Targeted Protein Degradation: A Promising Therapeutic Strategy for the Most Challenging Protein Targets

In an effort to expand the druggable space, researchers are developing novel therapeutic strategies that target challenging disease-causing proteins. In the last decade, targeted protein degradation (TPD) is getting a lot of attention as a new and promising modality to treat a variety of diseases from cancer to Alzheimer’s. By taking proteins down the cell’s own degradation pathway instead of the typical inhibition approach, TPD allows researchers to develop successful therapies that address previously undruggable targets due to the absence of defined binding sites.1 Inhibition involves blocking the active site on the POI Traditional small-molecule drug discovery focuses on the occupancy of the active site on the protein of interest (POI), directly inhibiting its function. This approach has shown limitations as many of the targets — such as scaffolding proteins, transcription factors, and other non-enzymatic proteins2,3,4 — don’t have available binding sites. Additionally, therapeutics that rely on the drug molecule binding to the active site of the POI, may require high systemic drug exposure to be successful, which can potentially increase the risk of off target effects.5 This infographic explains inhibition and degradation approaches in drug development, discusses the advantages and challenges related to each approach, and the key points you need to consider when designing your protein degraders. Ligand binding is blocked Competitive inhibitor Targeted protein degradation harnesses the body’s natural disposal system To ensure the quality of intracellular proteins, cells use the ubiquitin-proteasome pathway to eliminate misfolded, damaged or the toxic accumulation of certain proteins.6 The proteasome also plays a central role in the regulation of proteins that control cell-cycle progression and apoptosis.7 Proteins that enter this pathway first bind to the E3 ligase and its associated proteins. After this occurs, the ligase mediates the transfer of ubiquitin to the protein, tagging it for degradation by the proteasome. Researchers are developing bi-specific small molecules like PROteolysis TArgeting Chimeras (PROTACs) and molecular glues that bind to both the POI and E3 ligase to trigger the degradation of the POI. With this approach, researchers are finding a novel strategy for drugging targets that can’t be drugged via inhibition. Are protein degraders or inhibitors right for your drug discovery project? Besides their distinct ways to interact with the POI and mode of action, other important characteristics set small molecule degraders and inhibitors apart. Knowing them will help you determine which is best for your drug design and development project. Key considerations for designing your small-molecule PROTACs Here are four key questions that will help you understand the mechanisms involved in the protein degradation pathway and what makes an effective small molecule protein degrader. Is my PROTAC cell-permeable? A PROTAC has to cross the cell membrane to engage with the E3 ligase and the target protein inside the cell to do its job. However, PROTACs have relatively low membrane permeability due to their high molecular weight (MW > 800 Da) and all their hydrogen bond donors and acceptors. The good news is, PROTACs can be designed to be more membrane-permeable by understanding and improving their physicochemical properties. Does my PROTAC form a ternary complex? Once inside the cell, the degrader first binds either the E3 ligase or its target protein to form a binary complex. Then, the third component binds to it and they form the ternary complex. Formation of the ternary complex is essential for ubiquitination and subsequent degradation, making it perhaps the most critical step of the protein degradation pathway. The characterization of the ternary complex formation with bioanalytical tools provides valuable feedback for PROTAC development and rational lead optimization. Does my target become ubiquitinated? Once a stable ternary complex is formed, the target protein becomes poly-ubiquitinated by the UPS. It starts with a single ubiquitin moiety that attaches to lysine residues on the target protein, followed by the conjugation of additional ubiquitin molecules leading to the elongation of the ubiquitin chain. These steps can be tracked with ubiquitination assays in live cells or in cell-free systems. Does the ubiquitination trigger degradation of the protein by the UPS? Once the target protein is ubiquitinated it is recognized, recruited, and degraded by the 26S UPS, in a process known as proteolysis. To be considered a successful protein degrader, the PROTAC must trigger completely target degradation. Methods such as western blot or mass spectrometry-based proteomics are used to evaluate the degree of degradation accomplished. 1. Lai A, Crews C. Induced protein degradation: an emerging drug discovery paradigm.Nature Reviews Drug Discovery. 2016;16(2):101-114. doi:10.1038/ nrd.2016.211 2. Hopkins A, Groom C. The druggable genome.Nature Reviews Drug Discovery. 2002;1(9):727-730. doi:10.1038/nrd892 3. Lazo J, Sharlow E. Drugging Undruggable Molecular Cancer Targets.Annu Rev Pharmacol Toxicol. 2016;56(1):23-40. doi:10.1146/annurevpharmtox-010715-103440 4. Jin L, Wang W, Fang G. Targeting Protein-Protein Interaction by Small Molecules.Annu Rev Pharmacol Toxicol. 2014;54(1):435-456. doi:10.1146/ annurev-pharmtox-011613-140028 5. Adjei A. What Is the Right Dose? The Elusive Optimal Biologic Dose in Phase I Clinical Trials.Journal of Clinical Oncology. 2006;24(25):4054-4055. doi:10.1200/jco.2006.07.4658 6. Goldberg A. Protein degradation and protection against misfolded or damaged proteins.Nature. 2003;426(6968):895-899. doi:10.1038/nature02263 7. ADAMS J. The proteasome: structure, function, and role in the cell.Cancer Treat Rev. 2003;29:3-9. doi:10.1016/s0305-7372(03)00081-1 8. An S, Fu L. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018;36:553- 562. doi:10.1016/j.ebiom.2018.09.005 9. Adjei AA. What Is the Right Dose? The Elusive Optimal Biologic Dose in Phase I Clinical Trials. Journal of Clinical Oncology. 2006; 24:4054–4055. DOI: 10.1200/ jco.2006.07.4658 [PubMed: 16943522] 10. Scudellari M. Protein-slaying drugs could be the next blockbuster therapies. Nature.com. https://www.nature.com/articles/d41586-019-00879-3. Published 2019. Accessed April 1, 2021. 11. Lai AC, Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov. 2017 Feb;16(2):101-114. doi: 10.1038/ nrd.2016.211. Epub 2016 Nov 25. PMID: 27885283; PMCID: PMC5684876. 12. V L, Adil A.A M, Ahmed N, K.Rishi A, Jamal S. Small molecule inhibitors as emerging cancer therapeutics. Integr Cancer Sci Ther. 2014. doi:10.15761/ icst.1000109 13. Sasaki T, Koivunen J, Ogino A et al. A Novel ALK Secondary Mutation and EGFR Signaling Cause Resistance to ALK Kinase Inhibitors.Cancer Res. 2011;71(18):6051-6060. doi:10.1158/0008-5472.can-11-1340 14. Poulikakos P, Persaud Y, Janakiraman M et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).Nature. 2011;480(7377):387-390. doi:10.1038/nature10662 15. Zhang L, Riley-Gillis B, Vijay P, Shen Y. Acquired Resistance to BET-PROTACs (Proteolysis-Targeting Chimeras) Caused by Genomic Alterations in Core Components of E3 Ligase Complexes. Mol Cancer Ther. 2019;18(7):1302-1311. doi:10.1158/1535-7163.mct-18-1129 References POI A PROTAC has two binding regions. One region binds with the POI and the other binds to the E3 ligase. When ligase and POI are brought together by the molecular glue, ubiquitins are added to the POI tagging it for degradation by the proteasome. Molecular glues stabilize the interaction between E3 ligase and the POI. The E3-PROTACPOI complex causes the POI to be tagged with ubiquitin and directs the protein to the proteasome for degradation. 1. Ternary complex formation 2. Polyubiquitination 3. Degradation by the ubiquitinproteasome system (UPS) E3 ligase POI POI Molecular glue PROTAC Proteasome Proteasome Degraded POI Degraded POI POI ligand E3 ligand E3 ligase PROTAC Molecular glue Learn how researchers are using NanoTemper tools to evaluate the affinity and specificity of their PROTAC candidates here. Ligand PROTACs and molecular glues Small molecule inhibitors Low affinity is acceptable, although high affinity can be selected for PROTACs Any accessible site 700-1100 Da8 Due to their size, they present a challenge for oral delivery11 Potentially reduced systemic exposure since they can be recycled and re-used10 They have the potential to overcome drug resistance due to mutations in active site, but this may not always be the case8,15 High affinity Active site <500 Da12 Can be administered orally High systemic exposure to achieve sufficient siteoccupancy in vivo9 Susceptible to drug resistance as mutations can modify the active site13,14 Size Drug resistance Effective dosage Administration Affinity for POI Site specificity A promising therapeutic strategy for the most challenging protein targets