Targeted protein degradation (TPD) is an emerging protein degradation technology in recent decades that utilizes the cell’s own protein clearance mechanisms, such as lysosomal degradation and proteasomal degradation, to degrade target proteins. TPD encompasses various subfields, including molecular glue, PROTAC, LYTAC, AUTAC, AbTAC, and others. Compared to traditional small molecule inhibitors, TPD technology significantly expands the range of druggable protein targets. Moreover, it can eliminate all functions of the target protein upon degradation until the protein is resynthesized. Additionally, TPD can minimize potential drug resistance, is recyclable, and works at catalytic doses, enhancing drug safety. It is currently one of the hottest areas of research in the pharmaceutical industry.
Currently, the FDA has approved three molecular glue drugs: thalidomide (Contergan), lenalidomide (Revlimid), and pomalidomide (Pomalyst), for the treatment of multiple myeloma, myelodysplastic syndromes, and other conditions. In contrast, other subfields of TPD technology have not yet seen FDA-approved drugs. Among them, PROTAC molecules have made the most progress, with 29 PROTAC molecules advancing to clinical trials, including 21 in Phase I, 7 in Phase II, and 1 in Phase III as of September 2023.
While targeted protein degraders and traditional small molecule inhibitors both fall within the category of small molecule drugs, they still exhibit significant differences in certain characteristics. In theory, targeted protein degraders differ from traditional small molecule inhibitors in following key aspects:
Independent of Ligand Binding Mode
Targeted protein degraders do not rely on specific binding modes between ligands and their target proteins (e.g., agonism, partial agonism, antagonism). They only require binding to the target protein to effectively recruit ubiquitin ligases for protein degradation. This simplifies the ligand screening process for targeted protein degraders, eliminating the need to focus on whether the small molecule is an agonist or antagonist. For example, androgen receptor (AR) inhibitors on the market, such as enzalutamide, exert their anti-tumor effects by antagonizing AR receptor activity. However, in certain mutated tumor cells (e.g., H875Y, T878A), small molecule AR-targeting drugs can switch from inhibitory to partial or complete agonist activity, promoting tumor cell growth.
Arvinas has developed AR receptor degrader ARV-766, which effectively overcomes the limitations of AR inhibitors in patients with ligand-binding domain mutations (T878/H875/L702). In these patients, 42% responded significantly to the drug, leading to a reduction of blood PSA levels by over 50%.
Activity at High Target Protein Levels
Some small molecule inhibitors, when used to inhibit target activity, may lead to increased expression of the corresponding target protein within cells to adapt to the inhibition. This can result in a loss of efficacy for small molecule inhibitors. Intravenously administered MDM2 degrader KT-253, developed by Kymera Therapeutics, can overcome the limitations of MDM2 small molecule inhibitors in mouse models. It rapidly degrades MDM2 protein in vivo, leading to tumor cell death. A single dose of KT-253 treatment can achieve prolonged tumor regression.
Impact on Non-Catalytic Functions
Targeted protein degraders can completely eliminate the target protein, allowing them to affect the non-catalytic functions of the protein of interest (POI), such as scaffold functions and protein-protein interactions with other proteins. For example, BRAF protein degraders. BRAF is a commonly mutated oncogene in melanoma. Traditional small molecule therapies like vemurafenib inhibit tumor growth driven by mutant BRAF by inhibiting its kinase activity. However, existing BRAF inhibitors may cause mutant BRAF to act as a protein scaffold, recruiting wild-type RAF to form signaling complexes, driving tumor cell growth, reducing drug efficacy, and potentially triggering drug resistance mutations.
CFT1946, developed by C4 Therapeutics, is a BRAF degrader that directly induces BRAF degradation, eliminating its scaffold function. Therefore, it does not activate wild-type RAF, resulting in more prolonged efficacy than small molecule inhibitors.
No Need to Occupy All Target Proteins
Targeted protein degraders can catalyze the degradation of POIs and can re-bind to other POIs after the initial target protein degradation, inducing ubiquitination. Consequently, the binding affinity required between degraders and POIs can theoretically be significantly lower than that required for small molecule inhibitors. Furthermore, POI concentration decreases over time, allowing drug efficacy to accumulate. This enables long-term dosing with lower daily doses for more sustained efficacy, enhancing the safety of such drugs.
Longer Duration of Action
GSK’s RIPK2 degrader effectively degrades RIPK2 protein in mice and exhibits far longer-lasting anti-tumor effects than predicted by the PK data of this PROTAC molecule. This is mainly because the resynthesis of RIPK2 requires close to 240 hours. Additionally, when this PROTAC molecule is encapsulated in a polymer matrix, degradation of RIPK2 protein can still be observed after 60 days of administration.
Use of Alternative Binding Sites
Targeted protein degraders only need to bind to POIs to trigger protein ubiquitination, so POI ligands do not necessarily need to have a direct functional impact on the target protein. Any successful binding can suffice. Therefore, when screening POI ligands, inhibitors, antagonists, or allosteric modulators can all be considered as screening targets. C4 Therapeutics has developed an EGFR L858R degrader based on an allosteric modulator. The POI ligand of this PROTAC only binds to the L858R mutant of EGFR and spares the wild-type EGFR protein because there is no corresponding allosteric binding site in the wild-type EGFR.
Expression Differences of E3 Ligases Can Provide Tissue Selectivity
E3 ligases may have differential expression in tissues, providing PROTAC molecules with additional selectivity beyond POI ligands, enhancing safety. For example, E3 ligases are expressed at low levels in platelets but are normally or highly expressed in tumor cells. Based on this, Dialectic Therapeutics has developed tissue-selective Bcl-XL degrader DT2216. This compound can kill tumor cells while avoiding the platelet reduction side effects associated with BCL inhibitors like venetoclax.
Effective at Lower Tissue Distribution Concentrations
Unlike traditional small molecule inhibitors, TPD can function at lower concentrations through a catalytic mechanism and accumulate efficacy over time. This opens up the possibility of treating diseases that require crossing the blood-brain barrier, such as multiple sclerosis and brain metastases. For example, Nurix Therapeutics’ BTK degrader, NX-5948, reportedly has a low brain penetration rate of less than 5%. However, it still exhibits anti-tumor activity in microglial cells, demonstrating its efficacy.
Generation of Selectivity Based on Ternary Complexes
In melanoma, mutant and wild-type BRAF proteins have very similar structures. When using small molecule inhibitors, it is highly likely to result in off-target inhibition of wild-type BRAF. However, when forming ternary complexes of BRAF protein-degrader-E3 ligase, the ability of mutant BRAF protein to form complexes is much higher than that of wild-type BRAF protein. This grants C4 Therapeutics’ BRAF protein degrader CFT1946 higher selectivity against wild-type BRAF.
Indirect Degradation of Ligand-Free Proteins
CDK4/6 plays a critical regulatory role during the G1/S transition of the cell cycle. In the G1 phase, CDK4/6 is activated upon binding with cyclin D protein. Jian Jin from the Icahn School of Medicine at Mount Sinai reported in the JACS that the PROTAC molecule MS28, which targets CDK4/6, can induce ubiquitin ligases to approach Cyclin D1 and CDK4/6. This leads to the selective degradation of Cyclin D1, which currently lacks a reported small molecule ligand, and results in stronger anti-tumor proliferation effects.
As preclinical and clinical research into targeted protein degrader deepens, the differentiated advantages of targeted protein degraders over traditional small molecule inhibitors continue to be discovered and explored. This has attracted an increasing number of researchers to engage in this field. In the case of PROTAC technology, no molecules have been approved for market use yet, but PROTACs have progressed to Phase III clinical trials at the fastest rate. Targeted protein degraders have been extensively studied in the application of mature targets, and they are especially needed for challenging drug targets and intractable diseases. By developing new E3 ligases, new POI ligands, and brain-penetrant PROTACs, among other strategies, the full potential of TPD technology can be realized, addressing unmet clinical needs.
References:
- Zhao, L., et al., Targeted protein degradation: mechanisms, strategies and application. Sig Transduct Target Ther., 2022, 7, 113.
- Holderfield, M., Nagel, T., and Stuart, D., Mechanism and consequences of RAF kinase activation by small-molecule inhibitors, Br J Cancer., 2014, 111, 640-645.
- Xiong, Y., et al., Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets, J Am Chem Soc., 2022, 144(49), 22622-22632.