None

Targeted Protein Degraders

Developing and Optimizing Targeted Protein Degraders

David Fry* and Julien Dugal-Tessier
NJ Bio, 675 US Highway 1, Suite B129, North Brunswick, NJ 08902, U.S.A.

What is Targeted Protein Degradation?

Targeted Protein Degradation (TPD) represents a new class of drugs.1–5 These “two-headed” molecules usually involve a protein target binder at one end and an E3 ubiquitin ligase recruiter at the other, separated by a central linker.6 The result of this scaffold is forced proximity of the target protein to the E3 ligase, causing ubiquitin tagging for proteasomal destruction (Figure 1). This new approach provides unique benefits. For one, it is an indication agnostic platform. Another important advantage is that binding to the target protein can occur at any location and does not have to inhibit protein function. This TPD approach could expand targets that are currently considered “undruggable”, such as those with active sites that do not support binding of drug-like molecules, proteins with no active site, and some protein-protein interfaces. The TPD platform can also allow expansion and reuse of currently available molecules. These can include molecules that lack specificity or have resistance.7 Further, because its action is catalytic, the cellular concentration of the TPD needed is much lower than for conventional drugs.8

Figure 1: Depiction of the mechanism of Targeted Protein Degradation
The TPD concept and the first successful example were reported in 2001 from the labs of Sakamoto, Crews and Deshaies.9 These molecules were termed “PROTACs” (Proteolysis-Targeting Chimeras) and have been trademarked as such, thus the more general term targeted protein degradation (TPD) was developed. Since this landmark work, the field has steadily expanded, and there have now been many TPDs reported, with alternate names applied (e.g., SNIPERs)10. Other degradation methodologies are also being pursued: “molecular glue” (no linker)11, LYTAC (degradation by lysosome)12, and AUTAC (removal by autophagy)13. The chimera approach has also moved beyond protein degradation to such concepts as RIBOTAC (RNA targets degraded by ribonuclease)14 and PhoRC (directed dephosphorylation of proteins)15.

Start-up companies such as Arvinas, Nurix, C4 Therapeutics, Kymera Therapeutics, Cullgen, and Captor Therapeutics have focused on a TPD strategy to bring this concept to market. Many large pharma companies such as Amgen, AstraZeneca, Celgene, GSK, Roche and Novartis have also started programs in this area and have made significant investments. Two TPD compounds have made it as far as Phase I clinical trials, both from Arvinas: ARV-110 for metastatic castration-resistant prostate cancer16 and ARV-471 for ER-positive/HER2-negative breast cancer17.

How does one develop a TPD?

It is helpful to frame this question by considering the three components of a TPD and their roles: binder to the target protein, the E3 recruiter, and the linker joining these pharmacophores.

The Target Protein Binder

The target protein is one whose removal will alleviate the disease condition. Many of these proteins have been considered for conventional drug discovery efforts. Thus, the identification of a ligand for the target protein will fall into two classes: known and unknown binders.

  1. Known binders are molecules that are approved or ones that have clinical or preclinical data. If the protein of interest has an approved drug, making a TPD could improve that drug by increasing selectivity, bioavailability and/or eliminating resistance. If the protein of interest does not have an approved drug but has known binders, these could be rejuvenated through a TPD program. Examples include a molecule that bound to but did not inhibit protein function or bound to a site other than the active site.
  2. The second class includes proteins for which a ligand/binder has yet to be discovered. This would necessitate a hit discovery program followed by a lead generation TPD program.

Currently most lead discovery efforts involve screening a compound library and rationally designing a preliminary scaffold.18 Screening for a compound where the only requirement is binding is best done by a biophysical method since an activity-based screen would miss the compounds that do not have inhibitory activity. Such biophysical screening methods include surface plasmon resonance (or a version thereof), mass spectrometry, Tm-shift, fluorescence polarization, calorimetry, X-ray crystallography, and BioNMR spectroscopy. Most of these methods have been adapted for high-throughput robotic screening. The speed and reduced volumes of such screening usually result in a significant number of false positives. These false positives can lead to lengthy medicinal chemistry campaigns for hits that cannot be converted to leads. BioNMR spectroscopy and X-ray crystallography are lower throughput techniques but are gold standards for verifying binding. BioNMR is also the only method that can provide reliable proof of non-binding because it measures binding in solution.

The next question would be which screening library should be utilized to find a hit using BioNMR or other screening techniques. A common start is corporate and commercial libraries of compounds synthesized by medicinal chemists, but these libraries can have limited chemotypes (i.e. chemical diversity) due to the reliance on easier synthetic routes and/or focus on historic drug discovery targets. DNA-encoded and other “soup”-type libraries can provide vast numbers of compounds but require high affinity for hits to be isolated. The legacy of hit discovery suggests that it is not simply a “numbers game” but the quality of hits is important. Libraries are more likely to succeed if the components of the screening library are targeted toward the protein class, or if a fragment-based approach is employed. Fragment libraries represent an efficient and effective way to explore large chemical space, but they require a method that can reliably detect weak binding and grow the fragments for a novel lead. BioNMR is a well-suited method for fragment-based screening as it is very sensitive and provides immediate real hit validation.

The E3 Ubiquitin Recruiter

With over 600 E3 ligases in the human cell19, how does one select the proper E3 for a particular TPD? First, ubiquitination serves various purposes, not just tagging for proteolysis. Current knowledge identifies 270 of the known ubiquitin ligases as being involved in the ubiquitin-proteosome system, and of those only 10 are very widely expressed, while the others have specific tissue-distribution profiles.19 Therefore, it is critical to verify E3 function, and to match expression to the location being targeted but this could also be used to increase specificity of the E3 ligase towards only tissue of interest.

To date, the choice of E3 ligases has been dictated by whether an associated small molecule binder has been available and not because the optimal E3 ligase is being targeted. The vast majority of reported TPDs have utilized two E3 systems, identified by their target-protein recognition component – VHL (Von Hippel–Lindau tumor suppressor) and CBRN (Cereblon) – for both of which potent small molecule ligands are known. Other E3 ligases that have been used in TPDs include MDM2, cIAP, KEAP1, DCAF15, and DCAF16.

Involvement of new E3 ligases is an obvious area of high interest in the development of TPDs. The choice of E3 can endow a TPD with the critical attributes to make it a successful drug, such as higher effectiveness, better specificity and lower toxicity.19 However, with so many E3 ligases to choose from, it is helpful to have a rational framework to consider E3 options.20 E3 ligases can be divided into three classes, based on their target-recognition domain and ubiquitin transfer mechanism: RING domain (transfers ubiquitin directly), HECT domain (transfers ubiquitin via a thioester intermediate), and RBR (has a RING domain, but uses a thioester intermediate). The RING domain class can be further divided into those involving a single protein (such as MDM2 and the IAP family), and those involving a multi-subunit complex (such as VHL, CBRN, KEAP1, and the DCAFs). The multi-subunit systems are referred to as “Cullin-RING E3 ubiquitin ligases” (CRL), and have a generic arrangement that sequentially links a target protein recognition domain to one or more adaptor proteins, then to a Cullin domain, and then to a RING-Box (RBX) domain (Figure 2). For example, CRL2VHL is composed of the target-recognition domain VHL, adaptor proteins elongin B and elongin C, Cullin2, and RBX1. CRL4CRBN involves a complex of Cereblon, damaged DNA binding protein 1 (DDB1), Cullin-4A, and regulator of cullins 1 (ROC1).

Figure 2: Depiction of Cullin-Ring E3 Ubiquitin Ligases (CRL) for the common VHL and CRBN type TPDs.
For the purposes of constructing a TPD, the dominant considerations regarding choice of E3 are distribution location, expression level and existence of an effective ligand. Currently, the major hurdle appears to be obtaining an effective ligand. To date, reported TPDs have exploited only E3 recognition domains with well-established binders. Those binders only represent a small fraction of all available E3 recruiters (Table 1). The future evolution of the TPD field will be driven by exploration of the other known ligands for the established E3 recognition domains20, and the discovery of new ligands for as-yet-untried E3 ligases.

There are various peptide and small-molecule inhibitors known for VHL but reported TPDs utilizing this E3 ligase have almost exclusively employed one particularly potent small molecule which has come to be known as VH-298. Similarly, there are many TPDs reported utilizing CBRN, but almost all of them employ thalidomide or a close derivative as the E3 binder. TPDs based on MDM2 have so far largely utilized the small-molecule inhibitors called Nutlins, despite the existence of a wide variety of other potent small molecule inhibitors. TPDs based on cIAP have employed only a limited number of peptide-like inhibitors such as bestatin, however there are numerous drug-like inhibitors known for the IAP family, possessing many unique characteristics, such as selectivity for the BIR2 or BIR3 sub-domains which mediate ligand binding. As for more recently exploited E3 ligases, a TPD utilizing KEAP1 has been reported using a peptide as the binder, however there are now small drug-like inhibitors available for KEAP1. TPDs against DCAFs 15 and 16 are quite new and so far, have exploited individually identified small molecule binders.

It is expected that a rational screening program against novel E3 ligases will result in the discovery of ligands that can be used in the construction of the next generation of TPDs. At this stage, assessing the prospects for finding such a ligand for a new E3 ligase is challenging. A first step could be to examine the sequence of E3 ligases and see if it contains a domain that is known to support binding of small molecules.19 WDR domains are present in numerous E3 ligases, and these domains have been shown to bind small molecule ligands with nanomolar affinity. The same combination is true for Kelch domains and bromodomains. SPRY and WW domains are found in certain E3 ligases, and these domains have been shown to bind reasonably small linear and cyclic peptides. All these domains suggest that they could support binding of drug-like molecules and be applied to a TPD program. Still, the domain structures and ligand-binding abilities of many E3 ligases are still unknown and need to be rationally designed.

The Linker

Currently, the linker is the most under-exploited TPD component since the choice of E3 ligases is limited and fixed. The linker must be properly selected to allow the TPD to function, yet it is usually the only component of the platform that can be optimized for affinity, degradation efficiency, selectivity, and pharmacokinetic properties. From the few available X-ray structures of TPDs bound in a ternary complex 21–24 it appears that the target-binder and the E3 recruiter portions bind in the same manner as they do as standalone components. Even the available attachment points for the linker may be constrained by the way the target and E3 protein surround their respective binders. Therefore, the linker is the only portion of the TPD where there is freedom to try various chemistry strategies to engineer in specific attributes.

It is clear from X-ray structures that the TPD linker is not dumbbell-shaped, with the linker simply serving as a flexible connector between the target protein and the E3. Rather, the linker is folded into a particular conformation, and interacts with both the target protein and the E3. This pulls the two proteins into close proximity leading to novel inter-protein contacts. The new contacts are essential but cannot be predicted without structural information. In fact, for two proteins which would normally never encounter each other, it is surprising how willing they are to interact. What is driving this? Is it an inherent folding propensity of the linker, or are the inter-protein and linker-protein interactions so energetically favored that they attract each other? The answer is not clear. BioNMR can help answer the question because it can determine the inherent conformational preferences of the free TPD, although this has not been pursued yet. In addition, preliminary data indicate that the inter-protein and linker-protein interactions appear to be opportunistic as they involve mostly hydrogen bonding and some moderate hydrophobic clustering, but not shape recognition or significant penetration. However, within the hydrogen bonds formed, there is a trend toward capping the termini of helices, and this may suggest that longer-range effects, such as pole orientations, are influencing how the complex forms. Nevertheless, the structure of the complex is hard to predict a priori and the factors affecting activity are not well understood, thus creating a reliance on empirical findings. BioNMR is a rapid method for providing experimental information on the intermolecular contacts that are made and guiding rational design of TPDs.

How should one choose and optimize a TPD linker? It should first be stated that there are compact TPDs that have been called “molecular glues”, where a single organic scaffold brings together a target protein and a ubiquitin ligase without the need for what would be considered a “linker”. Ultimately, the target protein and E3 ligase must be brought together in a way that orients them properly for the ligase to catalytically attach a ubiquitin to a lysine on the protein of interest. However, the molecular details of this event are still a mystery and arriving at an optimal TPD linker is a stepwise process of trial and improvement.

Critical attributes that can be measured include degradation efficiency and druglikeness. For example, a project could start with an established set of 25 linkers consisting of methylene chains, polyethylene glycol chains, and three others with more rigidity, each varied in carbon atom length. These could be attached to the target and E3 binders at various attachment points. These candidates could then be ranked by comparing their degradation efficiencies. At this point, structural guidance would become very valuable since a hit structure has been found and it would need to be rationally developed into a lead molecule. Modeling the ternary complex is an option but can be imprecise without structural information (i.e. X-ray crystallography). However, BioNMR is an alternative where one can gain speed by obtaining information that is at lower resolution but can still improve the computational model. Through selective labeling of the target protein, the E3, and the TPD, BioNMR can indicate which regions of these components are participating in new interactions and can provide information on the bound conformation of the TPD. These data can be fed into modeling efforts to understand and visualize the ternary complex. BioNMR profiles can also be correlated with activity profiles to understand which attributes and overall structural consequences of the TPD are beneficial. With a better model, improved versions of the TPD can then be designed and increase confidence in the optimization campaign.

Another important attribute to consider is selectivity. It has been shown that the TPD context can endow target selectivity even if using a non-selective target protein binder.25 It is hard to predict what off-target effects to expect, but certain binder classes have clear selectivity issues. Structural information correlated to selectivity can help guide medicinal chemistry efforts toward compounds with enhanced specificity.26

The other set of attributes which must be optimized fall within the ADMET (absorption, distribution, metabolism, excretion, and toxicity) and pharmacokinetics properties. Druglikeness is a critical issue for TPDs because they tend to be large flexible molecules, which hampers their use as small molecule therapeutics.27 As stated, the front and back portions of the TPD are constrained by their necessary interactions with the target and E3 respectively, so any physiochemical liabilities they carry must be offset by the linker. To guide linker optimization, other key properties can be calculated, or experimentally measured, such as aqueous solubility, octanol/water partition coefficient (log P), and membrane permeability.28 Ultimately, the behaviors of TPDs that are serious drug candidates must further be assessed with regard to bioavailability, kinetics, metabolism and clearance. Despite their large size, it has been possible to develop TPDs that perform acceptably with regards to small molecule pharmacology. The clearest proof is the recent entry of two TPDs into clinical trials.

Larger issues such as toxicity are difficult to predict, but one obvious concern is that hijacking an E3 ligase to degrade a disease target will also degrade the protein in non-disease cells, and this may lead to unwanted side effects. One newly emerging idea, in which the linker plays a key role, is the attachment of antibodies to TPDs to target them to specific cells to avoid non-disease cell toxicity.29 Generally, the only portion of a TPD with accessible space for attaching such an appendage is the linker. In fact, a second linker leading to the antibody will almost surely be required. This reinforces the need for structural information, with regards to the optimal attachment point, length, rigidity, and chemotype of the second linker.

Summary

Targeted Protein Degraders are a completely new kind of drug that offer the opportunity of attacking targets that were heretofore considered undruggable and of reviving drugs that have shown liabilities with respect to effectiveness, selectivity, and resistance. In developing a TPD, it is helpful to conceptualize the molecule as having three components: a part that binds to the target protein, a part that binds to an E3 ligase, and a linker that joins these two parts together. All 3 components must work together as a unit to be effective and cannot be separately optimized. Structural information and rational design, and accessing new E3 ligases, will yield novel TPDs and increase the knowledge of this important class. Challenges in terms of ADME and pharmacokinetics are still mostly unaddressed and how much these molecules may deviate from normal drug discovery is yet to be ascertained. There are currently two compounds undergoing clinical trials which will help guide the next generation of TPDs. Based on the growing amounts of investment and publication in this area, the future of TPDs looks rich and exciting.

Table 1: Recent List of Targeted Protein Degradation Molecules

Target: ALK - E3 Ligase: CRBN

Zhang, C.; Han, X.-R.; Yang, X.; Jiang, B.; Liu, J.; Xiong, Y.; Jin, J. Proteolysis Targeting Chimeras (PROTACs) of Anaplastic Lymphoma Kinase (ALK). Eur. J. Med. Chem. 2018, 151, 304–314. https://doi.org/10.1016/j.ejmech.2018.03.071.

Target: ALK - E3 Ligase: VHL - TD-020

Kang, C. H.; Lee, D. H.; Lee, C. O.; Du Ha, J.; Park, C. H.; Hwang, J. Y. Induced Protein Degradation of Anaplastic Lymphoma Kinase (ALK) by Proteolysis Targeting Chimera (PROTAC). Biochem. Biophys. Res. Commun. 2018, 505 (2), 542–547. https://doi.org/10.1016/j.bbrc.2018.09.169.

Target: AR - E3 Ligase: MDM2

Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M. Targeted Intracellular Protein Degradation Induced by a Small Molecule: En Route to Chemical Proteomics. Bioorg. Med. Chem. Lett. 2008, 18 (22), 5904–5908. https://doi.org/10.1016/j.bmcl.2008.07.114.

Target: AR - E3 Ligase: VHL - ARC C-4

Salami, J.; Alabi, S.; Willard, R. R.; Vitale, N. J.; Wang, J.; Dong, H.; Jin, M.; McDonnell, D. P.; Crew, A. P.; Neklesa, T. K.; Crews, C. M. Androgen Receptor Degradation by the Proteolysis-Targeting Chimera ARCC-4 Outperforms Enzalutamide in Cellular Models of Prostate Cancer Drug Resistance. Commun. Biol. 2018, 1 (1), 100. https://doi.org/10.1038/s42003-018-0105-8.

Target: BcL-2/Mcl-1 - E3 Ligase: CRBN

Wang, Z.; He, N.; Guo, Z.; Niu, C.; Song, T.; Guo, Y.; Cao, K.; Wang, A.; Zhu, J.; Zhang, X.; Zhang, Z. Proteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding Ligands. J. Med. Chem. 2019, 62 (17), 8152–8163. https://doi.org/10.1021/acs.jmedchem.9b00919.

Target: BCR-ABL - E3 Ligase: CRBN

Lai, A. C.; Toure, M.; Hellerschmied, D.; Salami, J.; Jaime-Figueroa, S.; Ko, E.; Hines, J.; Crews, C. M. Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew. Chemie Int. Ed. 2016, 55 (2), 807–810. https://doi.org/10.1002/anie.201507634.

Target: BRD4 - E3 Ligase: CRBN - ZXH-3-26

Nowak, R. P.; DeAngelo, S. L.; Buckley, D.; He, Z.; Donovan, K. A.; An, J.; Safaee, N.; Jedrychowski, M. P.; Ponthier, C. M.; Ishoey, M.; Zhang, T.; Mancias, J. D.; Gray, N. S.; Bradner, J. E.; Fischer, E. S. Plasticity in Binding Confers Selectivity in Ligand-Induced Protein Degradation. Nat. Chem. Biol. 2018, 14 (7), 706–714. https://doi.org/10.1038/s41589-018-0055-y.

Target: BRD4 - E3 Ligase: CRBN - ARV-825

Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.; Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem. Biol. 2015, 22 (6), 755–763. https://doi.org/10.1016/j.chembiol.2015.05.009.

Target: BRD4 - E3 Ligase: CRBN - dBET1

Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza, A.; Dhe-Paganon, S.; Bradner, J. E. Phthalimide Conjugation as a Strategy for in Vivo Target Protein Degradation. Science 2015, 348 (6241), 1376–1381. https://doi.org/10.1126/science.aab1433.

Target: BRD4 - E3 Ligase: VHL - MZ1

Zengerle, M.; Chan, K.-H.; Ciulli, A. Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chem. Biol. 2015, 10 (8), 1770–1777. https://doi.org/10.1021/acschembio.5b00216.

Target: BRD4 - E3 Ligase: VHL - ARV-771

Raina, K.; Lu, J.; Qian, Y.; Altieri, M.; Gordon, D.; Rossi, A. M. K.; Wang, J.; Chen, X.; Dong, H.; Siu, K.; Winkler, J. D.; Crew, A. P.; Crews, C. M.; Coleman, K. G. PROTAC-Induced BET Protein Degradation as a Therapy for Castration-Resistant Prostate Cancer. Proc. Natl. Acad. Sci. 2016, 113 (26), 7124–7129. https://doi.org/10.1073/pnas.1521738113.

Target: BTK - E3 Ligase: CRBN - DD-04-016

Huang, H.-T.; Dobrovolsky, D.; Paulk, J.; Yang, G.; Weisberg, E. L.; Doctor, Z. M.; Buckley, D. L.; Cho, J.-H.; Ko, E.; Jang, J.; Shi, K.; Choi, H. G.; Griffin, J. D.; Li, Y.; Treon, S. P.; Fischer, E. S.; Bradner, J. E.; Tan, L.; Gray, N. S. A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-Kinase Degrader. Cell Chem. Biol. 2018, 25 (1), 88-99.e6. https://doi.org/10.1016/j.chembiol.2017.10.005.

Target: BRD4 - E3 Ligase: CRBN

Zhou, B.; Hu, J.; Xu, F.; Chen, Z.; Bai, L.; Fernandez-Salas, E.; Lin, M.; Liu, L.; Yang, C.-Y.; Zhao, Y.; McEachern, D.; Przybranowski, S.; Wen, B.; Sun, D.; Wang, S. Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. J. Med. Chem. 2018, 61 (2), 462–481. https://doi.org/10.1021/acs.jmedchem.6b01816.

Target: BRD4 - E3 Ligase: CRBN - QCA570

Qin, C.; Hu, Y.; Zhou, B.; Fernandez-Salas, E.; Yang, C.-Y.; Liu, L.; McEachern, D.; Przybranowski, S.; Wang, M.; Stuckey, J.; Meagher, J.; Bai, L.; Chen, Z.; Lin, M.; Yang, J.; Ziazadeh, D. N.; Xu, F.; Hu, J.; Xiang, W.; Huang, L.; Li, S.; Wen, B.; Sun, D.; Wang, S. Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression. J. Med. Chem. 2018, 61 (15), 6685–6704. https://doi.org/10.1021/acs.jmedchem.8b00506.

Target: BRD9 - E3 Ligase: CRBN - dBRD9

Remillard, D.; Buckley, D. L.; Paulk, J.; Brien, G. L.; Sonnett, M.; Seo, H.-S.; Dastjerdi, S.; Wühr, M.; Dhe-Paganon, S.; Armstrong, S. A.; Bradner, J. E. Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands. Angew. Chemie Int. Ed. 2017, 56 (21), 5738–5743. https://doi.org/10.1002/anie.201611281.

Target: ER - E3 Ligase: cIAP1

Itoh, Y.; Kitaguchi, R.; Ishikawa, M.; Naito, M.; Hashimoto, Y. Design, Synthesis and Biological Evaluation of Nuclear Receptor-Degradation Inducers. Bioorg. Med. Chem. 2011, 19 (22), 6768–6778. https://doi.org/10.1016/j.bmc.2011.09.041.

Target: BTK - E3 Ligase: CRBN - P13I

Sun, Y.; Zhao, X.; Ding, N.; Gao, H.; Wu, Y.; Yang, Y.; Zhao, M.; Hwang, J.; Song, Y.; Liu, W.; Rao, Y. PROTAC-Induced BTK Degradation as a Novel Therapy for Mutated BTK C481S Induced Ibrutinib-Resistant B-Cell Malignancies. Cell Res. 2018, 28 (7), 779–781. https://doi.org/10.1038/s41422-018-0055-1.

Target: CDK9 - E3 Ligase: CRBN - THAL-SNS-032

Olson, C. M.; Jiang, B.; Erb, M. A.; Liang, Y.; Doctor, Z. M.; Zhang, Z.; Zhang, T.; Kwiatkowski, N.; Boukhali, M.; Green, J. L.; Haas, W.; Nomanbhoy, T.; Fischer, E. S.; Young, R. A.; Bradner, J. E.; Winter, G. E.; Gray, N. S. Pharmacological Perturbation of CDK9 Using Selective CDK9 Inhibition or Degradation. Nat. Chem. Biol. 2018, 14 (2), 163–170. https://doi.org/10.1038/nchembio.2538.

Target: CRABP-II - E3 Ligase: cIAP1

Itoh, Y.; Ishikawa, M.; Naito, M.; Hashimoto, Y. Protein Knockdown Using Methyl Bestatin−Ligand Hybrid Molecules: Design and Synthesis of Inducers of Ubiquitination-Mediated Degradation of Cellular Retinoic Acid-Binding Proteins. J. Am. Chem. Soc. 2010, 132 (16), 5820–5826. https://doi.org/10.1021/ja100691p.

Target: RAR - E3 Ligase: cIAP1

Itoh, Y.; Kitaguchi, R.; Ishikawa, M.; Naito, M.; Hashimoto, Y. Design, Synthesis and Biological Evaluation of Nuclear Receptor-Degradation Inducers. Bioorg. Med. Chem. 2011, 19 (22), 6768–6778. https://doi.org/10.1016/j.bmc.2011.09.041.

Target: ER - E3 Ligase: VHL - ERD-308

Hu, J.; Hu, B.; Wang, M.; Xu, F.; Miao, B.; Yang, C.-Y.; Wang, M.; Liu, Z.; Hayes, D. F.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER). J. Med. Chem. 2019, 62 (3), 1420–1442. https://doi.org/10.1021/acs.jmedchem.8b01572.

Target: ERR - E3 Ligase: VHL

Bondeson, D. P.; Mares, A.; Smith, I. E. D.; Ko, E.; Campos, S.; Miah, A. H.; Mulholland, K. E.; Routly, N.; Buckley, D. L.; Gustafson, J. L.; Zinn, N.; Grandi, P.; Shimamura, S.; Bergamini, G.; Faelth-Savitski, M.; Bantscheff, M.; Cox, C.; Gordon, D. A.; Willard, R. R.; Flanagan, J. J.; Casillas, L. N.; Votta, B. J.; den Besten, W.; Famm, K.; Kruidenier, L.; Carter, P. S.; Harling, J. D.; Churcher, I.; Crews, C. M. Catalytic in Vivo Protein Knockdown by Small-Molecule PROTACs. Nat. Chem. Biol. 2015, 11 (8), 611–617. https://doi.org/10.1038/nchembio.1858.

Target: GCN5/PCAF - E3 Ligase: CRBN - GSK699

Bassi, Z. I.; Fillmore, M. C.; Miah, A. H.; Chapman, T. D.; Maller, C.; Roberts, E. J.; Davis, L. C.; Lewis, D. E.; Galwey, N. W.; Waddington, K. E.; Parravicini, V.; Macmillan-Jones, A. L.; Gongora, C.; Humphreys, P. G.; Churcher, I.; Prinjha, R. K.; Tough, D. F. Modulating PCAF/GCN5 Immune Cell Function through a PROTAC Approach. ACS Chem. Biol. 2018, 13 (10), 2862–2867. https://doi.org/10.1021/acschembio.8b00705.

Target: HDAC6 - E3 Ligase: CRBN

Yang, K.; Song, Y.; Xie, H.; Wu, H.; Wu, Y.-T.; Leisten, E. D.; Tang, W. Development of the First Small Molecule Histone Deacetylase 6 (HDAC6) Degraders. Bioorg. Med. Chem. Lett. 2018, 28 (14), 2493–2497. https://doi.org/10.1016/j.bmcl.2018.05.057.

Target: IRAK4 - E3 Ligase: VHL

Nunes, J.; McGonagle, G. A.; Eden, J.; Kiritharan, G.; Touzet, M.; Lewell, X.; Emery, J.; Eidam, H.; Harling, J. D.; Anderson, N. A. Targeting IRAK4 for Degradation with PROTACs. ACS Med. Chem. Lett. 2019, 10 (7), 1081–1085. https://doi.org/10.1021/acsmedchemlett.9b00219.

Target: PI3K - E3 Ligase: CRBN

Li, W.; Gao, C.; Zhao, L.; Yuan, Z.; Chen, Y.; Jiang, Y. Phthalimide Conjugations for the Degradation of Oncogenic PI3K. Eur. J. Med. Chem. 2018, 151, 237–247. https://doi.org/10.1016/j.ejmech.2018.03.066.

CTarget: Sirt2 - E3 Ligase: CRBN

Schiedel, M.; Herp, D.; Hammelmann, S.; Swyter, S.; Lehotzky, A.; Robaa, D.; Oláh, J.; Ovádi, J.; Sippl, W.; Jung, M. Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals). J. Med. Chem. 2018, 61 (2), 482–491. https://doi.org/10.1021/acs.jmedchem.6b01872.

Target: SMARCA2 - E3 Ligase: VHL

Farnaby, W.; Koegl, M.; Roy, M. J.; Whitworth, C.; Diers, E.; Trainor, N.; Zollman, D.; Steurer, S.; Karolyi-Oezguer, J.; Riedmueller, C.; Gmaschitz, T.; Wachter, J.; Dank, C.; Galant, M.; Sharps, B.; Rumpel, K.; Traxler, E.; Gerstberger, T.; Schnitzer, R.; Petermann, O.; Greb, P.; Weinstabl, H.; Bader, G.; Zoephel, A.; Weiss-Puxbaum, A.; Ehrenhöfer-Wölfer, K.; Wöhrle, S.; Boehmelt, G.; Rinnenthal, J.; Arnhof, H.; Wiechens, N.; Wu, M.-Y.; Owen-Hughes, T.; Ettmayer, P.; Pearson, M.; McConnell, D. B.; Ciulli, A. BAF Complex Vulnerabilities in Cancer Demonstrated via Structure-Based PROTAC Design. Nat. Chem. Biol. 2019, 15 (7), 672–680. https://doi.org/10.1038/s41589-019-0294-6.

Target: Wee1 - E3 Ligase: CRBN

Li, Z.; Pinch, B. J.; Olson, C. M.; Donovan, K. A.; Nowak, R. P.; Mills, C. E.; Scott, D. A.; Doctor, Z. M.; Eleuteri, N. A.; Chung, M.; Sorger, P. K.; Fischer, E. S.; Gray, N. S. Development and Characterization of a Wee1 Kinase Degrader. Cell Chem. Biol. 2020, 27 (1), 57-65.e9. https://doi.org/10.1016/j.chembiol.2019.10.013.

Target: FLT3 - E3 Ligase: CRBN - TL13-117

Huang, H.-T.; Dobrovolsky, D.; Paulk, J.; Yang, G.; Weisberg, E. L.; Doctor, Z. M.; Buckley, D. L.; Cho, J.-H.; Ko, E.; Jang, J.; Shi, K.; Choi, H. G.; Griffin, J. D.; Li, Y.; Treon, S. P.; Fischer, E. S.; Bradner, J. E.; Tan, L.; Gray, N. S. A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-Kinase Degrader. Cell Chem. Biol. 2018, 25 (1), 88-99.e6. https://doi.org/10.1016/j.chembiol.2017.10.005.

Target: TRIM24 - E3 Ligase: VHL - DFCI-4107

Gechijian, L. N.; Buckley, D. L.; Lawlor, M. A.; Reyes, J. M.; Paulk, J.; Ott, C. J.; Winter, G. E.; Erb, M. A.; Scott, T. G.; Xu, M.; Seo, H.-S.; Dhe-Paganon, S.; Kwiatkowski, N. P.; Perry, J. A.; Qi, J.; Gray, N. S.; Bradner, J. E. Functional TRIM24 Degrader via Conjugation of Ineffectual Bromodomain and VHL Ligands. Nat. Chem. Biol. 2018, 14 (4), 405–412. https://doi.org/10.1038/s41589-018-0010-y.

Target: TBK1 - E3 Ligase: VHL

Crew, A. P.; Raina, K.; Dong, H.; Qian, Y.; Wang, J.; Vigil, D.; Serebrenik, Y. V.; Hamman, B. D.; Morgan, A.; Ferraro, C.; Siu, K.; Neklesa, T. K.; Winkler, J. D.; Coleman, K. G.; Crews, C. M. Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1. J. Med. Chem. 2018, 61 (2), 583–598. https://doi.org/10.1021/acs.jmedchem.7b00635.

Target: RIPK2 - E3 Ligase: VHL

Bondeson, D. P.; Mares, A.; Smith, I. E. D.; Ko, E.; Campos, S.; Miah, A. H.; Mulholland, K. E.; Routly, N.; Buckley, D. L.; Gustafson, J. L.; Zinn, N.; Grandi, P.; Shimamura, S.; Bergamini, G.; Faelth-Savitski, M.; Bantscheff, M.; Cox, C.; Gordon, D. A.; Willard, R. R.; Flanagan, J. J.; Casillas, L. N.; Votta, B. J.; den Besten, W.; Famm, K.; Kruidenier, L.; Carter, P. S.; Harling, J. D.; Churcher, I.; Crews, C. M. Catalytic in Vivo Protein Knockdown by Small-Molecule PROTACs. Nat. Chem. Biol. 2015, 11 (8), 611–617. https://doi.org/10.1038/nchembio.1858.

Target: BcL-XL - E3 Ligase: CRBN

Zhang, X.; Thummuri, D.; Liu, X.; Hu, W.; Zhang, P.; Khan, S.; Yuan, Y.; Zhou, D.; Zheng, G. Discovery of PROTAC BCL-XL Degraders as Potent Anticancer Agents with Low on-Target Platelet Toxicity. Eur. J. Med. Chem. 2020, 192, 112186. https://doi.org/10.1016/j.ejmech.2020.112186.

References

Review Articles for Targeted Protein Degradation

(1)        Lai, A. C.; Crews, C. M. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nature Reviews Drug Discovery 2017, 16 (2), 101–114. https://doi.org/10.1038/nrd.2016.211

(2)        Zou, Y.; Ma, D.; Wang, Y. The PROTAC Technology in Drug Development. Cell Biochemistry and Function 2019, 37 (1), 21–30. https://doi.org/10.1002/cbf.3369.

(3)        Wang, Y.; Jiang, X.; Feng, F.; Liu, W.; Sun, H. Degradation of Proteins by PROTACs and Other Strategies. Acta Pharmaceutica Sinica B 2020, 10 (2), 207–238. https://doi.org/10.1016/j.apsb.2019.08.001.

(4)        An, S.; Fu, L. Small-Molecule PROTACs: An Emerging and Promising Approach for the Development of Targeted Therapy Drugs. EBioMedicine 2018, 36, 553–562. https://doi.org/10.1016/j.ebiom.2018.09.005.

(5)        Cromm, P. M.; Crews, C. M. Targeted Protein Degradation: From Chemical Biology to Drug Discovery. Cell Chemical Biology 2017, 24 (9), 1181–1190. https://doi.org/10.1016/j.chembiol.2017.05.024.

Specific References

(6)        Cromm, P. M.; Crews, C. M. Targeted Protein Degradation: From Chemical Biology to Drug Discovery. Cell Chemical Biology 2017, 24 (9), 1181–1190. https://doi.org/10.1016/j.chembiol.2017.05.024.

(7)        Arthur, R.; Beatriz Valle-Argos, B.; Steele, A. J.; Packham, G. Development of PROTACs to Address Clinical Limitations Associated with BTK-Targeted Kinase Inhibitors. Exploration of Targeted Anti-tumor Therapy 2020, 1 (3), 131–152. https://doi.org/10.37349/etat.2020.00009.

(8)        Salami, J.; Crews, C. M. Waste Disposal—An Attractive Strategy for Cancer Therapy. Science 2017, 355 (6330), 1163–1167. https://doi.org/10.1126/science.aam7340.

(9)        Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Protacs: Chimeric Molecules That Target Proteins to the Skp1-Cullin-F Box Complex for Ubiquitination and Degradation. Proceedings of the National Academy of Sciences 2001, 98 (15), 8554–8559. https://doi.org/10.1073/pnas.141230798.

(10)       Naito, M.; Ohoka, N.; Shibata, N. SNIPERs—Hijacking IAP Activity to Induce Protein Degradation. Drug Discovery Today: Technologies 2019, 31, 35–42. https://doi.org/10.1016/j.ddtec.2018.12.002.

(11)       Hughes, S. J.; Ciulli, A. Molecular Recognition of Ternary Complexes: A New Dimension in the Structure-Guided Design of Chemical Degraders. Essays in Biochemistry 2017, 61 (5), 505–516. https://doi.org/10.1042/EBC20170041.

(12)       Banik, S. M.; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, N. M.; Bertozzi, C. R. Lysosome-Targeting Chimaeras for Degradation of Extracellular Proteins. Nature 2020, 584 (7820), 291–297. https://doi.org/10.1038/s41586-020-2545-9.

(13)       Takahashi, D.; Moriyama, J.; Nakamura, T.; Miki, E.; Takahashi, E.; Sato, A.; Akaike, T.; Itto-Nakama, K.; Arimoto, H. AUTACs: Cargo-Specific Degraders Using Selective Autophagy. Molecular Cell 2019, 76 (5), 797-810.e10. https://doi.org/10.1016/j.molcel.2019.09.009.

(14)       Costales, M. G.; Matsumoto, Y.; Velagapudi, S. P.; Disney, M. D. Small Molecule Targeted Recruitment of a Nuclease to RNA. Journal of the American Chemical Society 2018, 140 (22), 6741–6744. https://doi.org/10.1021/jacs.8b01233.

(15)       Yamazoe, S.; Tom, J.; Fu, Y.; Wu, W.; Zeng, L.; Sun, C.; Liu, Q.; Lin, J.; Lin, K.; Fairbrother, W. J.; Staben, S. T. Heterobifunctional Molecules Induce Dephosphorylation of Kinases–A Proof of Concept Study. Journal of Medicinal Chemistry 2020, 63 (6), 2807–2813. https://doi.org/10.1021/acs.jmedchem.9b01167.

(16)       Neklesa, T.; Snyder, L. B.; Willard, R. R.; Vitale, N.; Pizzano, J.; Gordon, D. A.; Bookbinder, M.; Macaluso, J.; Dong, H.; Ferraro, C.; Wang, G.; Wang, J.; Crews, C. M.; Houston, J.; Crew, A. P.; Taylor, I. ARV-110: An Oral Androgen Receptor PROTAC Degrader for Prostate Cancer. Journal of Clinical Oncology 2019, 37 (7_suppl), 259–259. https://doi.org/10.1200/JCO.2019.37.7_suppl.259.

(17)       Lin, X.; Xiang, H.; Luo, G. Targeting Estrogen Receptor α for Degradation with PROTACs: A Promising Approach to Overcome Endocrine Resistance. European Journal of Medicinal Chemistry 2020, 206, 112689. https://doi.org/10.1016/j.ejmech.2020.112689.

(18)       Brown, D. G.; Boström, J. Where Do Recent Small Molecule Clinical Development Candidates Come From? Journal of Medicinal Chemistry 2018, 61 (21), 9442–9468. https://doi.org/10.1021/acs.jmedchem.8b00675.

(19)       Schapira, M.; Calabrese, M. F.; Bullock, A. N.; Crews, C. M. Targeted Protein Degradation: Expanding the Toolbox. Nature Reviews Drug Discovery 2019, 18 (12), 949–963. https://doi.org/10.1038/s41573-019-0047-y.

(20)       Blaquiere, N.; Villemure, E.; Staben, S. T. Medicinal Chemistry of Inhibiting RING-Type E3 Ubiquitin Ligases. Journal of Medicinal Chemistry 2020, 63 (15), 7957–7985. https://doi.org/10.1021/acs.jmedchem.9b01451.

(21)       Gadd, M. S.; Testa, A.; Lucas, X.; Chan, K.-H.; Chen, W.; Lamont, D. J.; Zengerle, M.; Ciulli, A. Structural Basis of PROTAC Cooperative Recognition for Selective Protein Degradation. Nature Chemical Biology 2017, 13 (5), 514–521. https://doi.org/10.1038/nchembio.2329.

(22)       Testa, A.; Hughes, S. J.; Lucas, X.; Wright, J. E.; Ciulli, A. Structure‐Based Design of a Macrocyclic PROTAC. Angewandte Chemie International Edition 2020, 59 (4), 1727–1734. https://doi.org/10.1002/anie.201914396.

(23)       Farnaby, W.; Koegl, M.; Roy, M. J.; Whitworth, C.; Diers, E.; Trainor, N.; Zollman, D.; Steurer, S.; Karolyi-Oezguer, J.; Riedmueller, C.; Gmaschitz, T.; Wachter, J.; Dank, C.; Galant, M.; Sharps, B.; Rumpel, K.; Traxler, E.; Gerstberger, T.; Schnitzer, R.; Petermann, O.; Greb, P.; Weinstabl, H.; Bader, G.; Zoephel, A.; Weiss-Puxbaum, A.; Ehrenhöfer-Wölfer, K.; Wöhrle, S.; Boehmelt, G.; Rinnenthal, J.; Arnhof, H.; Wiechens, N.; Wu, M.-Y.; Owen-Hughes, T.; Ettmayer, P.; Pearson, M.; McConnell, D. B.; Ciulli, A. BAF Complex Vulnerabilities in Cancer Demonstrated via Structure-Based PROTAC Design. Nature Chemical Biology 2019, 15 (7), 672–680. https://doi.org/10.1038/s41589-019-0294-6.

(24)       Chung, C.; Dai, H.; Fernandez, E.; Tinworth, C. P.; Churcher, I.; Cryan, J.; Denyer, J.; Harling, J. D.; Konopacka, A.; Queisser, M. A.; Tame, C. J.; Watt, G.; Jiang, F.; Qian, D.; Benowitz, A. B. Structural Insights into PROTAC-Mediated Degradation of Bcl-XL. ACS Chemical Biology 2020, 15 (9), 2316–2323. https://doi.org/10.1021/acschembio.0c00266.

(25)       Bondeson, D. P.; Smith, B. E.; Burslem, G. M.; Buhimschi, A. D.; Hines, J.; Jaime-Figueroa, S.; Wang, J.; Hamman, B. D.; Ishchenko, A.; Crews, C. M. Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell Chemical Biology 2018, 25 (1), 78-87.e5. https://doi.org/10.1016/j.chembiol.2017.09.010.

(26)       Smith, B. E.; Wang, S. L.; Jaime-Figueroa, S.; Harbin, A.; Wang, J.; Hamman, B. D.; Crews, C. M. Differential PROTAC Substrate Specificity Dictated by Orientation of Recruited E3 Ligase. Nature Communications 2019, 10 (1), 131. https://doi.org/10.1038/s41467-018-08027-7.

(27)       Edmondson, S. D.; Yang, B.; Fallan, C. Proteolysis Targeting Chimeras (PROTACs) in ‘beyond Rule-of-Five’ Chemical Space: Recent Progress and Future Challenges. Bioorganic & Medicinal Chemistry Letters 2019, 29 (13), 1555–1564. https://doi.org/10.1016/j.bmcl.2019.04.030.

(28)       Scott, D. E.; Rooney, T. P. C.; Bayle, E. D.; Mirza, T.; Willems, H. M. G.; Clarke, J. H.; Andrews, S. P.; Skidmore, J. Systematic Investigation of the Permeability of Androgen Receptor PROTACs. ACS Medicinal Chemistry Letters 2020, 11 (8), 1539–1547. https://doi.org/10.1021/acsmedchemlett.0c00194.

(29)       Pillow, T. H.; Adhikari, P.; Blake, R. A.; Chen, J.; del Rosario, G.; Deshmukh, G.; Figueroa, I.; Gascoigne, K. E.; Kamath, A. v.; Kaufman, S.; Kleinheinz, T.; Kozak, K. R.; Latifi, B.; Leipold, D. D.; Sing Li, C.; Li, R.; Mulvihill, M. M.; O’Donohue, A.; Rowntree, R. K.; Sadowsky, J. D.; Wai, J.; Wang, X.; Wu, C.; Xu, Z.; Yao, H.; Yu, S. F.; Zhang, D.; Zang, R.; Zhang, H.; Zhou, H.; Zhu, X.; Dragovich, P. S. Antibody Conjugation of a Chimeric BET Degrader Enables in Vivo Activity. ChemMedChem 2020, 15 (1), 17–25. https://doi.org/10.1002/cmdc.201900497.