Overview of The Development of PROTACs

Most of the drugs used in clinical practice are based on small molecules. Different from traditional small molecule inhibitors and antagonists, protein degradation technology has developed rapidly in recent years due to its ability to induce the degradation of therapeutic target proteins, which provides a new idea for the development of new drugs.

The concept of PROTACs (PROteolysis-TArgeting Chimeras) was first proposed by Crews et al. in 2001. PROTACs can use the natural protein cleaning system in the body to reduce protein level rather than inhibit protein function, so as to cure the disease. PROTAC is a heterobifunctional molecule that looks like a dumbbell, with one end of the molecule connected to a ligand that binds a target protein, one end to an E3 ubiquitin ligase, and a suitable linker in the middle. PROTAC degradation of target proteins is achieved through the ubiquitin proteasome system (UPS) : PROTAC molecules bind target protein (POI) and E3 ligase to form a teradical complex, which tags the target protein with ubiquitination, and the ubiquitinated protein is recognized and degraded by intracellular proteasome 26S.

Mechanism of PROTAC-mediated protein degradation

Figure 1. Mechanism of PROTAC-mediated protein degradation
(Signal Transduct Target Ther, 7(1): 181.)

On June 9, 2022, the team of Professor Rao Yu of Tsinghua University published a review article entitled "PROTACs: great opportunities for academia and industry (an update from 2020 to 2021)" in the journal Signal Transduction and Targeted Therapy under Nature, which detailed the research progress of PROTAC technology in the past two years, summarized the representative new targets of PROTACs targeting cancer, viral infections, immune diseases, neurodegenerative diseases.

Research Progress of New Targets of PROTACs

In the past 20 years, the field of PROTAC has entered a period of rapid development, especially since the successful degradation of BET protein by dBET1 PROTAC with Pomalidomide as E3 ligase ligand in 2015. In the past two years, research papers related to PROTAC have experienced explosive growth. In 2019, Rao Yu's team had summarized more than 40 protein targets that were reported to be degraded by PROTAC. In the past two years, about 90 protein targets that can be degraded by PROTAC have been added, covering cancer, immune disorders, viral infections, neurodegenerative diseases and other disease fields, among which cancer is the main application field.

The rapid development of PROTAC-related research

Figure 2. The rapid development of PROTAC-related research in the past two years
(Signal Transduct Target Ther, 7(1): 181.)

According to incomplete statistics, different degradation agents based on PROTAC technology can degrade about 54 kinases, accounting for 45% of the total target. Kinases have been preferred as targets for protein degradation, primarily because most kinases have known and potent inhibitors or ligands that can be easily modified to link the linkers and maintain sufficient binding affinity. In addition, kinases have deep binding pockets that facilitate the binding of PROTACs, which induce the interaction of the kinases with E3 ligases, which in turn ubiquitinate and ultimately degrade the kinases. Furthermore, despite the high homology of kinase proteins, PROTACs can selectively degrade different isoforms of kinases.

So far, 518 kinds of kinases have been discovered, which are involved in various physiological regulatory processes such as cell survival, proliferation, differentiation, apoptosis, and metabolism. As shown in the figure below, these kinases can be divided into nine categories according to their structure and function, namely receptor tyrosine kinases (RTKs), TKL kinases (TKLs), STE kinases (STEs), CAMK kinases (CAMKs), AGC kinases (AGCs) , CMGC kinases (CMGCs), atypical protein kinases, CK1 kinases (CK1) and others. Among them, those marked in red are human degradable kinases with existing PROTAC degraders. Receptor tyrosine kinases (RTKs) and CMGC kinases (CMGCs) have the most developed PROTAC degraders, with 19 and 14 respectively, accounting for more than half of the total. In contrast, CK1 kinase (CK1) and CAMK kinases (CAMKs) have not developed a PROTAC degrader.

Degradable human kinases and their classification

Figure 3. Degradable human kinases based on PROTAC technology and their classification
(Signal Transduct Target Ther, 7(1): 181.)

New Technology of PROTACs

Since PROTACs were developed on the basis of POI inhibitors, they still have a certain degree of off-target effects. Due to the large molecular weight of PROTAC, its poor cell membrane permeability and poor pharmacokinetic (PK) properties greatly reduce its biological and therapeutic effects. In addition, although some PROTACs can effectively induce the degradation of target proteins, their biological effects are weak and have no effective effect on disease. Finally, most proteins do not have corresponding small-molecule conjugates to design PROTACs, such as transcription factors that play an important role in disease development. Since there are few inhibitors of transcription factors, there is no binder available when designing PROTACs targeting transcription factors. This greatly limits the application of PROTAC technology. In order to solve the above problems, different types of PROTAC technologies have emerged in recent years, such as Antibody-PROTAC, Aptamer-PROTAC conjugates, Dual-target PROTACs, Folate-caged PROTACs and TF-PROTACs.

Antibody-PROTAC

Antibody-PROTAC is a new strategy to explore the assembly of new antibody-PROTAC conjugates in combination with antibodies. This technology enables specific degradation of proteins in different cells and tissues, thereby optimizing and maximizing the therapeutic window, reducing the side effects of broad-spectrum PROTACs, and increasing their potential as drugs or chemical tools.

Aptamer-PROTAC conjugates

Aptamers are single-stranded nucleic acids with complex three-dimensional structures, mainly including stems, loops, hairpins and G4 polymers. They bind to target proteins through special effects such as hydrogen bonds, van der Waals forces, base stacking forces, electrostatic effects, etc., with high specificity and affinity, and can improve the water solubility, membrane permeability, and tumor targeting of traditional PROTACs.

Dual-target PROTACS

In the occurrence and development of cancer, there are usually multiple factors that work together, including different kinds of kinases and growth factors, which can act independently or interfere with each other through signaling networks. This method is mainly to design a single molecule that binds two or more pharmacophore, and simultaneously targets two or more anti-tumor targets.

Folate-caged PROTACs

Folate receptor alpha (FOLR1) is low in normal tissues but highly expressed in many human cancers. Folate caged PROTACs are another technology to improve the targeting specificity of PROTACs. The basic principle is to introduce folate groups into PROTAC molecules to achieve release in target cells and tissues. In this technique, folic acid releases active PROTAC through the action of endogenous hydrolase in the cell, and then the degradant induces the degradation of the target protein.

TF-PROTACs

Transcription factors (TFs) are a class of proteins involved in gene expression and regulation, which are also potential targets for cancer therapy. Unlike conventional kinases, transcription factors do not possess the active pockets or allosteric regulatory sites commonly found in kinases or other enzymes, making them difficult to target by small molecule inhibitors. Since TFs can bind to specific DNA sequences and regulate the transcription process, it is theoretically possible to target TFs with different DNA sequences instead of small molecule inhibitors. Therefore, TF-PROTAC replaces the small-molecule ligand of the targeting protein with the corresponding DNA sequence, so that it forms TF-PROTAC, targets specific TF, and induces its degradation, thereby regulating the level and biological function of specific TF.

Development Potential of PROTACs

As an emerging and promising technology, PROTACs show great development potential in the following aspects.

First, PROTACs shows exceptional sensitivity to drug-resistant targets. Traditionally, chemotherapy has been the mainstay of cancer treatment, but the acquired drug resistance of chemotherapy drugs hinders clinical application and leads to disease recurrence. Later developed kinase inhibitors, immunotherapies have also exposed the problem of drug resistance. Since PROTACs affect protein function by clearing the entire target protein, including enzymatic active function and non-enzymatic function, this technology is expected to address the potential drug resistance faced by current treatments.

Second, PROTACs has the potential to target "undruggable targets". Most small-molecule drugs or large-molecule antibodies require binding to the active site of an enzyme or receptor to function; however, it is estimated that 80% of proteins in human cells lack such a site. And PROTACs can grab the target protein through any nook and cranny.

Third, PROTACs can affect non-enzymatic functions. Traditional small-molecule drugs generally work by eliminating the enzymatic activity of their targets. Accumulating studies have shown that PROTACs has the potential to expand the target "druggable space" and control protease and non-enzyme functions.

Comparison of PROTACs and other therapeutic techniques

Comparison of PROTACs and other treatment techniques
(Source: Signal Transduction and Targeted Therapy)

Arivinas, founded by Craig Crews, was the first to carry out the drug development of PROTAC technology. In the past two years, ARV-110, a PROTAC inhibitor targeting the androgen receptor (AR) and ARV-471, a PROTAC inhibitor targeting the estrogen receptor (ER), developed by Arivinas, have been clinically validated in prostate and breast cancer, respectively, which are milestones in the application of PROTAC technology.

Clinical Trial Study of PROTACs

In addition to being a research tool, PROTACs also have great potential for application in disease treatment. It has emerged as a new mode of drug discovery that has the potential to transform traditional drug discovery into a new blockbuster therapy. As of March 2022, as shown in the table below, more than ten PROTAC drugs have entered the clinical development stage worldwide. Among them, ARV-110 and ARV-471 of Arvinas have entered clinical phase II, which are the fastest clinical progress among PROTAC drugs.

summary of protein-degradation drug candidates

Table 1. The summary of protein-degradation drug candidates based on PROTAC technology in the global clinical and IND stages
(Signal Transduct Target Ther, 7(1): 181.)

Conclusion

Compared with other drugs and therapies, PROTACs have many potential advantages, such as wide tissue distribution and oral administration. Compared with other therapies (such as cell therapy, antibody drugs, etc.), the production process of PROTAC is simpler. Compared with small molecule drugs, PROTAC can target more targets that cannot be targeted by small molecule drugs, resulting in better effects. Therefore, PROTAC technology has received high attention from the industry, and has begun to be used in drug research and development for cancer, immune disorders, viral infections, neurodegenerative diseases, etc., among which the application in the field of cancer is the main one. Arivinas, founded by Craig Crews, was the first to carry out the drug development of PROTAC technology. In the past two years, ARV-110, a PROTAC inhibitor targeting the androgen receptor (AR) and ARV-471, a PROTAC inhibitor targeting the estrogen receptor (ER), developed by Arivinas, have been clinically validated in prostate and breast cancer, respectively, which are milestones in the application of PROTAC technology.

Although PROTAC has grown rapidly over the past 20 years, there are still many challenges to be addressed. These challenges mainly come from two aspects, namely, the optimization of PROTAC molecular design and druggability, and the comprehensive evaluation of biological activity. The first is about the molecular design and druggability of PROTAC, involving target protein ligands, new E3 ligase ligands and new linkers. The second is bioactivity evaluation, involving the screening, druggability evaluation and pharmacological evaluation of PROTAC molecules. There are no ready answers to these questions at present, but it is believed that with the development of more biological, pharmacological and clinical research, new evaluation methods and systems will be gradually established to solve these problems. It is believed that more and more PROTACs will enter preclinical and clinical studies in the future, which will further test the therapeutic effect of PROTACs. It is expected that PROTAC technology will bring benefits to human disease treatment and life health in the future.

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Reference:
PROTACs: great opportunities for academia and industry (an update from 2020 to 2021)