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“In the next few years, by targeting genomic variation, doctors will be able to cure a range of diseases such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer. In fact, we can now imagine that our grandchildren, cancer, A noun will become very strange to them.”


In June 2000, President Clinton made such a speech at a press conference completed by the sequencing of the human genome.


In fact, the sequencing of the genome was not completed at the time, but in 2000 the main work was almost completed. At the time, some researchers believed that after completing the sequencing of the human genome, we would be able to understand the genomic variation that led to the disease, as well as information about related proteins. And this information can help us find ways to cure the disease.


After President Clinton’s speech, 18 years have passed, and these diseases are still not cured, and it can be said that our means of treating these diseases are still very limited.


There are many reasons why we cannot effectively treat or cure these diseases. An in-depth understanding of the biological mechanisms of these diseases is not easy, and this is probably the biggest obstacle to the failure of new drug development.

In addition, even if we have found drug targets that seem to be viable, it is not easy to develop drugs for these targets.


With the constant iteration of technology, the cost of genome sequencing is currently very low. We seem to have been able to find a number of driving genes that cause disease, and a better understanding of the driving genes of cancer has led us to develop some successful drugs, such as imatinib, ochinib, and olaparib.

But more often, limited to the current state of the art, even if a driver gene is found, it is impossible to develop drugs for these targets.

Most traditional small molecule drugs as well as antibody drugs are targets for targeting proteins. But in fact, these targets that we are currently able to intervene through drugs only account for a small fraction of the protein in the human body, and about 80%-90% of the proteins are not effectively intervened by traditional drug development methods.


The main reason is that many of these protein targets lack a binding pocket that is directly involved in the regulation of protein function. At present, small molecule drugs mainly bind to the so-called active binding cavity in the protein, and competitively prevent endogenous substrates from entering the binding cavity, thereby achieving the purpose of blocking protein function (of course, there are other mechanisms of action).

The problem is that this drug development model is difficult to apply to proteins that lack the corresponding binding pocket. Moreover, it is currently difficult to develop corresponding antibody drugs for proteins existing inside cells.

Such targets are indeed very numerous, such as some scaffold proteins in the cell, transcription factors, or many non-enzymatic proteins.


We generally refer to such protein targets that are currently not intervened by traditional drug development methods as undruggable targets, that is, those that do not have drug-forming targets. Many people don’t like the idea of ​​undruggable, because if it is really impossible to intervene, there is no need for research. But at the moment it is really difficult to develop drugs for these targets.

Regarding undruggable targets, the first reaction of many people is KRAS.

But for biotech companies and venture capital institutions, these so-called impossible-to-drug targets have endless opportunities. In the past few years, pharmaceutical companies have invested hundreds of millions of dollars in this area to develop drugs that target these targets.


These pharmaceutical companies have adopted a variety of strategies, many of which are based on nucleic acid-based technologies such as antisense oligonucleotides (ASOs) or RNAi, and more recent CRISPR technology. By knocking out or knocking down these techniques, it is possible to achieve the goal of regulating intracellular target protein levels.


Although these techniques are widely used in basic research, they are faced with serious obstacles in the development of therapeutic treatments. For example, nucleic acid drugs are less stable in the blood, may be immunogenic, and may accumulate in the kidneys. and many more. In recent years, another interesting technology in the field has become popular: induced protein degradation technology.


Small molecule-induced protein degradation has very distinct differences from the mechanisms of action of nucleic acid drugs, gene editing, and traditional small molecule drugs. Inducing protein degradation also has unique advantages over these technologies.


Induced protein degradation

In fact, the induction of protein degradation is not a new concept, and researchers have had some attempts long ago.

Heat shock protein 90 (Hsp90) helps proteins fold correctly, and Hsp90 inhibitors have entered clinical research for the first time in the 1990s. Since Hsp90 plays an important role in maintaining the stability of proteins required for survival of a range of tumor cells, many researchers believe that Hsp90 inhibitors may be used in the treatment of cancer.

At that time, the research on HSP90 inhibitors was very hot, but many inhibitors had severe hepatotoxicity. The research on HSP90 inhibitors also experienced many setbacks. So far, no Hsp90 inhibitor has been approved for marketing.

And more importantly, Hsp90 can participate in the stability regulation of more than half of the kinases in the human body. Therefore, it is very difficult to ensure the safety and effectiveness of the drug by this non-specific way to affect the protein homeostasis. .

Unlike non-specific approaches to Hsp90 inhibitors, selective estrogen receptor-degrading drugs (SERDs) and selective androgen receptor-degrading drugs (SARDs) are capable of achieving specific target protein degradation.

Interestingly, however, these small molecule compounds were originally discovered during the development of modulators of protein function. They were not originally intended to achieve target protein degradation, but were later inadvertently found to be capable of degrading related proteins.

There are some drugs like SERDs/SARDs, but since the discovery process of these drugs is not based on rational design, this unintentional mechanism of action is not universal and difficult to apply to drug design at other targets.

But in the 1990s, Proteinin’s scientists used a very interesting way to specifically induce protein degradation. This approach is based on the intracellular ubiquitin-proteasome system.

The normal degradation process of most proteins is done by the intracellular proteasome, which is the main mechanism for most protein turnover and misfolded protein clearance in eukaryotic cells. These proteins are recognized by E3 ubiquitin ligase and then ubiquitinated, and then transported to the protease to degrade into multiple peptides.

With the deepening of protein homeostasis research, some scientists have begun to try to use the ubiquitin-proteasome system to achieve specific degradation of target proteins.


In 1999, the John Kenten team at Proteinex submitted a patent application to protect the bifunctional molecules they found to link the target protein to the ubiquitin ligase E3.

The bifunctional molecule comprises a ubiquitin recognition structure and a target protein binding structural fragment, which ubiquitinates the target protein and induces its degradation. The picture on the first page of the patent clearly depicts the mechanism of action of this class of molecules. But Kenten’s then boss was not interested in the new technology, and Kenten left the company afterwards.

It was probably at that time that scientists at academic institutions, including Craig Crews of Yale and Raymond Deshaies of Caltech, published bifunctional small molecules of peptides that could induce protein degradation to some extent. They call such small molecules PROTAC, Proteolysis-TArgeting Chimeras.

Although the first generation of PROTACs were able to induce ubiquitination of target proteins, these compounds have poor cell-level activity. This is probably due to the poor ability of the peptide to cross the cell membrane. These compounds are not drug-like, so pharmaceutical companies are not interested in these compounds.

Many of the second generation of PROTACs are designed based on the very short amino acid recognition sequence of VHL, and some compounds introduce cell-penetrating peptide sequences into the structure. But this is not a breakthrough in progress. In the years that followed, there has been no concern in this field.

For pharmaceutical chemists, they are already familiar with the design principles of small molecule drugs. Such bifunctional small molecules do not meet the previous chemist’s perception of small molecule drugs, and it is not easy for them to accept this compound design.

Although peptide-based PROTACs demonstrate a certain potential for this technique, at least in vitro assays are active. However, it is clear that peptide-based PROTACs still have many disadvantages. It is imperative to design PROTACs based entirely on small molecular ligands.

In 2008, Crews formed a team of chemists and biophysicists to explore non-peptide PROTACs. At the end of the year, they found a small molecule that recruited E3 ubiquitin ligase using nutlin ligands, but the activity of this compound was too weak to develop into a drug. In the following years, they synthesized hundreds of small molecule drugs and resolved many protein crystal structures.


For Crews Labs, the biggest challenge is indeed finding the right E3 ubiquitin ligase ligand. This is not a simple task. Finding such a ligand also means interfering with the interaction of multiple protein-complexed complexes, which is a historical problem.


In 2009, Alessio Ciulli, then a postdoctoral fellow at Crews Labs, began trying to solve this problem by finding a suitable E3 ubiquitin ligase small molecule ligand. After Ciulli set up his own research group, he was still conducting the research.

In early 2012, they finally found a small molecular ligand for VHL, a critical victory in the field of induced protein degradation. In that year, they reported HaloPROTACs based on VHL small molecule ligands.


At this time, pharmaceutical companies began to take an interest in the induction of protein degradation technology. GSK established its own protein degradation research group and began working with Crews Laboratories.


In 2015, two new discoveries set off a frenzy in the field of protein degradation. Bradner, a chemical biologist at the Dana-Farber Cancer Institute, published an important study using thalidomide to bind a ubiquitin ligase cereblon. Almost at the same time, Crews Labs published its own protein-degrading small molecules.

This is two studies in vivo, which also indicates the official opening of the era of induced protein degradation. And at this time, this technology has also attracted the attention of investment institutions. This technology investor who is likely to explore 80-90% of human protein targets will certainly not let go.


Crews later established a company, Arvinas, to commercialize PROTACs technology. In 2016, Bradner and other scientists founded C4 Therapeutics. In 2017, Atlas invested in Kymera.

These companies quickly established partnerships with large pharmaceutical companies, including Merck, Novartis, New Base, GSK, Takeda, and AstraZeneca.

With the advent of large pharmaceutical companies, pharmaceutical chemists are beginning to rethink the drug design of small molecules. These small molecule drugs do not follow the five principles of the Lipinski class of drugs. For example, the molecular weight of small molecules that induce protein degradation is too high, and the number of hydrogen bond receptors and donors is too large.


In addition to the unappealing structure of the compounds, these protein degradation agents differ significantly from traditional small molecule drugs in target binding, selectivity, and dosage.


For PROTACs, the tightness of binding of the molecule to the target is not the most important, and most importantly, the rate at which these bifunctional molecules link the target protein to the E3 ubiquitin ligase.


Moreover, such small molecule drugs only need to interact with the target protein, and do not need to enter the binding cavity of the protein as the traditional drug and directly affect the function of the protein. Since approximately 75% of proteins lack the active sites required for traditional small molecule inhibitors, the induced protein degradation technology has a huge advantage over traditional small molecule drug development models.


And there is already a lot of evidence that the selectivity of PROTACs is not as high as that of traditional small molecule drugs compared to traditional small molecule drugs.


New drug developers often hope that small molecule drugs only bind to target proteins, reducing the impact on other targets, thereby reducing the side effects caused by off-target effects of small molecule drugs. But scientists have found that some less selective small molecule inhibitors can selectively induce target protein degradation after making PROTACs.

Although the targets reported for the action of small molecule compounds that induce protein degradation are known targets, Arvinas is also a mature target for drug targets in clinical research, but there is no doubt that many people believe in this technology. potential. And researchers are also using a range of strategies to find small molecule ligands that can bind to difficult targets, such as screening methods that use NMR or DNA-encoding compound libraries to screen for lead compounds that bind to targets.

In fact, both cell level tests and animal model tests have proven that PROTACs technology is not limited to only one compounding tool, but there is a relatively large possibility to be developed as a new way of drug development.

Based on the unique nature of PROTACs, this technology will likely allow us to explore a broader range of drug targets, enabling us to conquer targets that have long been considered to be non-pharmaceutical.

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