Abstract 6407: A live cell method to assess E3 ligase and target protein occupancy for PROTACs

2020 ◽  
Author(s):  
James Vasta ◽  
Cesear Corona ◽  
Jennifer Wilkinson ◽  
Morgan R. Ingold ◽  
Chad Zimprich ◽  
...  
Keyword(s):  
E3 Ligase ◽  
Live Cell ◽  
Target Protein ◽  
Cell Method ◽  
Protein Occupancy

scholarly journals Predator: A novel method for targeted protein degradation

2020 ◽  
Author(s):  
Chuanyang Liu ◽  
Jingyu Kuang ◽  
Xinyuan Qiu ◽  
Lu Min ◽  
Wenying Li ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Expression ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Target Protein ◽  
Receptor Interaction ◽  
Physiological Status ◽  
Ubiquitin Proteasome ◽  
Bifunctional Fusion Protein ◽  
Predator System

AbstractProtein expression and degradation are fundamental to cell function and physiological status of organisms. Interfering with protein expression not only provides powerful strategies to analyze the function of proteins but also inspires effective treatment methods for diseases caused by protein dysfunction. Recently, harnessing the power of the ubiquitin-proteasome system for targeted protein degradation (TPD) has become the focus of researches. Over the past two decades, TPD technologies, such as E3 ligase modification, PROTACs, and the Trim-Away method, have successfully re-oriented the ubiquitin-proteasome pathway and thus degraded many pathogenic proteins and even "undruggable" targets. However, A low-cost, convenient, and modularized TPD method is currently not available. Herein, we proposed a synthetic biology TPD method, termed Predator, by integrating the classic function of E3 ligase Trim21 and the expression of a bifunctional fusion protein that links Trim21 and the target protein, which leads to the formation of a ternary complex inside mammalian cells and therefore induce the ubiquitination and subsequent proteasome-dependent degradation of the target protein. We first proved this concept by using nanobody and scFv as the targeting module for the Predator system to degrade free GFP and membrane protein ErbB3, respectively. Then, we give an example of how the engineered Predator system can be developed towards biomedical solutions in the context of diabetes mellitus. Ligands-receptor interaction and adenovirus-mediated gene delivery were introduced to the Predator system, and we found this bifunctional fusion protein, in which glucagon was selected to function as the targeting module, downregulated the endogenous glucagon receptor (GCGR) and attenuated glucagon-stimulated glucose production in primary hepatocytes. Although preliminarily, our results showed that this Predator system is a highly modularized and convenient TPD method with good potential for both fundamental researches and clinical usage.Graphic abstract

scholarly journals Enhancing Intracellular Accumulation and Target Engagement of PROTACs with Reversible Covalent Chemistry

2019 ◽  
Author(s):  
Wen-Hao Guo ◽  
Xiaoli Qi ◽  
Xin Yu ◽  
Yang Liu ◽  
Chan-I Chung ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Kinase Inhibitor ◽  
E3 Ligase ◽  
Target Protein ◽  
Binding Affinities ◽  
Target Engagement ◽  
Intracellular Accumulation ◽  
Molecular Weights ◽  
Dual Functional ◽  
Reversible Covalent

AbstractCurrent efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton’s Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

scholarly journals A beginner’s guide to PROTACs and targeted protein degradation

2021 ◽  
Author(s):  
Alessio Ciulli ◽  
Nicole Trainor
Keyword(s):  
Protein Degradation ◽  
Waste Disposal ◽  
E3 Ligase ◽  
Proteasomal Degradation ◽  
Target Protein ◽  
Keen Interest ◽  
Drug Candidates ◽  
The World ◽  
New Type ◽  
Chemical Tools

Those with a keen interest in targeting proteins, from chemical biologists to drug hunters alike, cannot help but take notice that a new type of molecule is making waves across this research space. Proteolysis Targeting Chimeras (or PROTACs) are protein degraders, which utilize the cell’s own waste disposal machinery to eliminate instead of inhibit a target protein. The key to PROTACs is their bifunctionality: they simultaneously bind a target protein and an E3 ligase protein, which then ubiquitylates the target, marking it for proteasomal degradation. This concept originated in the late 1990s and the first PROTAC was reported in 2001 by the laboratories of Craig Crews and Raymond Deshaies. However, interest in PROTACs did not pick up until 2015 when improved molecules were developed by the laboratories of Jay Bradner, Alessio Ciulli and Craig Crews. Ever since, PROTACs and the wider field of targeted protein degradation have expanded exponentially, with many groups around the world developing degraders as chemical tools to study proteins and as drug candidates for the treatment of diseases.

scholarly journals Small Molecule-Induced, Selective STAT3 Degradation Leads to Anti-Tumor Activity in STAT3-Dependent Heme Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3803-3803
Author(s):  
Fred Csibi ◽  
Nan Ji ◽  
Bin Yang ◽  
Karen Yuan ◽  
Michele Mayo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Small Molecule ◽  
Ternary Complex ◽  
Target Genes ◽  
E3 Ligase ◽  
Target Protein ◽  
Therapeutic Modality ◽  
Employment Equity ◽  
Equity Ownership ◽  
Anti Tumor Activity

Targeted protein degradation mediated by small molecule degraders represents a new and exciting therapeutic modality to target difficult-to-drug oncogenic proteins including transcription factors. These molecules bind to both the target protein and an E3 ligase, enabling the formation of a ternary complex that leads to ubiquitination and subsequent degradation of the target protein by the proteasome. STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family. In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated serine/threonine kinases, and phosphylated STAT3 (pSTAT3) then forms dimers that translocate into the nucleus, binds to DNA, and regulate transcription. STAT3 is frequently mutated and activated in numerous cancers including clinically aggressive hematologic malignancies with high unmet medical need. Mechanistically, aberrant activation of STAT3 has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, thus making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 heterobifunctional degrader, KYM-003, which displays strong anti-tumor activity in models of STAT3-dependent heme malignancies. KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. It strongly binds to STAT3 and a E3 ligase, leading to the formation of the a productive ternary complex, which leads to ubiquitination of STAT3 and subsequent proteasomal degradation. KYM-003 robustly degraded STAT3 in a number of primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in cell lines and human PBMCs. Degradation of STAT3 by KYM-003 led to significant downregulation of STAT3 target genes, such as SOCS3, MYC, and PIM1. Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme malignancies. Collectively, our data demonstrates that KYM-003 is a potent and selective STAT3 degrader that exhibited strong anti-tumor activity in vitro and in vivo. These data support STAT3 degraders as a new and exciting therapeutic opportunity in heme malignancies. Disclosures Csibi: kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Yang:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Mayo:kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Rusin:Kymera Therapeutics: Employment, Equity Ownership. Sharma:kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Townson:Kymera Therapeutics: Employment, Equity Ownership. Chen:kymera therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Walker:Kymera Therapeutics: Employment, Equity Ownership. Gollob:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

scholarly journals Perspectives on the development of first-in-class protein degraders

2021 ◽  
Author(s):  
Kalyn M Rambacher ◽  
Matthew F Calabrese ◽  
Masaya Yamaguchi
Keyword(s):  
Protein Degradation ◽  
E3 Ligase ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Target Protein ◽  
Protein Homeostasis ◽  
Recent Advances ◽  
Subsequent Degradation ◽  
Ubiquitin Proteasome

Targeted protein degradation is a broad and expanding field aimed at the modulation of protein homeostasis. A focus of this field has been directed toward molecules that hijack the ubiquitin proteasome system with heterobifunctional ligands that recruit a target protein to an E3 ligase to facilitate polyubiquitination and subsequent degradation by the 26S proteasome. Despite the success of these chimeras toward a number of clinically relevant targets, the ultimate breadth and scope of this approach remains uncertain. Here we highlight recent advances in assays and tools available to evaluate targeted protein degradation, including and beyond the study of E3-targeted chimeric ligands. We note several challenges associated with degrader development and discuss various approaches to expanding the protein homeostasis toolbox.

Progress on small-molecule proteolysis-targeting chimeras

2019 ◽  
Vol 11 (20) ◽  
pp. 2715-2734 ◽  
Author(s):  
Wenhai Huang ◽  
Beibei Wang ◽  
Zhimin Zhang ◽  
Chixiao Zhang ◽  
Shenxin Zeng ◽  
...  
Keyword(s):  
Small Molecule ◽  
Therapeutic Intervention ◽  
E3 Ligase ◽  
Ubiquitin Proteasome System ◽  
Target Protein ◽  
Research Progress ◽  
Target Proteins ◽  
Ubiquitin Proteasome

Proteolysis-targeting chimeras (PROTACs) have received much attention for their promising therapeutic intervention in recent years. These molecules, with the mechanism of simultaneous recruitment of target protein and an E3 ligase, can trigger the cellular ubiquitin–proteasome system to degrade the target proteins. This article systematically introduces the mechanism of small-molecule PROTACs, and summarized the research progress of small-molecule PROTACs. The prospect for further application and the problems to be solved are also discussed.

scholarly journals In vivo target protein degradation induced by PROTACs based on E3 ligase DCAF15

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Liang Li ◽  
Dazhao Mi ◽  
Haixiang Pei ◽  
Qiuhui Duan ◽  
Xinyue Wang ◽  
...  
Keyword(s):  
Protein Degradation ◽  
E3 Ligase ◽  
Target Protein

scholarly journals Rationalizing PROTAC-mediated ternary complex formation using Rosetta

2020 ◽  
Author(s):  
Nan Bai ◽  
Palani Kirubakaran ◽  
John Karanicolas
Keyword(s):  
Complex Formation ◽  
Ternary Complex ◽  
E3 Ligase ◽  
Computational Method ◽  
Target Protein ◽  
Binding Modes ◽  
Trial And Error ◽  
Protein Targets ◽  
E3 Ligases ◽  
Ternary Complex Formation

AbstractPROTACs are molecules that combine a target-binding warhead with an E3 ligase-recruiting moiety; by drawing the target protein into a ternary complex with the E3 ligase, PROTACs induce target protein degradation. While PROTACs hold exciting potential as chemical probes and as therapeutic agents, development of a PROTAC typically requires synthesis of numerous analogs to thoroughly explore variations on the chemical linker; without extensive trial and error, it is unclear how to link the two protein-recruiting moieties to promote formation of a productive ternary complex. Here, we describe a structure-based computational method for evaluating suitability of a given linker for ternary complex formation. Our method uses Rosetta to dock the protein components, then builds the PROTAC from its component fragments into each binding mode; complete models of the ternary complex are then refined. We apply this approach to retrospectively evaluate multiple PROTACs from the literature, spanning diverse target proteins. We find that modeling ternary complex formation is sufficient to explain both activity and selectivity reported for these PROTACs, implying that other cellular factors are not key determinants of activity in these cases. We further find that interpreting PROTAC activity is best approached using an ensemble of structures of the ternary complex rather than a single static conformation, and that members of a structurally-conserved protein family can be recruited by the same PROTAC through vastly different binding modes. To encourage adoption of these methods and promote further analyses, we disseminate both the computational methods and the models of ternary complexes.Significance StatementRecent years have brought a flood of interest in developing compounds that selectively degrade protein targets in cells, as exemplified by PROTACs. Fully realizing the promise of PROTACs to transform chemical biology by delivering degraders of diverse and undruggable protein targets has been impeded, however, by the fact that designing a suitable chemical linker between the functional moieties requires extensive trial and error. Here, we describe a structure-based computational method to predict PROTAC activity. We envision that this approach will allow design and optimization of PROTACs for efficient target degradation, selection of E3 ligases best suited for pairing with a given target protein, and understanding the basis by which PROTACs can exhibit different target selectivity than their component warheads.

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