PROTAC technology: Small molecule drug research and development mass murderer is coming! (Ⅱ)

Protein degradation targeted chimera (PROTAC)

PROTAC (PRO teolysis Targeting Chimera) is a technology that began 17 years ago (2001), using the intracellular “cleaner”, the ubiquitin-proteasome system (UPS). The normal physiological function of the ubiquitin-proteasome system is responsible for cleaning up denatured, mutated, or harmful proteins in cells. PROTAC, which uses the protein destruction mechanism of cells to remove specific carcinogenic proteins from cells, is an alternative to targeted therapy.

PROTAC can be said to be an improved and general platform technology for induced protein degradation. Prior to this, there have been some similar technologies and their prototypes, such as some of the earliest discovered selective hormone receptor inhibitors with degradation function, HyT technology and so on. At present, this technology is mainly used for target discovery and verification, and its ultimate goal is to achieve drugs, not just for target verification.

Dr. Craig Crews of Yale University is a pioneer in this field. However, this concept was first put forward by UCLA Sakamoto Laboratory when it published PNAS article in 2001. Craig is one of the authors. In this article, a molecule called Protac-1 was designed to degrade the target protein amino peptidase-2 (MetaP-2) in a Protac-1-dependent manner by recruiting the ubiquitin protein beta-TRCP, which clearly demonstrated the concept of PROTAC for the first time. In addition to recruiting ubiquitin proteins, HyT (Hydrophobic tagging) technology was subsequently developed. Using PROTAC technology, target proteins are labeled with large hydrophobic groups such as adamantane and Boc3Arg. The protein degradation machine mistook the target protein for incorrect folding and degraded it.

  • Technical principles of PROTAC.

The principle of PROTAC technology is actually very simple.  Its catalytic part is E3 ligase, but it needs a variety of proteins to obtain substrates to help find proteins that need to be degraded. This technique connects the target protein ligands to the ligands that help UPS find the substrate proteins through chemical bonds, which can degrade target proteins that do not need to be degraded.
The chimera molecule consists of three parts: one is the structure of the target protein; the other is the structure of the protein degradation system such as E3 Ligase, which is connected by the appropriate linker in the middle. This is similar to the bispecific monoclonal antibody technique and can be regarded as a bifunctional small molecule.

The ubiquitin is the death sentence of the protein, which is affixed to the proteasome and degraded by the proteasome. After entering the cell, the protein of interest (POI) ligand in the structure of PROTAC molecule can specifically bind to the corresponding target protein, while the other end can recruit E3 ligase to form POI-PROTAC-E3 ligase ternary complex. Among them, E3 ligase can mediate the ubiquitination of POI by ubiquitin binding enzyme E2.After the dissociation of the ternary complex, the ubiquitin “labeled” POI was recognized and degraded by the proteasome to selectively reduce the level of the target protein. In this process, the target protein ligands do not need to occupy the binding site for a long time, only the ternary complex can be formed for a short time to complete the ubiquitin of the target protein, and PROTAC can play a role in many cycles in the cell.

  • Technical advantages of PROTAC.

To a large extent, PROTAC combines the advantages of small molecular compounds and small molecular nucleic acids. It can not only effectively target the target protein, but also degrade and remove it, which has a very rich space for imagination and development. In theory, PROTAC only needs the amount of drugs, but also can selectively degrade different proteins expressed by the same gene after protein expression and modification, so it has certain theoretical advantages.

In theory, PROTAC only provides binding activity, and it is an event-driven “Event-driven”, which is different from the traditional possession-driven “Occupancy-driven”. It does not need to directly inhibit the functional activity of the target protein. Drugs do not need to bind to target proteins for a long time and high intensity, so they can target traditional targets that are difficult to make up, such as proteins with smooth surfaces that lack small molecular binding regions, and PROTAC related technologies can selectively degrade target proteins. Therefore, many targets that cannot be regulated by small molecules or cannot be reached by antibodies can be regulated by this technique. In addition, this process is similar to a catalytic reaction, and the drug can be reused to inhibit the target protein, and does not need to wait for moles of the drug, so it is possible to obtain a highly active drug. Therefore, the industry believes that

  • Technical disadvantages of PROTAC.

Just as even the most powerful martial arts have their own destiny, PROTAC technology certainly has its hard wounds. This kind of drug is a double-target drug, so its molecular weight, molecular rigidity, and water solubility are not very ideal, so oral absorption and transmembrane properties will be poor. PROTAC molecules are usually large, and competition is a major obstacle. Although active PROTAC compounds have been reported in vivo, they will even enter human clinical research this year. Overall, however, competition remains the main obstacle to most PROTAC small molecular proprietary drugs. Chemical synthesis is also much more difficult.

Miss toxicity should also be one of the biggest concerns. Traditionally, small molecules, macromolecular drugs, and even small nucleotides targeting protein activity generally do not inhibit protein activity completely, and most of them do not affect the expression of skeleton proteins, which certainly increases the probability of drug resistance. However, at the same time, the residual activity may also ensure the basic physiological activity of normal cells, tissues and organs and reduce the potential toxicity. As a more thorough target protein degrader, PROTAC may accidentally injure other miss proteins, even if the previously verified target will bring more serious toxicity, which needs to be closely monitored in future clinical trials.

Another hidden danger is that the miss effect of degradation is difficult to detect and track in preclinical toxicity screening, which increases the risk of drug development in the later stage.

In addition, the technique is clearly only effective for proteins that need to be suppressed, and is useless for finding agonists.

PROTAC related drugs. 

According to the Cortellis and Integrity databases, PROTAC-related drugs are in the stage of preclinical research and early detection. Among the fastest-growing are two PROTAC small molecule drugs from Arvinas, both of which Arvinas will target in clinical trials this year are proteins that have been fully studied. One is the androgen receptor, which is the target of prostate cancer, the other is the estrogen receptor, which is the target of breast cancer. In November 2017, it named ARV-110, the first clinical candidate drug that targets and induces androgen receptor protein degradation. In December 2017, ARV-378, the second candidate drug that targets and induces estrogen receptor protein degradation, was named. Arvinas hopes its PROTAC (ARV-110), which targets androgen receptors, will begin clinical trials in October. The candidate drug targeting estrogen receptor (ARV-378) is likely to enter clinical trials by the end of this year.

In the past two years, great progress has been made in the specific and efficient degradation of key carcinogenic proteins. BET family proteins, including BRD4, play an important role in the development of a variety of cancers, including acute myeloid leukemia, multiple myeloma, ovarian cancer and prostate cancer. The small molecule inhibition of multiple BET has entered the clinic, but because of the insufficient inhibition of downstream signaling pathway and the feedback mechanism can up-regulate the expression of BRD4 gene, the effectiveness of these drugs is general. The Craig Crews team at Yale University designed a small molecule of PROTAC, ARV-825, which completely degrades BRD4 proteins in cell experiments by linking BRD4 to CRBN. The team and Arvinas, a biotech company, also developed ARV-771, to degrade BET family proteins through VHL. Another team from the Dana Farber Cancer Institute designed another small molecule PROTAC drug, dBET1, based on JQ1, a small molecule inhibitor of BET. The drug showed better efficacy than JQ1 in a mouse model of acute myeloid leukemia.


It is estimated that only 10% of proteins can be regulated by small molecules, 10% can be regulated by biological macromolecules on the cell surface, and up to 80% of proteins cannot be regulated by existing drugs. The most attractive thing about targeted protein degradation is that it can target proteins that are traditionally considered untreatable, which may account for more than 80% of human proteome. Because the targeted protein degradation strategy can selectively degrade proteins by binding to almost any site, rather than the active site, this strategy can theoretically be used for any protein. In addition, another advantage of this strategy is that it can play a role in tumors that have developed drug resistance. Moreover, targeted protein degradation also has considerable potential in other diseases other than cancer.

Recent breakthroughs in the study of PROTAC small molecules have also focused on targeted targets. One of its greatest potential in the future is to target targets that can’t be targeted in the traditional sense, such as transcription factors, protein skeleton function, or KRAS mutation, which is the target of non-proprietary medicine.

Traditional small molecule drug research and development is facing the extrusion of emerging technologies, and deeper innovation is needed to continue to gain a foothold in the industry. PROTAC technology may cause 80% of proteome, which is not yet ready to be ready for medicine, to be regulated by small molecules of drugs. At a time when small molecular drugs are in urgent need of innovation, the emergence of PROTAC can be said to be a timely help. DNA coding technology significantly expands the space of small molecular chemistry, PROTAC makes drugs theoretically effective under the catalytic dose of drugs, although it may play an unexpected role in special circumstances, but it can’t subvert the whole field of new drug research and development in the short term. High and new technology will be integrated into more products, making the pharmaceutical industry a real high-tech industry. Although the specific events of new drug development are difficult to predict, it is not so important for the Central Plains to compete for deer, and the most important thing is how to benefit patients.

The development of new drugs based on ubiquitin-proteasome system represents a hot research field in the ascendant and has attracted the favor of a number of drug companies, academic institutions and investment institutions. The influx of venture capital and the participation of large pharmaceutical companies will help accelerate the development of PROTAC technology and related drug research and development. Gene Tek, Pfizer, Atlas, Novartis (NIBR), UC Berkeley and so on have all entered the field one after another, and have given birth to a number of new biotechnology companies related to it, in addition to the companies mentioned above. For example, Vividion Therapeutics, Warp Drive Bio and the newly founded Cullgen have their own unique small molecule drug development platforms based on chemical proteomics. All these indicate that the era of protein inhibition in the development of small molecules of drugs is about to die, and the era of protein degradation has quietly arrived.