Ten Ways Degraders Differentiate from Traditional Small Molecules

Targeted Protein Degradation (TPD) allows for elimination, rather than inhibition, of proteins by exploiting cells’ own protein clearance mechanisms. While traditional inhibitors can be very effective at preventing a protein from undertaking a particular function, blocking protein activity does not always have the same result as removing a protein entirely. This minireview highlights ten theoretical advantages of targeted protein degradation over traditional small molecule inhibition, with specific industry examples of bifunctional degraders demonstrating proof-of-concept for each. This follows a recent webinar we have held on the topic.

In contrast to traditional small molecule inhibitors, protein degraders are:

1. Agnostic to ligand mode of action (e.g. AR, ER degraders)
2. Able to overcome high protein levels (e.g. MDM2 degrader)
3. Able to affect non-catalytic functions (e.g. BRAF, IRAK4, BRD9 degraders)
4. Not dependent on complete target saturation (e.g. BTK, STAT3, BRAF degraders)
5. Able to show prolonged PK/PD for proteins with slow turnover (RIPK2, BTK degraders)
6. Able to use alternative binding sites (e.g. EGFR, Bcl-XL, PCSK9 degraders)
7. Able to show tissue selectivity based on E3 ligase expression (e.g. Bcl-XL)
8. Able to show selectivity based on selectivity for ternary complex formation (e.g. BRAF degrader)
9. Less dependent on tissue distribution (e.g. brain-penetrant BTK, EGFR degraders)
10. Able to degrade indirect substrates through bystander degradation (e.g. cyclin D1 degrader)

Protein degradation is a therapeutic broad area, and at Drug Hunter we have highlighted a number of degradation strategies including molecular glue degraders (e.g. IKZF1/3, IKZF2/4, GSPT1 degraders etc), monovalent degraders (e.g. SERDs, PI3Kα degrader), receptor internalization (e.g. S1P receptor modulators) and bifunctional degraders (PROTACs). There are also a number of emerging novel modalities including Lysosome-Targeting Chimaeras (LYTACs), Autophagy-Targeting Chimaeras (AUTACs) Antibody-based PROTACs (AbTACs) and ADC-degrader conjugates, and more modalities rapidly emerging. Given our recent coverage of other areas, this article will focus exclusively on bifunctional degraders.

A brief industry history of targeted protein degradation

Using modular, synthetic bifunctional molecules to recruit the ubiquitination system via E3 ligases to degrade proteins of interest was theorized as early as the late ‘90s (e.g. US6306663B1), but the first peer-reviewed reduction to practice with small molecule motifs wasn’t until Crews’ and Deshais’ proof-of-concept in 2001 with a SCF-MetAP-2 small molecule-peptide PROTAC (Proteolysis Targeting Chimera). In parallel, Celgene received approval for thalidomide in oncology in 1998, and discovered imides with distinct activities like lenalidomide (US2007021464A1, 1993 priority) and GSPT1 degraders like CC-885 whose patent applications were filed without knowledge of molecular mechanism as early as 2006 (WO2008027542A2), though they were not published until 2016. The publication of the identity of CRBN as an E3 ligase that interacts with thalidomide in 2010 catalyzed the simultaneous disclosures of the degradation mechanism of action of imide drugs (e.g. Celgene and Harvard 2014) and the structure of the CRBN E3 ligase complex with imide drugs (e.g. Celgene and FMI, Harvard, Novartis, 2014). Together with the discovery of drug-like VHL ligands in 2012 by the Crews and Ciulli labs (e.g. VH298), the imide scaffold offered a promising, drug-like starting point for bifunctional degrader discovery. 

This new mix of drug-like E3-recruiters, mechanistic understanding, and clinical validation from imide drugs, combined with a rapidly expanding biotech funding environment after 2013 was an explosive mixture for drug discovery. Companies like Arvinas (2013), Nurix (2014 pivot to TPD)  Kymera (2015), and C4 Therapeutics (2015) were funded to focus on targeted protein degradation. By 2015, several low molecular weight, small molecule bifunctional degraders were published (e.g. BET degraders from Bradner lab, Ciulli lab, and Arvinas and an ERRɑ degrader from Arvinas & GSK partnership). Arvinas took the first PROTACs into the clinic in 2019 (AR degrader ARV-110 and ER degrader ARV-471), with oral clinical proof-of-concepts reported in 2020, dispelling long-held concerns that bifunctional degraders would be challenging to render orally available. As of the publication of this article, at least 25 bifunctional protein degrader clinical candidates have been reported.

Protein degradation has been theorized to differentiate from traditional small molecule inhibition in several ways. In recent years, clinical and in vivo proof-of-concept have finally been demonstrated for many of these differences. 

Degraders are agnostic to ligand mode of action (e.g. ligand agonism vs. antagonism; AR, ER)

Unlike traditional small molecules, degraders do not depend on a specific mode of action on their target (e.g. agonism, partial agonism, antagonism, etc.) and only need to bind to their target of interest to be effective in recruiting the target for degradation. This removes a complication from screening for ligands, as with traditional small molecules, whether a molecule acts as an antagonist or agonist is not always predictable for certain targets. This angle for differentiation is most pronounced in the recent cases of androgen receptor degraders and estrogen receptor degraders. 

Androgen receptor degraders circumvent a sinister mechanism of cancer resistance

The androgen receptor (AR) is well-established target in prostate cancer, with numerous generations of AR antagonists on the market. However, the use of AR antagonists in the treatment of some prostate cancers can lead to difficulty due to a sinister mechanism of cancer drug resistance, with some mutations (e.g. H875Y) resulting in some antagonists undergoing a switch in MOA to become agonists, leading to potential acceleration in tumor growth. 

Use of an AR degrader can be advantageous as the mutation and/or functional effect is no longer as significant of an issue. For example, ARV-766, a second-generation AR degrader for prostate cancers, was optimized to have wider genotype coverage and improved stereochemical stability from its predecessor, ARV-110. In a recent trial, 42% of patients with AR ligand binding domain mutations achieved a significant response based on prostate antigen levels. 

Estrogen receptor degraders may avoid secondary cancers.

Estrogen receptor (ER) degradation is also an important angle for the treatment of breast cancer. Tamoxifen is an orally administered ER antagonist widely used within cancer therapy. However, tamoxifen is not an antagonist of the ER in all tissues and in some cases it can act as a partial agonist, contributing to secondary cancers after prolonged treatment. An alternative treatment is fulvestrant, an intramuscularly administered ER degrader that both antagonizes ER function and induces degradation, but it does not always reach target coverage concentration and has poor PK properties. There is therefore a push towards oral ER degraders with deeper ER coverage that are not susceptible to mutations leading to receptor agonism, such as Eisai/Menarini’s elacestrant, Genentech’s giredestrant, Sanofi’s amcenestrant, AstraZeneca’s AZD9833, and Arvinas’ PROTAC, ARV-471. 

Degraders can overcome high protein levels (e.g. increased expression due to feedback; MDM2)

Overcoming high protein levels due to the activation of unintended targets can be an issue, and one of the challenges with targeting MDM2 is the concomitant activation of P53, a tumor suppressor, in healthy tissues. Kymera Therapeutics has recently revealed their IV-administered MDM2 degrader, KT-253 (structure undisclosed). In contrast to small-molecule MDM2 inhibitors that increase MDM2 levels with exposure due to a biological feedback loop, this molecule rapidly degrades the target and spikes P53, therefore deeper responses can be seen quickly and intermittent dosing can be undertaken potentially leading to reduced side effects and allowing patients’ healthy cells to recover. 

Degraders can affect non-catalytic functions (e.g. scaffolding; BRAF, IRAK4, BRD9)

Since degraders can fully eliminate a target protein, they can also ablate their non-catalytic functions, such as their ability to scaffold larger complexes or signal through protein-protein interactions.

A BRAF degrader avoids paradoxical RAF activation by preventing scaffolding function.

BRAF is a commonly mutated oncogene in melanoma, and traditional small-molecule therapies such as vemurafenib inhibit the kinase activity of mutant BRAF to stop the growth of tumors driven by mutant BRAF. However, a phenomenon called ‘paradoxical RAF activation’ can occur where inhibitor-bound mutant BRAF can act as a scaffold, recruiting WT RAF to form a 14-3-3-mBRAF-WT RAF signaling complex and enhancing WT RAF signaling, contributing to secondary cancers. CFT1946 is a recently revealed BRAF degrader from C4 Therapeutics that does not cause this paradoxical RAF activation, since it degrades mutant BRAF, eliminating its scaffolding function.

A BRD9 degrader acts selectively on synovial sarcoma thanks to synthetic lethal dependence on BRD9 scaffolding.

A second example is showcased within BRD9 degraders. BRD9 is a histone reader and a component to some epigenetic complexes like BAF, which is required for chromatin remodelling and genetic functions. A number of companies have pursued inhibitors of BRD9 but its scaffolding function is important in some settings. For example, in synovial sarcoma the BAF complex is uniquely dependent on the scaffolding function of BRD9. The underlying cause of synovial sarcoma is an oncogenic gene translocation fusion between two proteins known as SS18 and SSX (SSX1 or SSX2, or rarely SSX4), which creates a synthetic lethal dependency on BRD9; cells with this fusion require BRD9 for oncogenic transcription and proliferation. Healthy tissues are not as sensitive to BRD9 degradation, therefore allowing for target of cancer cells specifically. 

An IRAK4 degrader demonstrates broader activity due to myddosome scaffolding role of IRAK4.

Traditional IRAK4 inhibitors have been of broad interest for immunology indications, but have seen limited clinical success (see our recent case study on GLPG2534 for a primer). Kymera Therapeutics’ argument for developing an oral IRAK4 inhibitor for immunology rests on the fact that IRAK4 also has scaffolding functions, helping form a key signalling complex, the myddosome. So far, their lead molecule KT-474 has shown preclinical differentiation including effects on IL-6 (which is not modulated by traditional IRAK4 kinase inhibitors like PF-06550833), and is in Ph. II to look for efficacy in humans. While its structure has not been disclosed, some potentially related patent applications have published (e.g. WO2022174269).

Complete target saturation may be unnecessary for degraders (e.g. BTK, STAT3, resistance) 

In TPD, full saturation of the target may not necessary due to the catalytic mechanism of action (each degrader molecule can degrade more than one target protein, and it is not necessary for the target to be fully occupied as a E3-degrader-target complex). Hence, the binding affinity needed between the degrader and the target of interest can theoretically be significantly lower than required for small-molecule inhibitors. Furthermore, since the concentration of the target is being reduced over time, the pharmacodynamic effect can accumulate over time, resulting in deeper efficacy on chronic dosing with a lower daily dose.

A BTK degrader is active on mutants with low binding affinity and oral bioavailability.

For example NX-2127, from Nurix Therapeutics, leads to full degradation of various strains of BTK mutants, even with low ligand affinity for certain mutants (~100 nM) relative to typical kinase inhibitors (often picomolar affinity). In addition, NX-2127 has poor bioavailability in higher species e.g. 1% for dogs. However, the human dose projection is 100 mg once daily and responses are still observed in human subjects. It is postulated that the mechanism of action is why relatively low sustained drug concentrations are apparently not required; the drug works catalytically, therefore the first dose leads to degradation of some proteins, and because the drug is administered chronically over several days, over time the level of protein continues to drop leading to durable effects. 

A STAT3 degrader is cellularly active despite low biochemical affinity.

A second example is evidenced by the emergence of degraders targeting proteins of targets that have traditionally been less ligandable using small molecules (e.g. transcription factors like STAT3, or proteins that act through PPIs like Bcl-XL). Kymera has taken a STAT3 degrader of undisclosed structure into the clinic (KT-333), and patented STAT3 degraders (e.g. US20230120381A1, I-174) have shown low biochemical affinity (K_d ~400 nM) but 10 nM cellular potency, again likely enabled by the catalytic mechanism.

Prolonged PK/PD due to degradation + catalytic mechanism can be observed (e.g. BTK, RIPK2)

For a long time it was assumed that the half-life of the small molecule was immaterial if the half-life of the protein was known, but thoughts around this are changing. GSK have recently published RIPK2 degraders, which are able to show prolonged pharmacology far beyond what would be expected based on their PK since RIPK2 requires a long period for re-synthesis (240 hours). In this case, degradation of RIPK2 is highly effective but there are also functional effects many days after dosing and drugs can be long-acting. GSK ‘PROTAC 6’ has been reported to continue degrading RIPK2 60 days after an initial dose when administered in a polymer matrix.

 

Alternative binding sites are usable (e.g. allosteric EGFR inhibitor, PPI inhibitors Bcl-XL, PCSK9)

Since degraders ultimately destroy the protein, it is not necessary to for the ligands to have a direct functional effect on the target of interest (e.g. inhibition, activation, conformational change, etc.). It can be sufficient if their binding simply triggers ubiquitination.

An allosteric EGFR degrader

The use of non-functional binding sites has been exploited by C4 Therapeutics and Roche, who had partnered to invent and develop a new, oral, allosteric binder EGFR L858R degrader. Within EGFR the L858R mutation creates an allosteric binding site that is not present on the WT protein, creating an opportunity to target the mutant without targeting the WT protein, but without needing to rely on the essentially identical active site. Interestingly, this degrader targets the EGFR mutant for degradation and is active in a brain metastasis model at 50 mpk PO/BID in rodents despite its massive size, with both excellent activity and selectivity over WT EGFR seen. 

A degrader of the extracellular protein, PCSK9

A second, less advanced example comes from a team led by Jason Imbroglio at Merck, who developed a PCSK9 degrader based on Liz Hedstrom’s Boc-Arg motif that had low affinity for PCSK9 (107 nM) but could still degrade the target. This appears to have been shelved, however, given Merck’s remarkable success with macrocyclic peptide MK-0616.

Tissue selectivity based on E3 ligase expression can be observed (e.g. avoid platelets or lungs)

Because the necessary E3 ligases for TPD can have differential expression across tissues, selectivity of degradation can be observed beyond what would be expected based on the target ligand’s intrinsic selectivity in terms of target binding. Arguably the best example at present is from the University of Florida spin-out Dialectic Therapeutics, who were able to modify an AbbVie Bcl-XL inhibitor to form a tissue-selective Bcl-XL degrader (DT2216). Bcl-XL is an anti-apoptotic protein, but drugging this target can lead to reduction in platelet levels. The ligase VHL has low expression in platelets, and by using the degrader, specific liquid or solid tumors can be targeted without having thrombocytopenia as a side-effect. While the extent of VHL expression and impact of DT2216 in platelet regeneration is not well-characterized yet, disclosed in vivo studies in mice so far are promising.

Selectivity based on ternary complexes possible (e.g. WT vs. mutant BRAF)

Mutant BRAF inhibitors are essential when targeting melanoma, but there are often only small differences in structure between mutant and WT BRAF proteins, leading to off-target effects attributable to WT BRAF inhibition. For example, the selectivity of vemurafenib for mutant BRAF vs WT BRAF is only 10 to 13-fold, which is likely driven in part by the differences in affinity for ATP. However, the degrader CFT1946 (which was derived from vemurafenib), also discussed earlier, is much more selective for mutant BRAF than WT BRAF, with no activity against WT BRAF detected in cells. This has been attributed to the fact that WT BRAF may not be recruited as efficiently to the necessary E3 ligase complex for ubiquitination and ultimate degradation.

Reduced tissue distribution may be needed (e.g. brain-active BTK degraders w/o high CNS penetration)

Reduced tissue distribution to certain compartments like the brain may be needed compared to traditional inhibitors because TPD does not require saturation of a drug and have accumulated pharmacology over time with lower ligand concentrations, opening up the possibility of treating diseases where crossing the blood-brain barrier is required despite the high molecular weight of the drugs (e.g. multiple sclerosis, brain metastases). For example, the second-generation BTK degrader from Nurix, NX-5948, is reported to have brain penetration of less than 5%, but is active in microglia.

Another example is C4 Therapeutics & Roche’s CFT8919, an EGFR L858R mutant degrader that appears to be brain penetrant (brain/plasma Kp ~ 0.45 in rodent at 9 h according to a graph in a recent presentation). The depth of in vivo responses observed in brain metastases models may be similarly enhanced by the degradation mechanism.

Difficult-to-ligand bystander substrates can be degraded indirectly

Finally, degraders can also lead to bystander degradation of targets that are traditionally challenging to find ligands for. A recent example comes from the Jin lab at Mount Sinai, where MS28 was intended to degrade CDK4/6 but actually degraded the bystander protein cyclin D1 before it turned over CDK4 or CDK6, leading to antiproliferative effects. 

Areas to Watch in the Future

As degraders continue to demonstrate differentiation from traditional small molecules clinically and in advanced preclinical studies, the use of TPD as an alternative to ‘traditional’ target inhibition using small molecules is an increasingly exciting area that continues to explode. New frontiers continue to open, with potentially brain-penetrant degraders now emerging, transmembrane and extracellular protein degraders appearing (e.g. for EGFR, PCSK9, and SLC transporters), and other difficult target classes showing proof-of-concept (e.g. STAT3). With new ligases being employed (e.g. DCAF1), new binding sites being identified (e.g. allosteric site for AR), and new warheads being used (e.g. covalent and reversible degraders), we will likely rapidly see new examples of degraders differentiating from small molecules along the lines discussed here. Many companies working in TPD including Arvinas have challenging-to-drug, “holy grail” targets in their pipelines (e.g. MYC, KRAS(G12D), AR-V7, Tau, alpha synuclein, mHTT), and we eagerly await progress in these areas.

Thanks to Our Sponsor, Revvity Signals

This article is made Open Access thanks to our sponsor, Revvity Signals (formerly PerkinElmer Informatics). Revvity Signals offers end-to-end cloud software solutions accelerating R&D and empowering scientists to make smarter decisions faster. The Signals™ Research Suite, powered by ChemDraw and TIBCO Spotfire, provides advanced capabilities for small and large molecule Drug Discovery.

Further Reading

1. For an excellent introduction to PROTACs and TPD, see this article (and references therein) from the Ciulli group: Ciulli, A.; Trainor, N.; A beginner’s guide to PROTACs and targeted protein degradation. The Biochemist, 2021, 43, 74 – 79.
2. The Crews lab also have a great article explaining the TPD/PROTAC landscape, as well as future directions: Békés, M.; Langley, D. R.; Crews, C. M.; PROTAC targeted protein degraders: the past is prologue, Nature, 2022, 21, 181–200 
3. For an excellent industry history from former Celgene leaders (Phil Chamberlain and Larry Hamann), see this article: Chamberlain, P.P., Hamann, L.G. Development of targeted protein degradation therapeutics. Nat. Chem. Biol., 2019, 15, 937–944.