Protein Splitters
"Shredders" for harmful proteins
Protein-killing drugs can become a powerful weapon in the fight against dangerous diseases. About these drugs, which are still in development, – in an article published in the journalNature.
When Craig Crews first managed to make proteins disappear "on command" thanks to a strange new compound, the biochemist called this disappearance a "strange trick", a "funny curiosity".
Today, this funny trick receives investments in billions of US dollars from major pharmaceutical companies such as Roche, Pfizer, Merck, Novartis and GlaxoSmithKline. "I think you can conclude that almost every company has research programs in this area," says Raymond Deshais, senior vice president of global research at the American biopharmaceutical company Amgen.
The "trick" in question – or rather, the therapeutic strategy – is called targeted protein degradation. The cell is cleansed of unwanted or damaged proteins by a special PROTAC molecule aimed at proteolysis, the process of protein hydrolysis (English PROteolysis–TArgeting Chimaeras). It can take a variety of forms.
This "protein cleaver" can bind to hard-to-reach proteins that other drugs cannot reach. They can be used as an effective tool in the fight against proteins that drug developers have long considered "unkillable": cancer-fueling villains, such as MYC protein, or tau protein, which clots in Alzheimer's disease.
The new area is very promising. In 2014, scientists discovered that lenalidomide, one of the world's best–selling drugs used in the treatment of myeloma, works similarly to protein splitters: the drug literally "chews" two previously untouchable proteins.
However, there is no published data in this area confirming that PROTAC and other similar compounds can lead to the disappearance of harmful proteins. And there are still questions about where and how these strange-looking molecules will work in the body.
Now the leading company that is working in a new direction is Arvinas, a biotech company in New Haven (USA, Connecticut) founded by Craig Crews. His team plans to start testing the action of PROTAC on prostate cancer soon, and try to attack the protein, which, however, is successfully affected by other drugs. "We are close to proving that these PROTACs can be drugs," says Ian Taylor, senior vice president of biology at Arvinas.
"Academic exercise"
PROTAC is often depicted in pictures as dumbbells. They are molecules consisting of two ends connected by a thin cable. The main force is concentrated on the edges of the "dumbbells". One catches a harmful protein, and the other captures ubiquitin ligase – a "cog" of the cell's natural waste disposal system, which marks defective or damaged proteins by applying another – small – protein called ubiquitin to them. These "ubiquitin tags" act as a kind of "Please remove" stickers that signal the protein "shredder" – the proteasome – to do its job.
By simply combining a ligase and a "harmful" protein, PROTAC ensures that the target is marked for destruction. Ligases are effective, and ubiquitin is abundant, so one PROTAC molecule must repeatedly perform its cell purification function and catch damaged proteins many times. At the same time, a small amount of the drug should be used for pronounced activity.
The first known published description of a molecule like PROTAC is given in a patent application filed in 1999 by two scientists from Proteinix, a biotech company in Gaithersburg (USA, Maryland). Authors John Kenten and Stephen Roberts proposed using a protein degradation system in a cell. Colleagues rejected this idea, saying that Kenten and Roberts complicate drug discovery by trying to bind two proteins – an unwanted protein and a ligase– simultaneously.
But on the other side of the United States, another pair of minds were pondering the same idea. During a field research seminar in 1998 in northwest Washington, Deshais stopped in front of a poster of Krues to listen to him talk about using small molecules to connect two proteins. Deshais, then a biochemist at the California Institute of Technology in Pasadena, was completely immersed in the study of ubiquitin ligase at the time. The human genome encodes approximately 600 similar enzymes-ligases, which should then "mark" harmful proteins. About a year ago, Deshais and Kruse discovered three families of proteins that contain 250 ubiquitin ligases.
At that time, Krews was developing a drug that acted the opposite of PROTAC. It blocked the ubiquitin system in cells, causing proteins to accumulate to dangerous levels and eventually cause cell death. The result of this work – the drug carfilzomib (or Kiprolis) – is now used for the treatment of multiple myeloma of blood cancer. "I thought that the work "on the contrary" would be just as interesting," says Kruse.
Soon, Krews and Deshais published an article describing the study, which shows that their first PROTAC, Protac-1, successfully captured and degraded the cancer-related protein METAP2 in extracts from eggs of spur frogs (Xenopus).
However, Protac-1 could not yet be presented as a real medicine, says Krews, who called the article an "academic exercise." PROTAC of the first generation had low activity in human cells, probably due to the fact that the compounds struggled to get inside. They relied on large, bulky peptides to bind to ligases. Scientists had to find a way to make the ligase-protein binding agent more like a medicine. "Something that could become a pharmaceutical drug," says Krews.
Having received financial and research support from the large British pharmaceutical company GlaxoSmithKline, Krews moved forward, focusing mainly on one specific ligase, the von Hippel-Lindau tumor suppressor (VHL syndrome). In 2012, Krews and colleagues in the laboratory reported that they had found small molecules that would bind the VHL ligase to the damaged protein. The scientist finally began to believe that PROTAC can really be made into a medicine.
Hunting for Small Molecules
Crews wasn't the only one chasing protein shredders. In 2010, while at the Institute of Oncology named after Dana-Farber (USA, Massachusetts), biochemist James Bradner read the report of a group of researchers from Japan led by Hiroshi Handa. Handa was trying to understand why the infamous drug thalidomide, approved in some countries in the late 1950s and early 1960s for the treatment of nausea during pregnancy, caused problems in newborn children with limb development. (Currently, the treatment of multiple myeloma and leprosy is approved with this medication.) Using thalidomide as a bait to search for proteins in cells, Handa discovered that the drug clings to and blocks the activity of one ubiquitin ligase, the cerablon protein. As experts from the Handa team found out, inhibition of this protein affects the growth and development of limbs in danio fish and chicks: fish and birds had the same defects as children whose mothers took medications with thalidomide during pregnancy.
Bradner realized that if thalidomide binds to ubiquitin ligase – which is not the easiest task, because such enzymes are notoriously difficult to obtain – then perhaps he could find a way to bind the drug to the same ligase, but target it to proteins involved in the disease. In 2013, Buckley joined Bradner's team, and they began searching for small molecules that bind to cereblone.
In May and June 2015, three teams – led by researchers Bradner, Chully and Krues – published five separate papers describing small PROTAC molecules with powerful activity, which is observed in drugs. Krews linked PROTAC to VHL and used it to reduce the number of several proteins to less than 10% in those cells that had not yet been treated with the drug. Bradner and his colleagues linked cereblone to their PROTAC molecule to lower levels of the cancer-causing protein, and Chully and his team at the same time decomposed the same protein using VHL as a ligase. Protein "shredders" worked properly both in the cells in the cup and in tumor cells in mice.
Along with the development of drug–like protein "destroyers", the Crews and Bradner teams have created two systems – HaloPROTACs10 and dTAG12, respectively - that allow researchers to apply the protein degradation method as a tool in the laboratory, using genetic tags to label proteins that need to be destroyed in cultured cells and in mice. With dTAG, "you can deplete protein in minutes or hours and keep track of what's going on," says Behnam Nabet, a chemical biologist who led the development of the system with Nathanael Gray at the Cancer Institute. Dana-Farber. – This gives you great opportunities to study oncogenes, kinases and proteins that have very fast activity." dTAG materials are currently freely available: more than 150 academic laboratories use a sample of the drug to investigate the effects of depletion of certain proteins in cells, says Nabet.
Bradner, who left Dana-Farber in 2016 to become president of the Novartis Institute for Biomedical Research, estimates that about 30 individual tools already include this technology.
Gold Rush
After a sharp rise in the popularity of low–molecular-weight PROTACs in 2015, Deshais wrote an article with the opinion that PROTAC has the potential to become a major new class of drugs that can surpass the two hottest areas of drug development of all time - protein kinase inhibitors (antitumor agents) and monoclonal antibodies. "The gold rush has begun!" the scientist wrote at the time.
Since then, according to Deshais, the fever has only intensified. He joined Amgen in 2017 and now oversees the company's work in the field of PROTAC.
According to Taylor, the Arvinas study, which is due to begin by mid-2019, will involve 28-36 men with metastatic prostate cancer, and it will last about nine months. During the study, scientists will test a new class of drugs with the PROTAC molecule, which destroys the androgen receptor, a protein that has previously been targeted by several approved drugs. The company hopes that by decomposing, rather than suppressing the receptor, its PROTAC will be able to treat people who no longer experience the benefits of existing drugs. And if the candidate succeeds, this field will finally have clinical data. Arvinas will show that PROTAC can be a cure.
This is very important because there have been many doubts about whether protein destructors can work in the human body. Fully assembled PROTACs violate generally accepted practice rules for medicines. The main one among them is the size. A good low molecular weight drug usually has a mass of less than 500 daltons. The current PROTAC molecules weigh more than 1000 daltons. However, the molecules can still penetrate the cells. According to Krews, this is because the molecule is probably recognized by the cell membrane as two smaller molecules that turn out to be bound together, and not as one large one.
Another advantage of PROTAC. Most low-molecular-weight drugs or monoclonal antibodies need to interact with the active site of an enzyme or receptor to do their job. But 80% of proteins in human cells do not have such a site. PROTAC can grab the protein anywhere, by any crack – they do not need to sit in the "active pocket" to work.
There is already some evidence to support this property. Last year, a team from the Institute of Cancer Research in London created a small molecule that can bind to a transcription factor regulator that does not have an active site. Scientists were able to create a powerful PROTAC by attaching a binding agent for cerablon ubiquitin ligase.
In this area, there is still no published data on the PROTAC molecule, which can target an important, drug-resistant protein and destroy it. The Arvinas team claims to have in vivo evidence that PROTAC molecules degrade tau protein in the brains of mice. On its website, the company reports that injection with a molecule that decomposes tau directly into the mouse hippocampus reduces the level of this protein by 50%.
By developing PROTAC for a range of diseases, including those that affect the brain, many researchers hope to show that the technology is "independent of the therapeutic area" (in Taylor's words). Various groups are also working to expand the pool of ligases that can recruit protein "disruptors". Currently, only four main ones are used, including VHL and cerablon, and a wider range of available ligases may allow drug developers to match the most effective combination of ligase-PROTAC with their cell type or protein of interest. "Potentially any ligase can be captured using this approach," says Chully, who collaborates with the German pharmaceutical company Boehringer Ingelheim in the development of PROTAC.
Building on new goals, improved PROTAC capabilities, and an upcoming clinical trial, the researchers are ready to prove that protein "cleavers" can be more than just a "trick." It's just a matter of time," says Chully.
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