraders, rational design and discovery remains challenging.

Where are we headed?

The field of targeted protein degradation using the UPS of our cells is rapidly growing and has a great potential to treat diseases though drugging  proteins that were difficult to target using traditional approaches. Currently only a handful of E3 ligases (mainly VHL and CBN) are being used to develop protein degraders. There is still a great space in this journey where other ligases from a pool of >600 can be utilized to develop PROTACs and molecular glues. E3 ligases living in different tissues, in different cells and different compartments of the cell can be used to target proteins co-localized in those places. A new field of targeted protein stabilization (TPS) is also emerging which utilizes induced proximity approaches, similar to PROTACs and MGs, to stabilize the desired proteins by bringing deubiquitinases close to the targets. The sky’s the limit!


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