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

Protein degradation targeted chimera (PROTAC)

PROTAC technology began in 2001 that uses the ubiquitin-proteasome system (UPS), the intracellular “cleaner” 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 is 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, PROTAC is mainly used for target discovery and verification, with its goal being drug discovery.

Dr. Craig Crews of Yale University is a pioneer in this field. However, the concept of PROTAC was clearly demonstrated and first published in a PNAS article in 2001 by the UCLA Sakamoto Laboratory. Craig is one of the authors. In this article, the molecule Protac-1 was designed to degrade the target protein amino peptidase-2 (MetaP-2) through Protac-1 by recruiting the ubiquitin protein beta-TRCP. In addition, 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 simple.  Its catalytic part is E3 ligase, but other proteins are needed to obtain substrates so that protein targets can be found for degradation.  This technique connects the target protein ligands to the ligands that help UPS find the substrate proteins through chemical bonds, which then degrade target.
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, connected by an appropriate linker. Similar to the bispecific monoclonal antibody technique, a chimera molecule can be regarded as a bifunctional small molecule.

Ubiquitination is the death sentence for proteins, affixing proteins to the proteasome for degradation. After entering the cell, the ligand of the protein of interest (POI) in the 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 using ubiquitin binding enzyme E2. After the dissociation of the ternary complex, the ubiquitin “labeled” POI is recognized and degraded by the proteasome to selectively reduce the level of the target protein. In this process, the target protein ligands do not occupy the binding site for a long time; the ternary complex is formed for a short time to complete target protein ubiquitination. PROTAC can play a role in many cell cycles.

  • Technical advantages of PROTAC.

To a large extent, PROTAC combines the advantages of small molecular compounds and small molecular nucleic acids. Not only it effectively targets the target protein, but PROTAC can also selectively degrade and remove different proteins expressed by the same gene after protein expression and modification.

In theory, PROTAC only provides binding activity, and it is event-driven, which is different from the traditional possession-driven, or “occupancy-driven”, meaning it does not directly inhibit the functional activity of the target protein. Drugs do not need to bind to target proteins for a long time and with high intensity, so they can target difficult traditional targets, such as proteins with smooth surfaces that lack small molecular binding regions. Therefore, many targets that cannot be regulated by small molecules or 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.

  • Technical disadvantages of PROTAC.

Drugs developed through PROTAC are double targeted, so its molecular weight, molecular rigidity, and water solubility are not ideal, causing poor oral absorption and transmembrane properties. PROTAC molecules are usually large, and competition is a major obstacle. Although active PROTAC compounds have been tested in vivo and the clinical trials are about to begin,  PROTAC small molecule proprietary drugs face many competitions. Chemical synthesis is also much more difficult.

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 target other 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 effect of degradation is difficult to detect and track in preclinical toxicity screening, contributing to the increased risk of drug development in the later stage. In addition, the technique is 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 preclinical research and early detection. Among the fastest development are two PROTAC small molecule drugs from a biotech company Arvinas, both will enter clinical trials this year. One is androgen receptor, which targets prostate cancer; the other is estrogen receptor that targets breast cancer. In November 2017, the first clinical candidate, ARV-110, targets and induces androgen receptor protein degradation. At the same time, a second candidate ARV-378 that targets and induces estrogen receptor protein degradation was introduced. Arvinas hopes to begin its ARV-110 clinical trial on October, followed by ARV-378 by the end of this year.

In the past two years, great progress has been made in making the degradation of key carcinogenic proteins efficiently. BET family proteins, including BRD4, play an important role in many 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 mediocre. 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 have 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 acute myeloid leukemia mouse model.


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. Target protein degradation is considered attractive because traditionally untreatable proteins can now be targeted. 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.

One of its greatest potential in the future is to target transcription factors, protein skeleton function, or KRAS mutation, which are targets of non-proprietary medicine.

Traditional small molecule drug research and development is facing the extrusion of emerging technologies, and deeper innovation is needed to gain a foothold in the industry. PROTAC technology may cause 80% of proteome, which is not yet 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 is 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. New technology will be integrated into more products, making the pharmaceutical field difficult to predict. Regardless, the most important thing is how to benefit patients.

The development of new drugs based on ubiquitin-proteasome system represents a popular field 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 many others have all entered the field one after another. 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 target protein degradation and small molecule drug development has quietly arrived.