Release date:2022/4/15 18:23:45

In the context of the increasing popularity of macromolecular drugs such as monoclonal antibody drugs, dual antibody drugs and ADCs, small molecule drugs have once again become the focus through PROTAC (Proteolysis targeting chimera), a breakthrough new technology. 

In the 20 years since the publication of the first PROTAC molecule, the technology has been transformed from academia into a preclinical and clinical phase new drug development project, and in 2019 the first clinical proof of concept was obtained in the field of oncology. After 20 years of development, PROTAC technology has attracted the attention of many pharmaceutical companies, such as Pfizer, Bayer, Novartis and other multinational pharmaceutical companies.

Professor Craig M. Crews of Yale University, co-founder of Arvinas, recently published a review in Nature Reviews Drug Discovery that summarized the development of PROTAC over the past 20 years. Four major development directions for PROTAC in the next 20 years are discussed. The authors point out that the field of targeted protein degradation is "ready to go" to challenge targets previously considered "undruggable". 

Introduce PROTAC

Many targets that play key roles in cancer and other diseases are notoriously difficult to become drug, some of which are difficult to bridge with small molecules due to their wide and shallow active sites. Others have "smooth" surfaces with few sites for small molecules to bind to.  

But with the continuous development of PROTAC technology, these undruggable targets have also become within reach.

PROTAC techonoly uses heterozygotic bifunctional small molecule compounds to bring the target protein and the intracellular E3 ubiquitin ligase closer, and specifically degrades the target protein in vivo through the ubiquitin-proteasome protein degradation pathway. The drug is composed of 3 parts, that is, one end is the specific E3 ubiquitin ligase ligand, the other end is the specific ligand of the target protein and the linker in the middle, thus forming the "target protein-PROTAC-E3 ubiquitin ligase".

Unlike traditional small-molecule drugs, PROTAC drugs do not need to bind tightly to the disease-causing target for long periods of time to degrade it.  By destroying protein targets rather than inhibiting them, problems of non-drugability and drug resistance are solved.

 PROTAC-targeted protein

Table 1. PROTAC-targeted protein degraders in clinical development

Four major trends in the future development of PROTAC

In the past few years, PROTAC technology has basically matured. The authors propose that the next milestones in the field of PROTAC will focus on the following four areas, namely, identify optimal protein degradation targets, expand the scope of clinical application of E3 ligase, expand the scope of clinical treatment beyond oncology and develop other PROTAC modes.  

1. Identify Optimal Protein Degradation Targets

The first batch of clinical phase proteolytic agents selected are clinically proven mature targets, such as the androgen and estrogen receptors selected by Arvinas. Targeted products have been developed with moderate success, and the potential of PROTACs as a therapeutic modality has been validated. However, the real potential of this treatment model lies in reaching targets that are difficult or undruggable with current treatment models. So which targets are ideal targets for PROTAC therapy? The review summarizes several common features of PROTAC targets, including:

(1). Overexpression, mutation, aggregation, isoform expression, or intracellular localization of proteins that lead to disease in a gain-of-function manner;
(2). Having a binding surface that is accessible to E3 ligase;
(3). Ideally, there is an unstructured region that can access the proteasome.

These are the "Principles of PROTAC targets" (see figure below) that proposed in this paper.

Proteins with drug resistance mutations to targeted therapy, proteins with backbone functions, and proteins that are "undruggable" by other therapeutic modalities may also be suitable PROTAC targets. 

PROTAC target

Principles of PROTAC Targets (Source: References)

While targets suitable for protein degradation using PROTACs do not require an enzymatic active site, they require a small-molecule binding site accessible to E3 ligases. The affinity of this site does not need to be particularly high, usually in the range of 1-500 nM, and the recruited E3 ligase must be able to reach the surface of the target.

2. Expansion of Available E3 Ligases

More than 600 E3 ubiquitin ligases are encoded in the human genome. However, only a few E3 ligases (VHL, CRBN, etc.) are currently used for PROTAC design. How to expand E3 ubiquitin ligases that can be used for PROTAC technology is also one of the challenges PROTAC faces. 

One of the advantages of PROTAC molecules is that protein degraders can be rationally designed according to the target, by selecting the molecules and linkers that recruit the ligase. Its modular feature means that the E3 ligase binding module can be replaced for different targets, and the E3 ligase can be recruited to achieve the best therapeutic effect.

What characteristics should a new E3 ligase suitable for clinical development have? Where should we look for new E3 ligases? The authors propose that new E3 ligases can be found in the following ways.

Widely used, ubiquitous ligase

A practical and valuable approach is to find broadly applicable, ubiquitous ligases, similar to CRBN and VHL. They can be paired with any target protein, allowing unrestricted application in multiple therapeutic indications. All current PROTAC molecules use only a few of these ligases, and their information can be queried in the online database PROTAC-DB.

​Interestingly, recruitment of VHL or CRBN for the same target protein may lead to different degradation efficiency. For example, in the development of protein degraders for KRAS G12C, previous studies have found that KRAS G12C can be targeted by the PROTAC system, but the degradation effect will be affected by whether the recruited E3 ligase is VHL or CRBN.

The review points out that some ligases worthy of PROTAC protein degradation can be identified based on the structural characteristics of ligases, and last year's breakthrough by DeepMind and RoseTTAFold in predicting the three-dimensional structure of proteins based on amino acid sequences may redefine which ligases can be used to develop protein degradation therapies.

Tissue and cell-specific E3 ligases

Another approach, based on key characteristics of ligases, such as tissue and cell specificity, tumor enrichment, and tumor necessity, could provide development opportunities for domain-specific protein degradation therapies. Previous studies have identified a number of E3 ligases expressed in specific tissues, such as the E3 ligases named RNF182 and TRIM9 that are specifically expressed in the central nervous system (CNS), and they may have important implications for targeting neuronal diseases. CNS specific targeted degradation can avoid systemic off-target and toxic side effects.           

Interestingly, some ligases exhibit "reverse specificity," in which they are expressed at particularly low levels in specific tissues and cells, a feature that may also present opportunities for protein degraders. An excellent example is that the expression level of VHL in platelets is low, so the PROTAC molecule DT2216 that recruits VHL can avoid damage to platelets when degrading BCL-XL, thereby reducing the toxicity caused by platelets and improving its therapeutic index. It is currently in Phase 1 clinical development.

Different types of E3 ubiquitin ligases

Different types of E3 ubiquitin ligases (Source: Reference [1])

E3 ligases enriched in diseased tissues or cells

​In addition, another new frontier in precisely targeting protein degradation is PROTAC molecules that specifically target tumor cells, which can be achieved by targeting tumor-specific or tumor-enriched E3 ligases. One strategy to achieve this goal is to target tumor-specific or tumor-enriched E3 ligases. In general, E3 ligases that are enriched in tumors coincide with tumor-dependent E3 ligases. This correlation can be used to discover E3 ligases that are necessary for many cancer cell types, and their advantage is that tumor cells are difficult to develop resistance to PROTAC molecules through mutations in the ligase.

Discovering, researching and developing tumor-specific E3 ligases for protein degradation is just one of the strategies to obtain tumor-specific PROTAC molecules. Another strategy is to take advantage of the specificity of antibodies for tumors by conjugating degradative molecules to tumor-specific antibodies. Studies by Genentech and its partners have demonstrated the tumor-targeting specificity of antibody-conjugated PROTAC molecules. However, antibody-conjugated PROTACs lose the oral characteristics of small-molecule PROTACs.

3. Expand the scope of clinical treatment beyond oncology Treating Diseases Beyond Oncology

So far, research on protein degraders has mainly focused on the oncology field, but since protein degraders may degrade any chosen target, their application can be broader. In fact, in recent years, protein degraders have gradually been used in fields other than oncology, such as neurodegenerative diseases. And there is a breakthrough in inflammation/immunology field.

Following the success of immune checkpoint inhibitors, the development of small molecule drugs that can stimulate the anticancer immune response is an important area of drug development.  PROTAC molecules can activate immune cells in the mode of small molecule drugs and mimic the effects of PD-1/PD-L1 targeted therapy, thus becoming a potential "first-in-class" therapy.  Recently, PROTAC molecules targeting MAP4K1 have shown promising preclinical activity. 

While small molecule inhibitors may serve the same purpose in this regard, PROTAC molecules may be better tolerated and have lower toxic side effects.

One of the key features of PROTAC molecules is their ability to degrade proteins that are not targeted by traditional small-molecule inhibitors because they lack an active site. This feature makes proteins that accumulate in various neurodegenerative diseases (such as Tau) become potential targets.

4. Develop innovative PROTAC modes

In addition to PROTAC, various novel protein degradation technologies have been developed, further expanding the range of targets that can be targeted by this technology. Innovative PROTAC strategies, including biology-based PROTACs (bioPROTACs) and hybrid PROTACs (Hybrid PROTACs), may further expand the range of targets that PROTACs can target.

The researchers define three types of PROTACs: traditional small-molecule PROTACs, PROTACs of peptides or other biological products ( bioPROTACs ), and PROTACs containing both peptide and traditional small-molecule "warheads" (hybrid PROTACs).

Biological product PROTACs use peptides, fusion proteins, and oligonucleotides as ligands for recognizing targets. PROTACs based on fusion proteins or peptides have successfully degraded HER2, MYC, KRAS and other cancer-related targets in research. Oligonucleotide-based PROTACs can be used to target transcription factors and RNA-binding proteins. Mutations in these proteins lead to many types of cancer, as well as obesity, cardiovascular and neurological diseases.

These biologic PROTACs have the potential to become a new treatment modality, but their delivery and administration modes are hurdles to overcome. These therapies may need to be delivered in the manner of gene therapy, utilizing precisely regulated promoters to control expression levels within cells. The development of nanoparticle based delivery technologies and viral vector based gene delivery systems will play an important role in the advancement of this therapeutic model.

The past 20 years is prologue, and the field of targeted protein degradation has been ""poised to challenge", challenging targets that were previously considered "undruggable". Based on the advances in targeted protein degradation over the past 20 years and the interest and investment in it from academia and industry, it is clear that this technique could become a key therapeutic model. Although there is no drug approved by the FDA so far, we believe that this treatment model has the potential to provide innovative treatment options for patients with a wide range of diseases.

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Related articles:
[1] PROTACs VS. Tranditional Small Molecule Inhibitors
[2] PROTAC And Other Protein Degradation Technology 
[3] Summary of PROTAC Degraders in Clinical Trials

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