tumor suppressor
Rather than neutralize disease-causing proteins by deploying small-molecule inhibitors, some developers would destroy protein targets by ushering them to the proteasome, the cell’s garbage disposal system. This alternative approach, which applies tags to proteins in order to consign them to oblivion, could be effective against currently undruggable targets. [Juan Gaertner/Science Photo Library/Getty Images]

Researchers have learned to hijack cells’ natural protein turnover mechanism to degrade disease-causing proteins throughout the human body. Engineered small-molecule drugs recruit the ubiquitin proteasome system (UPS) to the target protein with a high degree of selectivity, providing advantages over other targeted therapies.

A handful of targeted protein degrader drugs have already reached clinical application, with many additional therapies entering the pipeline as companies and academic researchers continue to develop new molecules and mechanisms. So far, the field has primarily focused on cancer therapeutics because aberrant proteins in cancer cells are obvious targets. However, targeted protein degradation technology could eventually be applied to myriad diseases and disorders, including those affecting the central nervous system.

Designing/optimizing degrader molecules

“We’re about to translate awesome new modalities that have really incredible pharmacology into drugs that we believe will make a huge difference to patients,” says Stewart L. Fisher, PhD, chief scientific officer, C4 Therapeutics.

C4 Therapeutics is one of many biotech companies developing and testing PROteolysis TArgeting Chimeras, known as PROTACs. These are small-molecule drugs that recruit specific E3 ubiquitin ligases to transfer polyubiquitin chains onto target proteins, thereby marking them for degradation by the cell’s native proteasome.

C4 Therapeutics
C4 Therapeutics is advancing monofunctional and bifunctional degradation-activating compounds—MonoDACs™ and BiDACs™, respectively. MonoDACs, or “glue degraders,” bind to and create a new surface on E3 ligases to enhance the binding of E3 ligases to target proteins. BiDACs, or “heterobifunctional degraders,” are designed so that one end binds to the disease-causing target protein and the other end binds to the E3 ligase.

Unlike many existing drugs, such as inhibitors, targeted protein degraders need not bind to an active site on a protein. So long as the degrader forms a ternary complex with the E3 ligase, the protein can be ubiquinated, and degradation will occur. “This is opening up space where we can go after traditionally undruggable proteins,” notes Fisher. “We’re not limited to just that binding site.”

Most existing PROTACs rely on bifunctional degradation activation compounds (biDACs), which are heterobifunctional molecules featuring one site that binds a target protein and another that binds an E3 ligase. However, targeted protein degradation can also be achieved using monofunctional degradation activation compounds (monoDACs), also known as molecular glues or glue degraders. These bind to either the E3 ligase or the target protein and chemically modify the surface, prompting protein-protein interactions that ultimately cause the E3 ligase to bind to the target protein, resulting in degradation.

Fisher says that monoDACs tend to be smaller molecules than biDACs, which could simplify optimization with respect to bioavailability and compliance with drug development guidelines. However, monoDACs’ reliance on protein-protein interactions can weaken their ability to select the target of interest.

“One of the things that I think differentiates us at C4 Therapeutics is that we use both of these approaches as the targets present opportunities,” maintains Fisher. “I’m not aware of other biotech firms in this space that have the capability to focus on both monoDACs and biDACs.” C4 Therapeutics currently has four PROTACs in preclinical trials: an IKZF1-targeting monoDAC for treating hematologic malignancies, and a BRD9-targeting biDAC for treating sarcoma.

All of C4 Therapeutics’ degrader molecules recruit the same E3 ligase, known as cereblon. Along with the Von Hippel–Lindau (VHL) ligase, cereblon is currently one of the most commonly recruited E3 ligases in targeted protein degradation. Fisher says that his company decided to invest deeply in cereblon because it is involved in the molecular action of well-known drugs such as thalidomide. Clinical history indicates that the ligase’s action is well tolerated and unlikely to cause severe side effects.

Expanding the E3 ligase toolbox

Although PROTACs have been on the scene for a relatively short time, resistance mechanisms have already cropped up in preclinical trials. Most frequently, cancer cells evolve to downregulate the E3 ligases that the PROTACs depend on for polyubiquination of their target proteins. Furthermore, some proteins of interest are not effectively degraded using cereblon or VHL. Developing PROTACs that recruit E3 ligases other than VHL or cereblon could help bypass resistance mechanisms and expand the range of viable targets. In particular, some scientists have highlighted the potential advantages of focusing on E3 ligases that serve essential pathway roles. Taking this approach could make it harder for cells to downregulate them in response to PROTAC application.

“The creation of novel E3 ligands is the future of targeted protein degradation,” says Jing Liu, PhD, executive director of medical chemistry, Cullgen. The company has identified multiple ligands that bind E3 ligases not previously exploited for targeted protein degradation, and has confirmed that these ligands can be incorporated into bifunctional degrader molecules.

ubiquitin-mediated, small molecule–induced target elimination (uSMITE) technology
Cullgen is developing ubiquitin-mediated, small molecule–induced target elimination (uSMITE) technology. This image shows the chemical structure of one of Cullgen’s selective degraders. Notice that the structure consists of three moieties: one for binding the target (in this case, tropomyosin receptor kinase A); one for binding cereblon (which forms part of the E3 ubiquitin ligase complex); and one for linking the other two moieties.

Liu says that roughly 50% of Cullgen’s research and development efforts are aimed at developing novel E3 ligands, whereas the remaining 50% of these efforts focus on developing the company’s existing internal pipeline of targeted protein degraders. Cullgen has built a library of linkers with different chemical and physical properties, allowing for efficient drug optimization.

For example, Cullgen previously reported on its creation of potent and selective degraders for tropomyosin receptor kinase A (TRKA), a key target for cancer treatments. However, the initial degrader molecules—CG416 and CG428—showed low oral bioavailability, so company scientists returned to the laboratory and created second-generation degraders—CG1037 and CG1054—for the same targets. These second-generation molecules showed higher oral bioavailability in mouse models without sacrificing efficacy or causing significant side effects.

Liu points to this process of developing TRKA degraders as a proof-of-concept example, saying, “We can utilize such pinpoint targeting capabilities to develop degraders with unique selectivity profiles to treat different diseases.”

Implementing location specificity

Suresh Kumar, PhD, senior director and head of discovery, Progenra, says that avoiding resistance isn’t the only reason to develop PROTACs that recruit E3 ligases other than cereblon and VHL.

“If your target is a membrane protein, and you want to degrade that protein with a PROTAC,” Kumar says, “that job is better done with a membrane-targeted E3 ligase than with a nuclear-located ligase.”

For example, K-Ras, a well-known yet famously elusive oncological therapeutic target, is a membrane protein. Kumar says that to his knowledge, Progenra is “currently the only company that has a membrane-targeted ligase.” At multiple conferences in September and October 2020, Kumar and his colleagues presented experimental results from the development of a potent, membrane-targeted PROTAC capable of degrading K-Ras with high specificity.

Over the past 15 years, Progenra has developed and utilized a proprietary platform that it calls UbiPro, which consists of a series of enzyme activity assays that “closely replicate physiological milieu.” Progenra uses this platform for drug discovery involving both E3 ligases and deubiquitinases, another group of enzymes in the UPS. The platform has the capacity for high-throughput screening, with a panel of over 30 purified E3 ligases that can be applied for profiling and selectivity.

Kumar expects that Progenra and the rest of the PROTAC field will eventually expand far beyond cancer therapeutics. “Our ligases have extremely high relevance to human biology, with implications in diseases ranging from cancer to Parkinson’s disease, Alzheimer’s disease, and inflammatory disorders,” he insists. “All these human diseases have an underlying problem at the fundamental cellular level in terms of degrading proteins—either lack of degradation or excessive degradation.” Progenra is currently evaluating novel PROTACs as anti-inflammatory agents but has not yet made further details public.

Bypassing E3 ligases entirely

Even as PROTAC technology continues to advance, there remain limitations. Recruiting specific E3 ligases means relying on a relatively narrow set of chemical structures that bind those ligases. Those molecules can present design challenges and limit target scope. Amphista Therapeutics decided to let other companies tackle PROTACs, and instead secured funding to pursue novel methods of targeted protein degradation.

“The ubiquitin proteasome system is, in many ways, one of the most rubbish enzyme systems there is, because it’s really very poorly selective,” says Ian Churcher, PhD, chief scientific officer, Amphista Therapeutics. “If you get substrates close enough for long enough to the ubiquitin proteasome system, they will be degraded. And that’s really what we set out to do.”

Amphista has designed multiple “magnet” ligands that are believed to recruit multiple UPS proteins and activate multiple parallel degradation pathways that depend on critical cellular components. This approach makes it more difficult for cancers to develop resistance. Researchers incorporate these magnet ligands into bifunctional molecules that bind to target proteins and bring them into proximity with the UPS.

“We don’t believe anyone has ever used these molecules before in this way,” remarks Churcher. “We often think of it as next-generation targeted protein degradation. It’s an amazing field with huge potential, but we want to expand that potential into more drug targets, better profiles, and better dosing routes for patients.”

Amphista is currently developing two types of small-molecule drugs that utilize these novel mechanisms. One recruits deubiquitinase enzymes, whereas the other recruits the proteasome directly. Amphista plans to bring its first molecule into the clinic in 2023.

Initiating protein self-sabotage

Jian Jin, PhD, director of the Mount Sinai Center for Therapeutics Discovery, is the lead author on a February 2020 paper in Nature Chemical Biology describing MS1943, a first-in-class selective degrader for histone methyltransferase EZH2. This degrader relies on a technique known as hydrophobic tagging, which Jin says has been understudied by the biomedical community thus far.

At Mount Sinai’s Icahn School of Medicine, the laboratory of Jian Jin, PhD, used a hydrophobic tagging approach to generate MS1943, a first-in-class degrader of EZH2, a protein that is overexpressed in multiple types of cancer. Importantly, MS1943 has a profound cytotoxic effect in multiple triple-negative breast cancer cells.

Hydrophobic tagging involves attaching a bulky hydrophobic chemical group to a small-molecule binder of the target protein—in this case, EZH2 inhibitor C24. Jin indicates that the mechanism of degrader action is not yet fully elucidated, but scientists believe that the presence of the large hydrophobic group causes the protein to misfold, which ultimately triggers its degradation via the natural action of the UPS.

MS1943 showed high potency in triple-negative breast cancer cells while sparing normal cells. Furthermore, the compound showed high oral bioavailability, which Jin hypothesizes may be due in part to the molecule’s smaller size compared to most PROTACs.

“Now that we have published this approach, I think more and more research groups will explore this technology,” predicts Jin. “We are actively optimizing these compounds and will hopefully progress into clinical studies.”

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