Degrader Digest - Issue 1

Last updated April 1, 2021. Dear Protein Degradation Friends, Welcome to the Targeted Protein Degradation Digest! Targeted Protein Degradation (TPD) is an emerging modality of medicine that is evolving at an incredibly quick pace. When I co-founded C4 Therapeutics with Jay Bradner, MD, Ken Anderson, MD, and Nathanael Gray, PhD, in January 2016, there were only a few companies dedicated to TPD. Today there are dozens of companies pushing the state-of-the-art, setting up a potential tsunami of exciting new drugs in the next 3 to 5 years. TPD Digest is a no-cost, dedicated one-stop resource that is constantly updated with the latest public information on TPD. It is designed for all audiences from established thought leaders and scientists to neophytes. I hope that TPD Digest will be your go-to resource to deep dive into this rapidly advancing and exciting technology. I look forward to your feedback on how we can continuously improve resources for the TPD community. This TPD Digest contains the following sections: - Intro - Video Animations - Presentations - Industry Events - Companies Working on Protein Degradation - Media Articles - Scientific Papers Yours, Marc Cohen Cover art: proteasome illustration

  • Introduction to TPD

    Proteins are large and complex molecules that play essential roles in the human cell. In the nucleus, DNA is transcribed into RNA, which acts as the intermediary messenger of the genetic code in a form that is readable by the ribosome. The ribosome then translates the RNA by lining up amino acids according to the RNA code, creating a successive chain that is then folded to become a functional protein. Protein interactions have a central role in biological function, and control the mechanisms that lead to healthy or diseased states. Mutations that alter the normal function of proteins lead to protein dysfunction, and in turn, disease. Scientific advances continue to implicate specific proteins in multiple disease states. The human body has a highly conserved homeostasis system, which relies on protein degradation machinery to break down aged or damaged proteins into their amino acid components. E3 ligases mediate this process to maintain stable equilibrium by acting as a quality control inspector, identifying old, damaged, or misfolded proteins that are ready for degradation. When an E3 ligase identifies a target protein for degradation, it attaches a molecular tag, ubiquitin, to the target protein. This ubiquitination process typically tags the target protein with multiple ubiquitin proteins, which is known as poly-ubiquitination. The E3 ligase releases the target protein once it is poly-ubiquitinated to be quickly identified by the proteasome, the cell's recycling plant. Once the proteasome degrades the poly-ubiquitinated proteins into their amino acid components, they can be re-used to form new proteins or can be excreted by the cell. Approximately 5% of the human genome is dedicated to the ubiquitin-proteasome protein degradation system. Furthermore, proteins of therapeutic interest are often regulated by E3 ligases, which function to regulate protein concentrations across multiple steady states. All of these factors collectively emphasize the critical role…

Video Animations

Video exemplifications are incredibly helpful. If you have good examples to share, please forward the link to be included in the TPD Digest.

Presentations

  • Deshaies (Amgen) 1: A primer on the ubiquitin-proteasome system

    https://www.ibiology.org/cell-biology/primer-ubiquitin-proteasome-system/ Part 1: A primer on the ubiquitin-proteasome system: The ubiquitin-proteasome system is one of the principal means of degrading misfolded, mutated, or unwanted proteins in the cell. Part 2: Cullin-RING ubiquitin ligases: Over 200 types of cullin-RING ubiquitin ligases are formed by interchanging subunits. This carefully regulated system allows for great substrate specificity. Part 3: Targeting the ubiquitin-proteasome system in cancer: The ubiquitin-proteasome system is overworked in mutation-ridden cancer cells making it a potential target for anti-cancer drugs. https://www.ibiology.org/ibioseminars/cell-biology/primer-ubiquitin-proteasome-system.html Talk Overview: Neurons, blood cells and muscle cells have distinct morphologies and functions because they have different proteomes or collections of proteins. The proteome is maintained through the synthesis and degradation of different proteins. In his first talk, Dr. Deshaies explains that the ubiquitin-proteasome system (UPS) is the principal means of degrading proteins in the cytosol and therefore is key to maintaining the proteome. Ubiquitin is a small protein that can be added to, or removed from, other proteins altering their function or targeting them for degradation. Deshaies describes the multiple mechanisms for regulating ubiquitin ligase activity and the importance of protein degradation in many cell signaling pathways and in the removal of defective proteins. In Part 2, Deshaies delves more deeply into a specific group of ubiquitin ligases, the cullin-RING ligases (CRLs). By rearranging the subunits of CRLs in a combinatorial process, it is possible to generate more than 200 of these enzymes each with a specific substrate. The formation and activity of CRL complexes in the cell is regulated by the conjugation/deconjugation (Nedd8) or binding/dissociation (Cand1) of other proteins to the CRL complex, and this, in turn, is…

  • New Frontiers in Targeted Protein Degradation and Degrader Development

    Learn More about Targeted Protein Degradation: https://www.tocris.com/product-type/targeted-protein-degradation The use of heterobifunctional small molecule Degraders (e.g. PROTACs, SNIPERs etc) to elicit targeted protein degradation (TPD) is an area of increasing interest in chemical biology. They offer an attractive approach for inducing selective protein knockdown in a reversible and tuneable manner, without requiring genetic modification of cells. This is currently an area of tremendous interest, offering a potential new approach to tackle the ‘undruggable’ proteome and overcome common resistance mechanisms to current therapies. This webinar provides an introduction to Targeted Protein Degradation, discussing design and optimization of Degrader molecules and how structural biology and rational design can shed light on the Degrader development process. The second section of the webinar will present the dTAG platform for target validation. dTAG molecules specifically recognize and degrade FKBP12F36V fusion proteins, presenting a new platform for target validation studies.

TPD Industry Events

  • Dana-Farber Targeted Protein Degradation - The Webinar Series

    Bi-weekly Seminars exploring different mechanisms of small molecule mediated protein degradation for the development of chemical probes and/or drugs. The Webinar Series will take place bi-weekly on: Thursdays 12 pm noon (Eastern Standard Time) 9 am (Pacific Standard Time) 5 pm (British Summer Time) 6 pm (Central European Summer Time) You can join webinar by Zoom. https://dfci.zoom.us/webinar/register/WN_m7JJaw52T8yZYt8-ykL6UQ ​

  • Drug Discovery Chemistry Virtual: Ubiquitin-Induced Targeted Protein Degradation

    MAY 18-19, 2021 | ALL TIMES EASTERN DAYLIGHT (UTC-04:00) This conference brings together experts who can discuss the potential, as well as the challenges underlying targeted protein degradation as a new approach for therapeutic intervention.

  • Virtual Keystone Symposia: Targeted Protein Degradation: From Small Molecules to Complex Organelles

    June 7-8, 2021 | 10:00AM EDT | 2:00PM UTC | 4:00PM CEST* This conference will bring together researchers from these different fields, who do not typically interact, to build a holistic and integrated vision of protein degradation. Such an integrative conference highlighting the connections between the different branches of protein degradation research does not yet exist, so this Keystone Symposia conference will be the first of its kind to reshape how these fields interact and collaborate to yield transformative insights into both basic science and disease processes.

  • European Protein Degradation Congress 2021 | Kisaco Research

    SEPTEMBER 22-23, 2021 BASEL, SWITZERLAND Leading industry players from biotechs and big pharma will come together to exchange thoughts on how to best overcome obstacles to achieving reliable in vitro and in vivo data. Academic experts will be present their latest research to help build a deeper understanding of the mechanisms involved in the Ubiquitin pathway, providing a clearer picture on what makes a good, or not good, target for degradation.

  • Discovery on Target

    September 27-30, 2021 Discovery on Target (DOT) is the industry’s preeminent event on novel drug targets and technologies for drug discovery professionals. DOT highlights advances in current and emerging “hot” targets and technologies, as well as target validation strategies for the discovery and development of novel therapeutic agents, ranging from biologics to small molecules.

  • Undruggable Leaders Forum

    SEPTEMBER 2021 | EST The Undruggable Leaders Virtual Forum, the pan-undruggable meeting serving senior decision-makers from pharma and biotech who are striving to discover and advance undruggable targets.

  • Targeted Protein Degradation Summit

    October 2021 The 4th Targeted Protein Degradation Summit returns in October 2021 to once again explore the latest approaches, perspectives, and data-drive case studies that will continue to advance PROTACs, molecular glues, and other degrader modalities towards clinical success.

Public Companies

Working on Protein Degradation Candidates

  • Arvinas | Inspiring a New Pharmaceutical Paradigm

    Arvinas is a pharmaceutical company focused on developing new small molecule strategies aimed at degrading disease-causing cellular proteins. We are translating these innovative protein degradation approaches into novel drugs for the treatment of cancer and other diseases.

  • C4 Therapeutics

    C4 Therapeutics® is committed to transforming the treatment of cancer, neurological, and other important diseases with novel early stage therapies that eliminate disease-causing proteins. Our approach to medicine harnesses the innate machinery of the cell to attack disease and potentially bring deep and durable responses to patients.

  • Kymera

    Kymera is a biopharmaceutical company developing novel protein degrader therapeutics to treat disease in powerful new ways, which harness the body’s natural protein degradation system to selectively degrade disease causing proteins with the goal of completely removing them from the body. Kymera is driven to invent transformative medicines for a wide variety of diseases where traditional drug modalities have failed to sufficiently unlock the appropriate biological solution.

  • Nurix Therapeutics

    Nurix Therapeutics discovers drugs that harness the body’s natural process to control protein levels. Our drugs control ubiquitin E3 ligases, the key enzymes responsible for protein breakdown in human cells, as a unique therapeutic approach called targeted protein modulation to treat a broad range of diseases. Our focus is on developing targeted therapies to treat cancer including novel, small molecule immuno-oncology agents.

Private Companies

Working on Protein Degradation Candidates

  • A-Alpha Bio | Measure more protein interactions

    AlphaSeq is a synthetic biology platform used to accelerate drug development by enabling library-on-library measurements of protein interactions Discover biologics with defined specificity to many targets Optimize affinity and specificity rapidly in one workflow Characterize library binding to many targets simultaneously What can you do with AlphaSeq? Discover, optimize, and characterize biologics Screen antagonists against full interaction networks Screen agonists in a flexible library format Learn about opportunities to partner with A-Alpha Bio

  • Amphista Therapeutics

    Amphista Therapeutics is developing medicines for hard to treat diseases using next generation targeted protein degradation approaches. Our portfolio uses our own novel protein degrading mechanisms which build on the strengths of current protein degradation approaches whilst directly addressing their limitations. or why not drop us an email info@amphista.com Amphista Therapeutics is an exciting spin out company founded by Advent Life Sciences and built on groundbreaking science from the laboratory of Professor Alessio Ciulli (University of Dundee), a world leader in the field of targeted protein degradation.

  • BioTheryX

    We are a clinical-stage company harnessing our deep expertise in Targeted Protein Degradation to restore protein homeostasis in order to treat a wide range of diseases.

  • Captor Therapeutics

    Captor Therapeutics is a high-tech pharmaceutical company focused on targeting the undruggable proteome.

  • Cedilla Therapeutics

    Cedilla is harnessing intrinsic protein stability mechanisms to broaden the reach of small molecule therapeutics. We work on cancer and other diseases caused by protein dysregulation.

  • Civetta Therapeutics

    Civetta Therapeutics was founded to advance new medicines through small molecule targeting of propeller domains with the goal of developing important therapeutics for cancers and other diseases.

  • Coho Therapeutics

    Coho Therapeutics is a nascent biotechnology company developing medicines within a new class of therapeutics called protein degraders.

  • Cullgen

    Cullgen is a privately held biopharmaceutical company dedicated to the development of first-in-class new chemical entities (NCEs) for the treatment of diseases lacking effective therapeutic approaches.

  • Dialectic Therapeutics

    Dialectic Therapeutics (DT) is a pre-clinical biotechnology company dedicated to the development of unique, impactful anti-cancer drugs that effectively treat patients with few to no other options.

  • Dunad Therapeutics

    Dunad Therapeutics is focusing on the development of next-generation targeted protein degradation therapies. Dunad’s platform enables selective degradation via direct target modification using tuneable mono-valent small molecules, unlocking new target space and allowing for the development of more drug-like, orally bioavailable and CNS-accessible protein degraders.

  • FIMECS, Inc.

    FIMECS is a protein degradation technology-based drug discovery biotech focused on developing first-in-class protein degradation therapeutics for cancers and other difficult-to-treat diseases.

  • Frontier Medicines

    Frontier Medicines’ integrated approach leverages chemoproteomics and machine learning to accelerate the development of medicines that drug the “undruggable” protein targets that drive human disease.

  • Hinova Pharmaceuticals

    Hinova Pharmaceuticals Inc. is a high-tech innovative drug discovery and development company dedicated to bring new medicines to patients worldwide.

  • Lycia Therapeutics

    Developing first-in-class therapeutics that degrade extracellular proteins to address difficult-to-treat diseases

  • Monte Rosa Therapeutics

    Our drug discovery platform is tailored to promote specific protein ubiquitination and subsequent proteasomal degradation of disease-causing, undruggable proteins, in essence leading to pharmacological protein knockout with high efficiency and accuracy.

  • Neomorph

    Advancing the science of targeted protein degradation to destroy ‘undruggable’ proteins and cure disease.

  • Orionis Biosciences

    Orionis Biosciences is a life sciences company pioneering technological innovation in genome-scale drug discovery and tunable molecular design of novel therapeutic drug modalities.

  • PhoreMost

    PhoreMost’s SITESEEKER® platform enables the inception of First-in-class drug discovery programmes. Using this unique approach, PhoreMost is developing an internal asset pipeline in oncology and other diseases of ageing.

  • Pin Therapeutics

    Pin Therapeutics aims to develop targeted protein degradation hijacking ubiquitin and/or UBL (ubiquitin-like) biology.

  • Plexium | A Comprehensive Drug Development Methodology

    At Plexium, our mission is to reinvent the way medicines are discovered. We are implementing novel technologies and clinical approaches that enable the rapid and scalable development of first-in-class therapeutics. Funds will help advance Plexium’s DELPhe platform to discover novel E3 ligase-modulating therapeutics to treat cancer... Plexium CEO Swamy Vijayan to present at the North American Protein Degradation Congress in San Diego, CA on February... Plexium CEO Swamy Vijayan to present at the Targeted Protein Degradation Summit in Boston on October 23, 2019.

Media Articles

Scientific Papers

  • Targeting oncoproteins with a positive selection assay for protein degraders

    February 5, 2021 Vidyasagar Koduri, Leslie Duplaquet, Benjamin L. Lampson, Adam C. Wang, Amin H. Sabet, Mette Ishoey, Joshiawa Paulk, Mingxing Teng, Isaac S. Harris, Jennifer E. Endress, Xiaoxi Liu, Ethan Dasilva, Joao A. Paulo, Kimberly J. Briggs, John G. Doench, Christopher J. Ott, Tinghu Zhang, Katherine A. Donovan, Eric S. Fischer, Steven P. Gygi, Nathanael S. Gray, James Bradner, Jeffrey A. Medin, Sara J. Buhrlage, Matthew G. Oser, William G. Kaelin Jr. Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9–based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

  • Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function

    January 28, 2021 Mai Luo, Jessica N. Spradlin, Lydia Boike, Bingqi Tong, Scott M. Brittain, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle, Thomas J. Maimone, Daniel K. Nomura The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation—a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

  • Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based Assay

    January 14, 2021 Samuel Maiwald, Max Planck, Christopher Heim, Birte Hernandez Alvarez, Marcus D. Hartmann Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN).

  • High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

    January 11, 2021 Jeffrey R. Simard, Linda Lee, Ellen Vieux, Reina Improgo, Trang Tieu, Andrew J. Phillips, Stewart L. Fisher, Roy M. Pollock, Eunice Park The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

  • Gluing Proteins for Targeted Degradation

    January 11, 2021 Mikihiko Naito, Shigeo Murata Targeted protein degradation is an emerging technology for drug development. An article published in Nature reported a novel mechanism of targeted protein degradation triggered by small-molecule-induced polymerization of the oncogenic transcription factor BCL6.

  • The therapeutic potential of PROTACs

    October 20, 2020 Andrew B. Benowitz, Katherine L. Jones, John D. Harling Introduction PROTACs represent a novel class of heterobifunctional molecules that simultaneously bind to a target protein and to an E3 ligase complex, resulting in the transfer of ubiquitin and initiating a process ultimately causing the proteasomal degradation of the target protein. This mechanism of action imbues PROTACs with the ability to modulate target biology in unique ways compared to inhibitors, and the development of PROTACs as therapeutic agents is expected to result in new medicines to treat multiple diseases.

  • The Rise of Molecular Glues

    January 7, 2021 Stuart L. Schreiber 2021 marks the 30th anniversary of the revelation that cyclosporin A and FK506 act in a way previously not seen—as “molecular glues” that induce neo-protein–protein associations. As a torrent of new molecular-glue probes and medicines are fueling interest in this field, I explore the arc of this story.

  • Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide

    January 6, 2021 Max Jan, Irene Scarfò, Rebecca C. Larson, Amanda Walker, Andrea Schmidts, Andrew A. Guirguis, Jessica A. Gasser, Mikołaj Słabicki, Amanda A. Bouffard, Ana P. Castano, Michael C. Kann, Maria L. Cabral, Alexander Tepper, Daniel E. Grinshpun, Adam S. Sperling, Taeyoon Kyung, Quinlan L. Sievers, Michael E. Birnbaum, Marcela V. Maus, Benjamin L. Ebert Cell-based treatments such as chimeric antigen receptor (CAR) T cells have been finding increasing applications against cancer and other disorders. However, these treatments can have side effects, and it is difficult to stop the activity of an overactive cell-based therapy once it is inside a patient. To improve regulation of CAR T cell treatment, Jan et al . developed a switch-based system for activating and inactivating these cells. T cells with an OFF switch were deactivated using the drug lenalidomide, whereas those with an ON switch required both lenalidomide and a target antigen for activation, allowing control over the timing of T cell activation. Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous “living drugs,” these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify “super-degron” tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic…

  • Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins

    January 5, 2021 Ryosuke Shirasaki, Geoffrey M. Matthews, Sara Gandolfi, Ricardode Matos Simoes, Dennis L. Buckley, Joseline Raja Vora, Quinlan L.Sievers, Johanna B. Brüggenthies, Olga Dashevsky, Haley Poarch, Huihui Tang, Megan A. Bariteau, Michal Sheffer, Yiguo Hu, Sondra L. Downey-Kopyscinski, Paul J.Hengeveld, Brian J. Glassner, Eugen Dhimolea, Constantine S. Mitsiades Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological “degraders” of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

  • Targeted degradation of the enhancer lysine acetyltransferases CBP and p300

    January 4, 2021 Raghu Vannam, Jan Sayilgan, Samuel Ojeda, Barbara Karakyriakou, Eileen Hu, Johannes Kreuzer, Robert Morris, Xcanda Ixchel, Herrera Lopez, Sumit Rai, Wilhelm Haas, Michael Lawrence, Christopher J.Ott The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

  • Targeted Protein Degradation: An Emerging Therapeutic Strategy in Cancer

    2021 Samir H Barghout Drug discovery in the scope of cancer therapy has been focused on conventional agents that nonselectively induce DNA damage or selectively inhibit the activity of key oncogenic molecules without affecting their protein levels. An emerging therapeutic strategy that garnered attention in recent years is the induction of Targeted Protein Degradation (TPD) of cellular targets by hijacking the intracellular proteolysis machinery. This novel approach offers several advantages over conventional inhibitors and introduces a paradigm shift in several pharmacological aspects of drug therapy. While TPD has been found to be the major mode of action of clinically approved anticancer agents such as fulvestrant and thalidomide, recent years have witnessed systematic endeavors to expand the repertoire of proteins amenable to therapeutic ablation by TPD. Such endeavors have led to three major classes of agents that induce protein degradation, including molecular glues, Proteolysis Targeting Chimeras (PROTACs) and Hydrophobic Tag (HyT)-based degraders. Here, we briefly highlight agents in these classes and key advances made in the field with a focus on clinical translation in cancer therapy.

  • Preclinical and Clinical Advances of Targeted Protein Degradation as a Novel Cancer Therapeutic Strategy: An Oncologist Perspective

    December 28, 2020 Xinrui Yang, He Yin, Richard D. Kim, Jason B. Fleming & Hao Xie PROteolysis Targeting Chimeras (PROTACs) are a family of heterobifunctional small molecules that specifically target cellular proteins for degradation. Given that their mode of action is distinct from that of small-molecule inhibitors widely used in clinical practice, PROTACs have the potential to improve current cancer therapies. Multiple studies have suggested that PROTACs exhibit enhanced pharmacodynamics and reduced toxicity both in vitro and in vivo compared to clinically relevant small-molecule kinase inhibitors. In addition, PROTACs have been reported to be less prone to mutation-mediated drug resistance in specific disease settings. Since its development in 2001, the field of targeted protein degradation, in which PROTACs are used, has expanded rapidly. However, earlier studies focused on the advancement of the technology itself, while preclinical and clinical data on the disease-modifying effect of PROTACs have only recently been reported. As preclinical and clinical evidence accumulates, the efficacy of PROTACs as targeted therapeutics—distinct from that of small-molecule kinase inhibitors—suggests potential translational benefit in the clinical setting. In this short review, we aim to describe translational potentials of PROTACs. We offer our perspectives as practicing oncologists on the preclinical and clinical data on PROTACs as novel therapeutics for both solid and hematological malignancies.

  • The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation

    December 18, 2020 Charlie Y. Shi, Elena R. Kingston, Benjamin Kleaveland, Daniel H. Lin, Michael W. Stubna, David P. Bartel Abstract MicroRNAs help to regulate many genes in animal cells. Each microRNA associates with an Argonaute (AGO) protein, forming a complex in which the microRNA pairs with a messenger RNA (mRNA) target and AGO recruits factors that accelerate mRNA decay. However, for some unusual targets, the reverse occurs: Pairing to the target recruits factors that accelerate microRNA decay rather than degradation of the mRNA. Working independently, Shi et al. and Han et al. elucidate the mechanism of this phenomenon. They found that pairing to the unusual targets recruits a ubiquitin ligase that causes degradation of AGO, thereby exposing the microRNA to cellular nucleases. Mutating the ubiquitin ligase in diverse animals and cell types deregulates numerous microRNAs, implying that this phenomenon is widely deployed to sculpt microRNA levels. Science , this issue p. [eabc9359][1], p. [eabc9546][2] ### INTRODUCTION MicroRNAs (miRNAs) are short RNAs that direct widespread gene repression in the cells of humans and other animals. Each miRNA associates with an Argonaute (AGO) protein to form a silencing complex in which the miRNA pairs to sites within target mRNAs, and AGO recruits machinery that causes destabilization or reduced translation of the targeted transcript. In the aggregate, miRNAs reduce the output of most human genes, and most of the 90 broadly conserved miRNA families found in mammals are required for viability or proper development. Most miRNAs are quite long-lived because association with AGO protects them from cellular nucleases. However, some miRNAs are relatively unstable. A potential explanation for the instability of these miRNAs comes from the observation that interactions with certain target sites can promote miRNA destruction, thereby inverting the typical regulatory logic. This phenomenon of target-directed miRNA degradation…

  • PROTACs: New method to degrade transcription regulating proteins

    December 1, 2020 Beichen Hu, Yirong Zhou, Dejuan Sun, Yueying Yang, Yang Liu, Xingzhou Li, Hua Li, Lixia Chen Abstract Transcription is the fundamental process in all living organisms. A variety of important proteins, such as NRs, BETs, HDACs and many others are involved in transcription process. In general, overexpression of these proteins would cause many diseases. Some approved therapeutics employed inhibitors to regulate the transcription process, however, the results are far from satisfying. Therefore, it is in high demand to develop new technology to improve the therapeutic effects. In recent years, proteolysis-targeting chimaera (PROTAC) turned out to be a novel efficient therapeutic method to treat various diseases which were caused by proteins overexpression. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and subsequent degradation of the target protein by the proteasome. In contrast to traditional inhibitors, PROTACs showed higher efficiency to tackle the diseases which were caused by protein overexpression due to their excellent performance for degrading target proteins in transcription regulation. In this review, 29 kinds of PROTACs targeting transcription regulator proteins are summarized, and meanwhile the advantages of PROTACs are highlighted. Furthermore, several examples of PROTACs regulating the transcription for the treatment of diseases and functioning as tools for biological research are also disscussed.

  • Targeted Protein Degradation Tools: Overview and Future Perspectives - PubMed

    November 26, 2020 Yuri Prozzillo, Gaia Fattorini, Maria Virginia Santopietro, Luigi Suglia, Alessandra Ruggiero, Diego Ferreri, Giovanni Messina Abstract Targeted protein inactivation (TPI) is an elegant approach to investigate protein function and its role in the cellular landscape, overcoming limitations of genetic perturbation strategies. These systems act in a reversible manner and reduce off-target effects exceeding the limitations of CRISPR/Cas9 and RNA interference, respectively. Several TPI have been developed and wisely improved, including compartment delocalization tools and protein degradation systems. However, unlike chemical tools such as PROTACs (PROteolysis TArgeting Chimeras), which work in a wild-type genomic background, TPI technologies require adding an aminoacidic signal sequence (tag) to the protein of interest (POI). On the other hand, the design and optimization of PROTACs are very laborious and time-consuming. In this review, we focus on anchor-away, deGradFP, auxin-inducible degron (AID) and dTAG technologies and discuss their recent applications and advances. Finally, we propose nano-grad, a novel nanobody-based protein degradation tool, which specifically proteolyzes endogenous tag-free target protein.

  • Cereblon Modulators Target ZBTB16 and Its Oncogenic Fusion Partners for Degradation via Distinct Structural Degrons - PubMed

    November 18, 2020 Mary E Matyskiela, Jinyi Zhu, Joshua M Baughman, Thomas Clayton, Michelle Slade, Hon Kit Wong, Kristina Danga, Xinde Zheng, Mark Labow, Laurie LeBrun, Gang Lu, Philip P Chamberlain, Joel W Thompson Abstract There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate. We also report two new Cereblon modulators, CC-3060 and CC-647, that promote ZBTB16 degradation. Unexpectedly, CC-3060 and CC-647 target ZBTB16 for degradation by primarily engaging distinct structural degrons on different zinc finger domains. The reciprocal fusion proteins, ZBTB16-RARα and RARα-ZBTB16, which cause a rare acute promyelocytic leukemia, contain these same structural degrons and can be targeted for proteasomal degradation with Cereblon modulator treatment. Thus, a targeted protein degradation approach via Cereblon modulators may represent a novel therapeutic strategy in acute promyelocytic leukemia where ZBTB16/RARA rearrangements are critical disease drivers.

  • Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes

    November 16, 2020 James Schiemer, Reto Horst, Yilin Meng, Justin I. Montgomery, Yingrong Xu, Xidong Feng, Kris Borzilleri, Daniel P. Uccello, Carolyn Leverett, Stephen Brown, Ye Che, Matthew F. Brown, Matthew M. Hayward, Adam M. Gilbert, Mark C. Noe & Matthew F. Calabrese Abstract Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton’s tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

  • CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle-Dependent Degradation of CDK2 - PubMed

    November 16, 2020 Kristin M Riching, Marie K Schwinn, James D Vasta, Matthew B Robers, Thomas Machleidt, Marjeta Urh, Danette L Daniels Abstract Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

  • High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds using HiBiT CRISPR Cell Lines

    November 9, 2020 Kristin M. Riching, Sarah D. Mahan, Marjeta Urh, Danette L. Daniels Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

  • E3 Ligase Ligands for PROTACs: How They Were Found and How to Discover New Ones

    November 3, 2020 Tasuku Ishida, Alessio Ciulli Abstract Bifunctional degrader molecules, also called proteolysis-targeting chimeras (PROTACs), are a new modality of chemical tools and potential therapeutics to understand and treat human disease. A required PROTAC component is a ligand binding to an E3 ubiquitin ligase, which is then joined to another ligand binding to a protein to be degraded via the ubiquitin–proteasome system. The advent of nonpeptidic small-molecule E3 ligase ligands, notably for von Hippel–Lindau (VHL) and cereblon (CRBN), revolutionized the field and ushered in the design of drug-like PROTACs with potent and selective degradation activity. A first wave of PROTAC drugs are now undergoing clinical development in cancer, and the field is seeking to extend the repertoire of chemistries that allow hijacking new E3 ligases to improve the scope of targeted protein degradation. Here, we briefly review how traditional E3 ligase ligands were discovered, and then outline approaches and ligands that have been recently used to discover new E3 ligases for PROTACs. We will then take an outlook at current and future strategies undertaken that invoke either target-based screening or phenotypic-based approaches, including the use of DNA-encoded libraries (DELs), display technologies and cyclic peptides, smaller molecular glue degraders, and covalent warhead ligands. These approaches are ripe for expanding the chemical space of PROTACs and usher in the advent of other emerging bifunctional modalities of proximity-based pharmacology.

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