The PROTAC was first proposed by the team of Professor Craig Crews of Yale University in 2001. In 2008, the team reported the first small molecule PROTAC to achieve the degradation of androgen receptor, which became an important turning point in the development of this field. Recently, the team of Professor Craig Crews published a review article entitled Protein degraders enter the clinic — a new approach to cancer therapy in Nature Reviews Clinical Oncology, reviewed the technical progress of PROTAC and analyzed the future development direction and potential challenges.
Figure 1. Major milestones in the development of protein degraders (Source: References )
In 2019, two PROTAC degraders from Arvinas, entered clinical trials, namely ARV-110 (NCT03888612) and ARV-471 (NCT04072952), which target AR and ER respectively. This has also caused PROTAC technology to receive extensive attention from the pharmaceutical industry and academia.
Table 1: PROTAC-targeted protein degraders in clinical development
Table 2. Molecular glue degraders in clinical development
1. Mechanism of Action of PROTACs
A PROTAC is composed of three parts: an E3 ligand that binds to an E3 ligase, a ligand that binds to the protein of interest, and a linker connecting the two ligands. E3 ubiquitin ligases mark target proteins as defective or damaged by attaching a small protein (i.e., ubiquitin). The cell's protein shredder (i.e., the proteasome) then recognizes and degrades the labeled target protein. PROTACs achieve degradation through "hijacking" the cell's ubiquitin–proteasome system (UPS) by bringing together the target protein and an E3 ligase. First, the E1 ligase activates and conjugates the ubiquitin to the E2 ligase. The E2 ligase then forms a complex with the E3 ligase.
Figure 2. Mechanism of action of protein degraders vs. small molecule inhibitors in cancer (Source: References )
PROTAC protein depressors can eliminate, rather than merely inhibit, target proteins, promising to improve many of the limitations of traditional inhibitors. Its main advantages include event-driven activity, targeting non-patent proteins, overcoming resistance, and low doses.
The mode of action of traditional small molecule inhibitors needs to "occupy" the active site of the target protein and block the transduction of downstream signaling pathways under the condition of high drug concentration. Also, "druggable" proteins need to have binding pockets that small molecules can occupy, and 80% of proteins without binding pockets are generally considered "undruggable".
The mode of action of PROTAC is completely different. It is not "occupancy-driven" to affect the function of the protein, but "event-driven" to mediate the degradation of the target protein. It does not need to act on the active site of the protein, but only needs to have a certain binding rate with the target protein. Therefore, protein degradation agents have more target protein binding partners to choose from, which is expected to overcome the "undruggable" target in the traditional sense.
2. Target Selection of PROTACs
The molecular weight of PROTAC is larger, current suitable target selection for PROTACs can have several characteristics:
- ▶Traditional non-druggable targets: such as KRAS and STAT3 that are already entering clinical practice;
- ▶Targets that have developed resistance to existing therapies: such as BTK C481S mutation;
- ▶Protein expression deviates from the natural state, such as overexpression, aggregation, isoform expression; such as AR;
- ▶Scaffold proteins (proteins without active sites): such as IRAK3, IRAK4
3. Selection of E3 Ligase
There are over 600 E3 ligases in the human body, and only about 10 have been developed so far for use as protein degraders (CRBN, VHL, IAP, MDM2, DCAF15, DCAF16, RNF114, etc.).
CRBN and VHL, which are the most widely used E3 ligases, have the highest scope and can degrade a wide range of target proteins flexibly and efficiently, and achieve a high level of systemic degradation with relatively wide expression.
In addition, there is growing interest in tissue - or tumor-specific ligases, which are less toxic and therefore have a wider therapeutic window.
New discovery platforms based on covalent ligand screening have shown potential for rapid identification of novel E3 ligand ligands, and several covalent ligands for RNF4, RNF114, DCAF16, KEAP1, DCAF11, and FEM1B have been identified.
4. Clinical And Preclinical Progress of PROTACs
Protein degraders have shown beneficial properties both preclinical and clinical. In the field of oncology, preclinical data show that compared with small molecule inhibitors, PROTACs show better target specificity and efficacy in inhibiting tumor growth, and they are active against drug resistance mutations generated after small molecule inhibitor treatment.
As of now, there are at least 20 PROTAC projects in the clinical stage around the world, of which the fastest progress is ARV-471, a collaboration between Arvinas and Pfizer, which started phase III clinical trials in 2022. Now, the mode and therapeutic activity of PROTACs have been verified in the clinic by ARV-110, ARV-471 and NX-2127, etc. Preliminary data on protein degraders targeting androgen receptor, estrogen receptor and BTK show encouraging clinical activity in patients with prostate cancer, breast cancer and chronic lymphocytic leukemia, respectively, with more expected results of ongoing clinical studies.
Figure 3. Clinical effects of PROTACs (Source: Reference )
5. Future Directions of PROTAC
A key next step for PROTACs is to determine whether this approach can actually break through the degradation of undruggable targets. At present, KT-333 (STAT3 degrader) and ASP3082 (KRAS G12D degrader) have obtained preliminary data of phase I clinical research. In addition, targeted delivery has also shown promise in protein degraders to minimize potential toxicity. A variety of targeted delivery strategies have been developed, including antibody-conjugated protein degraders, light-controlled protein degraders, etc. In addition to tumors, protein degradation technology has broad therapeutic potential and may be applied in other diseases in the future (for example, IRAK4 degraders for autoimmune diseases).
In the future, we expect more clinical transformation results, and explore how to find better PROTAC molecules to solve the current research and development pain points. Specifically, in the next 20 years, PROTAC will develop in the following directions:
- ▶Identify and clinically validate the target types that are more suitable for degradation technology;
- ▶Expand the range of E3 ligase to achieve precision therapy;
- ▶Expanding the field of clinical treatment beyond cancer;
- ▶Clinical validation of targeted protein degradation patterns beyond molecular glues and PROTACs.
6. Potential Challenges of PROTACs
One unresolved question with this technology is whether patients will develop resistance to protein degraders.
Most drug resistance reported in preclinical studies occurred due to alterations in the ubiquitin-proteasome system rather than the target protein. A preclinical study suggests that upregulation of the multidrug resistance-1 (MDR1) gene is a mechanism for resistance to protein degraders; coadministration with MDR1 inhibitors may help overcome this resistance.
Evaluation of patients on long-term treatment with protein degraders is also needed to confirm whether the resistance mechanisms observed in preclinical settings also occur in the clinical setting.
In addition to possible drug resistance, another challenge in PROTAC development is the design of new molecules. For example, transmembrane protein targets are often bound extracellularly, such as GPCRs, without physical access to the cytoplasmic ubiquitin-proteasome machinery that drives protein degradation mechanisms.
In addition, although undruggable targets are a potential application of PROTACs, it is still difficult to identify the binding sites of degraders. However, in the molecular design of protein degraders, the DEL technology mentioned above and new technologies complementary to protein degraders may overcome these obstacles.
Since PROTAC was discovered by Craig Crews twenty years ago, the technology has now entered the stage of practical application. Although PROTAC is a small molecule innovative drug with high research and development difficulty, its marketing regulation and supply chain have been relatively mature, and the competition pattern is good. With druggability and other challenges overcome, PROTAC is highly likely to replicate or even surpass the successful development cycle of small molecule inhibitors and immunotherapy, and embrace a golden age of explosion in the next decade.
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.Protein degraders enter the clinic — a new approach to cancer therapy.
.Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase.
.PROTACs and Targeted Protein Degradation
.Overview of New Targets And Technologies of PROTAC
.Summary of PROTAC Degraders in Clinical Trials
.Four Major Trends In The Development of PROTAC
. PROTACs VS. Traditional Small Molecule Inhibitors