Imperial College London



-635bottomDepartment of Chemistry, Imperial College London, Molecular Sciences Research Hub,?White City Campus, Wood Lane, London?W12 0BZ, UK. E-mail: e.tate@imperial.ac.ukCellzome GmbH, a GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, GermanyMerck Sharp & Dohme, 1st floor, 2 Pancras Square, London NC1 4AG, UKGlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK. ? Electronic Supplementary Information (ESI) available. See DOI:?10.1039/x0xx00000x00Department of Chemistry, Imperial College London, Molecular Sciences Research Hub,?White City Campus, Wood Lane, London?W12 0BZ, UK. E-mail: e.tate@imperial.ac.ukCellzome GmbH, a GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, GermanyMerck Sharp & Dohme, 1st floor, 2 Pancras Square, London NC1 4AG, UKGlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK. ? Electronic Supplementary Information (ESI) available. See DOI:?10.1039/x0xx00000xReceived 00th January 20xx,Accepted 00th January 20xxDOI: 10.1039/x0xx00000xA Caged E3 Ligase Ligand for PROTAC-mediated Protein Degradation with Light Cyrille S. Kounde,a Maria M. Shchepinova,a Charlie N. Saunders,a Marcel Muelbaier,b Mark D. Rackham,c John D. Harling d and Edward W. Tate *aWith the intent of achieving greater spatiotemporal control of PROTAC-induced protein degradation, a light-activated degrader was designed by photocaging an essential E3 ligase binding motif in a BRD4 targeting PROTAC. Proteolysis was triggered only after a short irradiation time, the kinetics of which could be monitored by live-cell video microscopy.Heterobifunctional degraders known as Proteolysis Targeting Chimeras (PROTACs) are well-established as powerful tools for small molecule-directed protein degradation.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nrd.2016.211","ISSN":"14741784","abstract":"Small-molecule drug discovery has traditionally focused on occupancy of a binding site that directly affects protein function, and this approach typically precludes targeting proteins that lack such amenable sites. Furthermore, high systemic drug exposures may be needed to maintain sufficient target inhibition in vivo, increasing the risk of undesirable off-target effects. Induced protein degradation is an alternative approach that is event-driven: upon drug binding, the target protein is tagged for elimination. Emerging technologies based on proteolysis-targeting chimaeras (PROTACs) that exploit cellular quality control machinery to selectively degrade target proteins are attracting considerable attention in the pharmaceutical industry owing to the advantages they could offer over traditional small-molecule strategies. These advantages include the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.","author":[{"dropping-particle":"","family":"Lai","given":"Ashton C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Reviews Drug Discovery","id":"ITEM-1","issue":"2","issued":{"date-parts":[["2017"]]},"page":"101-114","publisher":"Nature Publishing Group","title":"Induced protein degradation: An emerging drug discovery paradigm","type":"article-journal","volume":"16"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1021/acs.jmedchem.7b01272","ISSN":"15204804","abstract":"Los microorganismos han sido considerados como agentes causantes de biodeterioro de obras de arte, sin embargo, en la actualidad se empiezan a utilizar para la limpieza de restos de compuestos orgánicos y costras salinas difíciles de eliminar por los métodos tradicionales de restauración. Este trabajo desarrolla el uso de Pseudomonas stutzeri para la “biolimpieza” de pinturas murales, con la intención de eliminar restos de materia orgánica de antiguas restauraciones o eflorescencias salinas insolubles. Para ello se ensayan distintas cepas de Pseudomonas stutzeri de colecciones tipo y distintos soportes que facilite su aplicación directa sobre obra real. Los métodos obtenidos en las pruebas previas se aplican de manera experimental sobre fragmentos de las pinturas murales de la Iglesia de los Santos Juanes de Valencia.","author":[{"dropping-particle":"","family":"Churcher","given":"Ian","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Medicinal Chemistry","id":"ITEM-2","issue":"2","issued":{"date-parts":[["2018"]]},"page":"444-452","title":"Protac-Induced Protein Degradation in Drug Discovery: Breaking the Rules or Just Making New Ones?","type":"article-journal","volume":"61"},"uris":[""]}],"mendeley":{"formattedCitation":"^1,2","plainTextFormattedCitation":"1,2","previouslyFormattedCitation":"^1,2"},"properties":{"noteIndex":0},"schema":""}1,2 These two-headed molecules consist of a ligand for a protein of interest joined to an E3 ligase-recruiting motif via a linker that plays an important role in directing the formation of a non-physiological ternary complex between the E3 ligase and the protein of interest in cells.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.ddtec.2019.01.001","ISSN":"17406749","abstract":"Targeted protein degradation mediated by small molecule degraders represents an exciting new therapeutic opportunity to eliminate disease-causing proteins. These molecules recruit E3 ubiquitin ligases to the protein of interest and mediate its ubiquitination and subsequent proteolysis by the proteasome. Significant advancements have been made in the discovery and development of clinically relevant degraders. In this review we will focus on the recent progress in understanding ternary complex formation and structures, ubiquitination, and other critical factors that govern the efficiency of degraders both in vitro and in vivo. With deeper knowledges of these areas, the field is building guiding principles to reduce the level of empiricism and to identify therapeutically relevant degraders more rationally and efficiently.","author":[{"dropping-particle":"","family":"Zhang","given":"Yi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Loh","given":"Christine","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"Jesse","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mainolfi","given":"Nello","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Drug Discovery Today: Technologies","id":"ITEM-1","issue":"xx","issued":{"date-parts":[["2019"]]},"page":"53-60","title":"Targeted protein degradation mechanisms","type":"article-journal","volume":"31"},"uris":[""]}],"mendeley":{"formattedCitation":"³","plainTextFormattedCitation":"3","previouslyFormattedCitation":"³"},"properties":{"noteIndex":0},"schema":""}3 PROTACs thereby promote intracellular polyubiquitination and subsequent proteasome-dependent degradation of the targeted protein in a unique post-translational mode of action, which complements traditional gene and siRNA protein silencing strategies.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/s00018-019-03112-6","ISBN":"0123456789","ISSN":"14209071","abstract":"Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems suffer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Targeted proteolysis has the potential to overcome some of these limitations. The field of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specific proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the different targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics.","author":[{"dropping-particle":"","family":"R?th","given":"Sascha","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fulcher","given":"Luke J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sapkota","given":"Gopal P.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Cellular and Molecular Life Sciences","id":"ITEM-1","issue":"14","issued":{"date-parts":[["2019"]]},"page":"2761-2777","publisher":"Springer International Publishing","title":"Advances in targeted degradation of endogenous proteins","type":"article-journal","volume":"76"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴","plainTextFormattedCitation":"4","previouslyFormattedCitation":"⁴"},"properties":{"noteIndex":0},"schema":""}4 Moreover, the catalytic mechanism of action,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nchembio.1858","ISSN":"15524469","abstract":"The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.","author":[{"dropping-particle":"","family":"Bondeson","given":"Daniel P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mares","given":"Alina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Smith","given":"Ian E.D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ko","given":"Eunhwa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Campos","given":"Sebastien","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Miah","given":"Afjal H.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mulholland","given":"Katie E.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Routly","given":"Natasha","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Buckley","given":"Dennis L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gustafson","given":"Jeffrey L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zinn","given":"Nico","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Grandi","given":"Paola","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shimamura","given":"Satoko","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bergamini","given":"Giovanna","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Faelth-Savitski","given":"Maria","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bantscheff","given":"Marcus","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cox","given":"Carly","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gordon","given":"Deborah A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Willard","given":"Ryan R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Flanagan","given":"John J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Casillas","given":"Linda N.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Votta","given":"Bartholomew J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Besten","given":"Willem","non-dropping-particle":"Den","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Famm","given":"Kristoffer","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kruidenier","given":"Laurens","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carter","given":"Paul S.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Harling","given":"John D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Churcher","given":"Ian","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Chemical Biology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2015"]]},"page":"611-617","title":"Catalytic in vivo protein knockdown by small-molecule PROTACs","type":"article-journal","volume":"11"},"uris":[""]}],"mendeley":{"formattedCitation":"⁵","plainTextFormattedCitation":"5","previouslyFormattedCitation":"⁵"},"properties":{"noteIndex":0},"schema":""}5 versatility of the approachADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.ddtec.2019.01.002","ISSN":"17406749","abstract":"The majority of currently used therapeutics are small molecule-based and utilize occupancy-driven pharmacology as the mode of action (MOA), in which the protein function is modulated via temporary inhibition. New modalities that operate using alternative MOAs are essential for tapping into the “undruggable” proteome. The PROteolysis Targeting Chimera (PROTAC) technology provides an attractive new approach that utilizes an event-driven MOA. Small molecule-based heterobifunctional PROTACs modulate protein target levels by hijacking the ubiquitin-proteasome system to induce degradation of the target. Here, we address important milestones in the development of the PROTAC technology, as well as emphasize key findings from this previous year and highlight future directions of this promising drug discovery modality.","author":[{"dropping-particle":"","family":"Pettersson","given":"Mariell","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Drug Discovery Today: Technologies","id":"ITEM-1","issue":"xx","issued":{"date-parts":[["2019"]]},"page":"15-27","publisher":"Elsevier Ltd","title":"PROteolysis TArgeting Chimeras (PROTACs) — Past, present and future","type":"article-journal","volume":"31"},"uris":[""]}],"mendeley":{"formattedCitation":"⁶","plainTextFormattedCitation":"6","previouslyFormattedCitation":"⁶"},"properties":{"noteIndex":0},"schema":""}6 and potential to degrade targets lacking enzymatic activityADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/anie.201706529","ISSN":"15213773","abstract":"Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt), and removal of toxic mHtt is expected to be an effective therapeutic approach. We designed two small hybrid molecules (1 and 2) by linking a ligand for ubiquitin ligase (cellular inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt aggregates, anticipating that these compounds would recruit cIAP1 to mHtt and induce selective degradation by the ubiquitin-proteasome system. The synthesized compounds reduced mHtt levels in HD patient fibroblasts and appear to be promising candidates for the development of a treatment for HD.","author":[{"dropping-particle":"","family":"Tomoshige","given":"Shusuke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nomura","given":"Sayaka","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohgane","given":"Kenji","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hashimoto","given":"Yuichi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ishikawa","given":"Minoru","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Angewandte Chemie - International Edition","id":"ITEM-1","issue":"38","issued":{"date-parts":[["2017"]]},"page":"11530-11533","title":"Discovery of Small Molecules that Induce the Degradation of Huntingtin","type":"article-journal","volume":"56"},"uris":[""]}],"mendeley":{"formattedCitation":"⁷","plainTextFormattedCitation":"7","previouslyFormattedCitation":"⁷"},"properties":{"noteIndex":0},"schema":""}7 has prompted major drug discovery efforts both in academia and industry.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acschembio.6b01068","ISSN":"15548937","abstract":"? 2017 American Chemical Society. Until recently, the only ways to reduce specific protein signaling were to either knock down the target by RNAi or to interfere with the signaling by inhibiting an enzyme or receptor within the signal transduction cascade. Herein, we review an emerging class of small molecule pharmacological agents, called PROTACs, that present a novel approach to specifically target proteins and their respective signaling pathways. These heterobifunctional molecules utilize endogenous cellular quality control machinery by recruiting it to target proteins in order to induce their degradation.","author":[{"dropping-particle":"","family":"Ottis","given":"Philipp","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Chemical Biology","id":"ITEM-1","issue":"4","issued":{"date-parts":[["2017"]]},"page":"892-898","title":"Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy","type":"article-journal","volume":"12"},"uris":[""]}],"mendeley":{"formattedCitation":"⁸","plainTextFormattedCitation":"8","previouslyFormattedCitation":"⁸"},"properties":{"noteIndex":0},"schema":""}8 32296108637270Figure 1. PROTAC caging concept. Attachment of a caging group leads to an inactive degrader. Post-translational modification of the protein of interest (POI) with ubiquitin (Ub) followed by subsequent degradation by the proteasome occurs only after uncaging with a light-emitting diode (LED). 00Figure 1. PROTAC caging concept. Attachment of a caging group leads to an inactive degrader. Post-translational modification of the protein of interest (POI) with ubiquitin (Ub) followed by subsequent degradation by the proteasome occurs only after uncaging with a light-emitting diode (LED). We hypothesized that PROTAC-mediated protein knockdown could be achieved with an additional level of precision if placed under a conditional stimulus such as light. Since protein function is often regulated in time and space, light would represent a suitable tool to activate or deactivate degradation as light itself can be controlled both spatially and temporally. Besides, the combination of PROTACs with light to degrade a disease-relevant protein may offer some applications in the field of photopharmacology.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acs.chemrev.8b00037","ISSN":"15206890","PMID":"29985590","abstract":"Synthetic photoswitches have been known for many years, but their usefulness in biology, pharmacology, and medicine has only recently been systematically explored. Over the past decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.","author":[{"dropping-particle":"","family":"Hüll","given":"Katharina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Morstein","given":"Johannes","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Trauner","given":"Dirk","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Chemical Reviews","id":"ITEM-1","issue":"21","issued":{"date-parts":[["2018"]]},"page":"10710-10747","title":"In Vivo Photopharmacology","type":"article-journal","volume":"118"},"uris":[""]}],"mendeley":{"formattedCitation":"⁹","plainTextFormattedCitation":"9","previouslyFormattedCitation":"⁹"},"properties":{"noteIndex":0},"schema":""}9 Light-mediated small molecule regulation of mammalian proteins has not been explored extensively until very recently.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acschembio.5b00069","ISSN":"15548937","abstract":"The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control. (Figure Presented).","author":[{"dropping-particle":"","family":"Delacour","given":"Quentin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Li","given":"Chenge","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Plamont","given":"Marie Aude","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Billon-Denis","given":"Emmanuelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Aujard","given":"Isabelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Saux","given":"Thomas","non-dropping-particle":"Le","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jullien","given":"Ludovic","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gautier","given":"Arnaud","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Chemical Biology","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2015"]]},"page":"1643-1647","title":"Light-Activated Proteolysis for the Spatiotemporal Control of Proteins","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁰","plainTextFormattedCitation":"10","previouslyFormattedCitation":"¹⁰"},"properties":{"noteIndex":0},"schema":""}10 For example, whilst this manuscript was in preparation, a number of reports have described the optical control of protein degradation with PROTACs bearing photoswitchable or photolabile groups.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acscentsci.9b00713","ISSN":"2374-7943","abstract":"Off-target effects are persistent issues of modern inhibition-based therapies. By merging the strategies of photopharmacology and small molecule degraders, we introduce a novel concept for persistent spatiotemporal control of induced protein degradation that potentially prevents off-target toxicity. Building on the successful principle of bifunctional all-small molecule Proteolysis Targeting Chimeras (PROTACs), we designed photoswitchable PROTACs (photoPROTACs) by including ortho-F4-azobenzene linkers between both warhead ligands. This highly bistable yet photoswitchable structural component leads to reversible control over the topological distance between both ligands. The azo-cis-isomer is observed to be inactive because the distance defined by the linker is prohibitively short to permit complex formation between the protein binding partners. By contrast, the azo-trans-isomer is active because it can engage both protein partners to form the necessary and productive ternary complex. Importantly, due to the bistable nature of the ortho-F4-azobenzene moiety employed, the photostationary state of the photoPROTAC is persistent, with no need for continuous irradiation. This technique offers reversible on/off switching of protein degradation that is compatible with an intracellular environment and, therefore, could be vastly useful in experimental probing of biological signaling pathways – especially those crucial for oncogenic signal transduction. Additionally, this strategy may be suitable for therapeutic implementation in a wide variety of disease phenotypes. By enabling reversible activation and deactivation of protein degradation, photoPROTACs offer advantages over conventional photocaging strategies that irreversibly release active agents.","author":[{"dropping-particle":"","family":"Pfaff","given":"Patrick","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Samarasinghe","given":"Kusal T. G.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carreira","given":"Erick M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Central Science","id":"ITEM-1","issued":{"date-parts":[["2019"]]},"title":"Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs","type":"article-journal"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1126/sciadv.aay5064","ISSN":"23752548","abstract":"PROTACs (PROteolysis TArgeting Chimeras) are bifunctional molecules that target proteins for ubiquitylation by an E3 ligase complex and subsequent degradation by the proteasome. They have emerged as powerful tools to control the levels of specific cellular proteins. We now introduce photoswitchable PROTACs that can be activated with the spatiotemporal precision that light provides. These trifunctional molecules, which we named PHOTACs (PHOtochemically TArgeting Chimeras), consist of a ligand for an E3 ligase, a photoswitch, and a ligand for a protein of interest. We demonstrate this concept by using PHOTACs that target either BET family proteins (BRD2,3,4) or FKBP12. Our lead compounds display little or no activity in the dark but can be reversibly activated with different wavelengths of light. Our modular approach provides a method for the optical control of protein levels with photopharmacology and could lead to new types of precision therapeutics that avoid undesired systemic toxicity.","author":[{"dropping-particle":"","family":"Reynders","given":"Martin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Matsuura","given":"Bryan S.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bérouti","given":"Marleen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Simoneschi","given":"Daniele","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Marzio","given":"Antonio","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pagano","given":"Michele","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Trauner","given":"Dirk","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Science Advances","id":"ITEM-2","issue":"8","issued":{"date-parts":[["2020"]]},"title":"PHOTACs enable optical control of protein degradation","type":"article-journal","volume":"6"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.1021/jacs.9b06422","ISSN":"15205126","abstract":"Induction of protein degradation is emerging as a powerful strategy to modulate protein functions and alter cellular signaling pathways. Proteolysis-targeting chimeras (PROTACs) have been used to degrade a range of diverse proteins in vitro and in vivo. Here we present a type of photo-caged PROTACs (pc-PROTACs) to induce degradation activity with light. Photo-removable blocking groups were added to a degrader of Brd4, and the resulting molecule pc-PROTAC1 showed potent degradation activity in live cells only after light irradiation. Furthermore, this molecule efficiently degraded Brd4 and induced expected phenotypic changes in zebrafish. Additionally, this approach was successfully applied to construct pc-PROTAC3 of BTK. Thus, a general strategy to induce protein degradation with light was established to augment the chemists' toolbox to study disease-relevant protein targets.","author":[{"dropping-particle":"","family":"Xue","given":"Gang","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Kun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhou","given":"Danli","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhong","given":"Hanbing","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pan","given":"Zhengying","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-3","issue":"46","issued":{"date-parts":[["2019"]]},"page":"18370-18374","title":"Light-Induced Protein Degradation with Photocaged PROTACs","type":"article-journal","volume":"141"},"uris":[""]}],"mendeley":{"formattedCitation":"^11–13","plainTextFormattedCitation":"11–13","previouslyFormattedCitation":"^11–13"},"properties":{"noteIndex":0},"schema":""}11–13 The caging approach is based on the inactivation of a PROTAC with a photocleavable appendage that prevents key binding interactions with either the protein of interest or the E3 ligase, and has been applied to both Cereblon and Von Hippel Lindau (VHL) E3 ligase recruiting PROTACs.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/jacs.9b12718","ISSN":"15205126","abstract":"As an emerging approach to protein perturbation, small molecule-induced protein degradation has gained significant attention as both a chemical tool and a potential therapeutic. To enable discrete control over its function, we have developed a broadly applicable approach for the optical activation of small molecule-induced protein degradation. By installing two different photolabile protecting groups, so-called caging groups, onto two different ligands recruiting Von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ubiquitin ligases, our strategy enables light-triggered protein degradation for any small molecule warhead.","author":[{"dropping-particle":"","family":"Naro","given":"Yuta","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Darrah","given":"Kristie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Deiters","given":"Alexander","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2020"]]},"page":"2193-2197","title":"Optical Control of Small Molecule-Induced Protein Degradation","type":"article-journal","volume":"142"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1126/sciadv.aay5154","ISSN":"23752548","abstract":"By hijacking endogenous E3 ligase to degrade protein targets via the ubiquitin-proteasome system, PROTACs (PRoteolysis TArgeting Chimeras) provide a new strategy to inhibit protein targets that were regarded as undruggable before. However, the catalytic nature of PROTAC potentially leads to uncontrolled degradation that causes systemic toxicity issues, limiting the application of PROTAC in the clinic. Here, we introduce a light-inducible switch on PROTACs, thereafter termed as opto-PROTAC, to enable the degradation of protein targets in a spatiotemporal manner. By adding a photolabile caging group on pomalidomide as a parental compound and two additional PROTACs, dBET1 and dALK, we demonstrated light-inducible protein degradation. These opto-PROTACs display no activities in the dark, while the restricted degradation can be induced at a specific time and rate by ultraviolet A irradiation. Our approach provides a generalizable platform for the development of light-controlled PROTACs and enables PROTAC to be a precision medicine.","author":[{"dropping-particle":"","family":"Liu","given":"Jing","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"He","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ma","given":"Leina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"He","given":"Zhixiang","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Dong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Liu","given":"Yi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lin","given":"Qian","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhang","given":"Tinghu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gray","given":"Nathanael","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kaniskan","given":"H. ?mit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jin","given":"Jian","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wei","given":"Wenyi","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Science Advances","id":"ITEM-2","issue":"8","issued":{"date-parts":[["2020"]]},"page":"1-12","title":"Light-induced control of protein destruction by opto-PROTAC","type":"article-journal","volume":"6"},"uris":[""]}],"mendeley":{"formattedCitation":"^14,15","plainTextFormattedCitation":"14,15","previouslyFormattedCitation":"^14,15"},"properties":{"noteIndex":0},"schema":""}14,15 Herein, we expand the caged PROTAC toolbox by attaching the photocleavable 4,5-dimethoxy-2-nitrobenzyl (DMNB) group to a VHL E3 ligase-recruiting ligand and produce a degrader that can be activated on demand using light (Figure 1). 590556738620-6358756650Figure 3. Evaluation of caged PROTAC activity. (A) HeLa cells were treated with PROTAC 4 or uncaged PROTAC 2 for 24?h. Cells were lysed, and lysates collected for Western blot analysis. (B) Effect of irradiation: cells were incubated with DMSO, PROTAC 4, PROTAC 2 or PROTAC 3 for 2?h then irradiated for 1?min at 365 nm. After 22?h, lysates were collected for Western blot analysis. (C) Time-course experiment: cells were lysed at 60?min intervals to evaluate onset of BRD4 degradation. For PROTAC 3, t0 represents time immediately following irradiation. (D) Washout experiment: cells were incubated with DMSO, PROTAC 4, 3 or 2 (1 μM) for 2?h, then washed 3 times with PBS before irradiation. Cells were lysed after 6?h, and lysates collected for Western blot analysis.00Figure 3. Evaluation of caged PROTAC activity. (A) HeLa cells were treated with PROTAC 4 or uncaged PROTAC 2 for 24?h. Cells were lysed, and lysates collected for Western blot analysis. (B) Effect of irradiation: cells were incubated with DMSO, PROTAC 4, PROTAC 2 or PROTAC 3 for 2?h then irradiated for 1?min at 365 nm. After 22?h, lysates were collected for Western blot analysis. (C) Time-course experiment: cells were lysed at 60?min intervals to evaluate onset of BRD4 degradation. For PROTAC 3, t0 represents time immediately following irradiation. (D) Washout experiment: cells were incubated with DMSO, PROTAC 4, 3 or 2 (1 μM) for 2?h, then washed 3 times with PBS before irradiation. Cells were lysed after 6?h, and lysates collected for Western blot analysis.71120640016500The optimized VHL ligand 1, which is derived from Hypoxia-Inducible FactorADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/ja209924v","ISSN":"00027863","abstract":"E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein-protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel-Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1alpha, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia.","author":[{"dropping-particle":"","family":"Buckley","given":"Dennis L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Molle","given":"Inge","non-dropping-particle":"Van","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gareiss","given":"Peter C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tae","given":"Hyun Seop","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Michel","given":"Julien","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Noblin","given":"Devin J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jorgensen","given":"William L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ciulli","given":"Alessio","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crews","given":"Craig M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"10","issued":{"date-parts":[["2012"]]},"page":"4465-4468","title":"Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction","type":"article-journal","volume":"134"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁶","plainTextFormattedCitation":"16","previouslyFormattedCitation":"¹⁶"},"properties":{"noteIndex":0},"schema":""}16 was functionalized with a DMNB moiety at the essential hydroxyl group to block the recruitment of VHL E3 ligase (Figure 2A). We used pan-bromodomain inhibitor JQ1 as a warhead and coupled it to the caged version of 1 through a short PEG linker (Scheme S2, ESI?). The final bifunctional molecules 2 (uncaged) and 3 (caged) are structurally related to the previously described PROTAC MZ1.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acschembio.5b00216","ISSN":"15548937","abstract":"The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra- BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.","author":[{"dropping-particle":"","family":"Zengerle","given":"Michael","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chan","given":"Kwok Ho","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ciulli","given":"Alessio","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Chemical Biology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2015"]]},"page":"1770-1777","title":"Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁷","plainTextFormattedCitation":"17","previouslyFormattedCitation":"¹⁷"},"properties":{"noteIndex":0},"schema":""}17 Additionally, another reported BRD4 degrader, compound 4, was included as a control for biological evaluation.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.cell.2018.02.030","ISSN":"10974172","abstract":"Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed “multiplexed proteome dynamics profiling” (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems. Tracking both protein synthesis and degradation across thousands of proteins yields insights into functional regulation by protein degradation.","author":[{"dropping-particle":"","family":"Savitski","given":"Mikhail M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zinn","given":"Nico","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Faelth-Savitski","given":"Maria","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Poeckel","given":"Daniel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gade","given":"Stephan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Becher","given":"Isabelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Muelbaier","given":"Marcel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wagner","given":"Anne J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Strohmer","given":"Katrin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Werner","given":"Thilo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Melchert","given":"Stephanie","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Petretich","given":"Massimo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Rutkowska","given":"Anna","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Vappiani","given":"Johanna","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Franken","given":"Holger","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Steidel","given":"Michael","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sweetman","given":"Gavain M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gilan","given":"Omer","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lam","given":"Enid Y.N.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dawson","given":"Mark A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Prinjha","given":"Rab K.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Grandi","given":"Paola","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bergamini","given":"Giovanna","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bantscheff","given":"Marcus","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Cell","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2018"]]},"page":"260-274.e25","publisher":"Elsevier Inc.","title":"Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis","type":"article-journal","volume":"173"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁸","plainTextFormattedCitation":"18","previouslyFormattedCitation":"¹⁸"},"properties":{"noteIndex":0},"schema":""}18Cleavage of the DMNB caging group using a 365?nm 25?mW LED was assessed by UV-Vis spectroscopy (Figure S1, ESI?) and monitored by LCMS (Figure 2B). Complete uncaging of compound 3 to release PROTAC 2 was achieved after a 50?μM solution was irradiated for 180 seconds with over half of the initial concentration of 3 found as uncaged 2 in just 60 seconds (Figure S2, ESI?). Furthermore, compound 3 showed good stability over the course of several days in solution when protected from light (Figure S3, ESI?).-6355143500Figure 2. Caged PROTAC design and photocharacterization. (A) Chemical structures of pan-bromodomain inhibitor JQ1, Von Hippel Lindau E3 ligase ligand 1, uncaged PROTAC 2 with free hydroxyl group, caged PROTAC 3 with 4,5-dimethoxy-2-nitrobenzyl moiety, and previously reported BRD4 degrader 4. (B) Uncaging of PROTAC 3 following irradiation with a 25 mW 365 nm LED. A 50?μM solution of 3 in acetonitrile-water (1:1) was irradiated for 1, 3 or 5?mins. LCMS area under the curve (AUC) was extracted from the chromatogram full UV spectrum.00Figure 2. Caged PROTAC design and photocharacterization. (A) Chemical structures of pan-bromodomain inhibitor JQ1, Von Hippel Lindau E3 ligase ligand 1, uncaged PROTAC 2 with free hydroxyl group, caged PROTAC 3 with 4,5-dimethoxy-2-nitrobenzyl moiety, and previously reported BRD4 degrader 4. (B) Uncaging of PROTAC 3 following irradiation with a 25 mW 365 nm LED. A 50?μM solution of 3 in acetonitrile-water (1:1) was irradiated for 1, 3 or 5?mins. LCMS area under the curve (AUC) was extracted from the chromatogram full UV spectrum.11747583502500We first assessed the ability of the uncaged PROTAC 2 to degrade a bromodomain-containing protein targeted by JQ1, BRD4. Consistent with the reported degraders 4 and MZ1,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/acschembio.5b00216","ISSN":"15548937","abstract":"The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra- BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.","author":[{"dropping-particle":"","family":"Zengerle","given":"Michael","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chan","given":"Kwok Ho","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ciulli","given":"Alessio","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ACS Chemical Biology","id":"ITEM-1","issue":"8","issued":{"date-parts":[["2015"]]},"page":"1770-1777","title":"Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁷","plainTextFormattedCitation":"17","previouslyFormattedCitation":"¹⁷"},"properties":{"noteIndex":0},"schema":""}17 compound 2 efficiently knocked down BRD4 at concentrations above 100?nM (Figure 3A). Expression of the transcription factor c-Myc was also affected by BRD4 knockdown with 2 (Figure S4, ESI?). Subsequently, we tested the ability of the caged PROTAC 3 to degrade BRD4 following activation with light. HeLa cells were incubated with 3 for 2?h prior to irradiation for 60?seconds at 80?mm from a 25?mW 365?nm LED. Dose-dependent degradation of BRD4 was observed only upon irradiation, with complete knockdown seen at 1?μM (Figure 3B). Moreover, caged PROTAC 3 showed good stability in the cellular environment since no degradation was observed in non-irradiated cells even after a 24 h-incubation period.We next asked if degradation with 3 after irradiation could be observed within the same timeframe as the parent PROTAC 2, 346710084772500given that the caged molecule 3 requires an activation step before it can act as a degrader. BRD4 level was monitored for the first few hours following incubation with uncaged and caged PROTACs (Figure 3C). Interestingly, while PROTAC 4 took about 4h to fully knockdown BRD4, complete loss of the protein was seen just after 1?h for both 2 and 3. This result demonstrates that our caging strategy allows degradation to proceed within the same timeframe as for the parent uncaged molecule 2 and rules out uncaging as a rate-limiting step. Next, to ensure that caged PROTAC 3 enters the cell before being uncaged, a washout experiment was conducted (Figure 3D). After a 2?h-incubation with compounds, HeLa cells were thoroughly washed to remove residual PROTAC then were irradiated at 365?nm for 60 seconds. Western blot analysis suggests that 3 effectively entered the cells within the 2?h-treatment period since degradation of BRD4 was still observed after irradiation. Incomplete knockdown of BRD4 was noticed with PROTAC 4 after a 2-h incubation and correlated with the slower degradation rate seen in the time-course experiment. In addition, we also confirmed that our caging strategy did not affect the mode of action of the degrader 3 which operates in an E3 ligase and proteasome-dependent manner only after being uncaged (Figure S5, ESI?).32321555803800008808085Figure 5. Light-induced GFP-BRD4 degradation by caged PROTAC measured by?live-cell fluorescence imaging of HEK293 cells transfected with pEGFP-BRD4-C1 plasmid. Cells were incubated with?(A)?1?μM PROTAC 3 for 1?h and UV irradiated for 60 seconds, or with (B) 1 μM PROTAC 3,?(C) 1?μM PROTAC 2 or?(D) DMSO vehicle (0.1% v/v), and UV irradiated for 60 seconds. GFP fluorescence was monitored over 3?h, with images taken every 20 min. Scale bar = 20?μm. (E)?Quantification of GFP signal degradation over time following PROTAC (1?μM) or DMSO (0.1% v/v) treatment, with or without UV irradiation. Each data point represents background-subtracted 0 time-normalized mean fluorescence from n = 10 single cells, error bars represent SEM.00Figure 5. Light-induced GFP-BRD4 degradation by caged PROTAC measured by?live-cell fluorescence imaging of HEK293 cells transfected with pEGFP-BRD4-C1 plasmid. Cells were incubated with?(A)?1?μM PROTAC 3 for 1?h and UV irradiated for 60 seconds, or with (B) 1 μM PROTAC 3,?(C) 1?μM PROTAC 2 or?(D) DMSO vehicle (0.1% v/v), and UV irradiated for 60 seconds. GFP fluorescence was monitored over 3?h, with images taken every 20 min. Scale bar = 20?μm. (E)?Quantification of GFP signal degradation over time following PROTAC (1?μM) or DMSO (0.1% v/v) treatment, with or without UV irradiation. Each data point represents background-subtracted 0 time-normalized mean fluorescence from n = 10 single cells, error bars represent SEM.BRD4 inhibition is reported to significantly impact cell proliferation,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nature09504","ISSN":"00280836","abstract":"Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family. ? 2010 Macmillan Publishers Limited.","author":[{"dropping-particle":"","family":"Filippakopoulos","given":"Panagis","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Qi","given":"Jun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Picaud","given":"Sarah","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shen","given":"Yao","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Smith","given":"William B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fedorov","given":"Oleg","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Morse","given":"Elizabeth M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Keates","given":"Tracey","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hickman","given":"Tyler T.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Felletar","given":"Ildiko","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Philpott","given":"Martin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Munro","given":"Shonagh","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"McKeown","given":"Michael R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Yuchuan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Christie","given":"Amanda L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"West","given":"Nathan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cameron","given":"Michael J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Schwartz","given":"Brian","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Heightman","given":"Tom D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Thangue","given":"Nicholas","non-dropping-particle":"La","parse-names":false,"suffix":""},{"dropping-particle":"","family":"French","given":"Christopher A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wiest","given":"Olaf","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kung","given":"Andrew L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Knapp","given":"Stefan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bradner","given":"James E.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature","id":"ITEM-1","issue":"7327","issued":{"date-parts":[["2010"]]},"page":"1067-1073","title":"Selective inhibition of BET bromodomains","type":"article-journal","volume":"468"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁹","plainTextFormattedCitation":"19","previouslyFormattedCitation":"¹⁹"},"properties":{"noteIndex":0},"schema":""}19 and we therefore examined the overall phenotypic effect of 3 using live-cell imaging analysis before and after irradiation (Figure 4 and Figure S6, ESI?).33648652279650Figure 4. Effect of PROTACs on cell proliferation. HeLa cells were treated with DMSO, PROTAC 2 (1?μM) or 3 (1?μM) either without initial irradiation or with a 60-second irradiation time. Cell count was followed over 6 days, normalized to cell count at t0, and fold changes plotted.00Figure 4. Effect of PROTACs on cell proliferation. HeLa cells were treated with DMSO, PROTAC 2 (1?μM) or 3 (1?μM) either without initial irradiation or with a 60-second irradiation time. Cell count was followed over 6 days, normalized to cell count at t0, and fold changes plotted.HeLa cells were monitored for up to 6 days after treatment with 1?μM of caged and uncaged PROTACs. The cell proliferation profile without irradiation highlights the different mode of action between PROTAC 2 and 3 (Figure 4). While the caged compound 3 displayed a cytostatic profile over time (average proliferation fold change = 1), the uncaged PROTAC 2 reduced cell count by half (average proliferation fold change < 0.5) after 48 h. Most importantly, uncaging of compound 3 was characterized by a striking change in the growth curve profile that exactly overlaid with 2, confirming mechanistically the release of the active degrader upon irradiation. We attribute the cytostatic effect of PROTAC 3 before irradiation to interaction of the JQ1 moiety with BRD4 (Figure S6B, ESI?). An in-cell target engagement study using a cellular thermal shift assayADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Molina","given":"Daniel Martinez","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jafari","given":"Rozbeh","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ignatushchenko","given":"Marina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Seki","given":"Takahiro","non-dropping-particle":"","parse-names":false,"suffix":""}],"id":"ITEM-1","issue":"July","issued":{"date-parts":[["2013"]]},"page":"84-88","title":"D ( 4 ).","type":"article-journal","volume":"341"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁰","plainTextFormattedCitation":"20","previouslyFormattedCitation":"²⁰"},"properties":{"noteIndex":0},"schema":""}20 (CETSA) confirmed PROTAC 3 binding to BRD4 (Figure S7, ESI?).Finally, to complement our previous assessment of BRD4 degradation by Western blots, we investigated photoinduced BRD4 knockdown in real time using live-cell fluorescence imaging (Figure 5). For this purpose, a GFP-BRD4 expression construct was generated for transient expression in HEK293 cells. Prior to?transfection, we also confirmed that PROTAC 3 degraded BRD4 in HEK293 cells when irradiated (Figure S8, ESI?). Similar to the data gathered from HeLa cells, 1?μM compound 2 delivered significant GFP-BRD4 knockdown within 2?h as shown by the rapid decrease in fluorescence as compared to the DMSO control (Figure 5C, 5D and 5E). On the other hand, PROTAC 3 had no effect on GFP-BRD4 levels until it was uncaged to deliver an active degrader that removed GFP-tagged BRD4 within the same timeframe as 2 (Figure 5A, 5B and 5E). A comparable degradation profile was obtained with 5?μM PROTAC 3 following irradiation (Figure S9 and S10, ESI?).In summary, we have demonstrated conditional PROTAC-directed protein degradation using a caged VHL ligand and light as a conditional stimulus. This method relies on the straightforward attachment of the DMNB group onto a VHL E3 ligase ligand, enabling intracellular activation of the caged degrader with a short irradiation time (60 seconds) followed by VHL- and proteasome-dependent removal of BRD4. Overall, this study establishes caged VHL PROTACs as useful tools for the spatiotemporal control of protein stability, whereby E3 ligase activity is rendered conditional on light, independent of the ligand against the protein of interest. We anticipate this caging strategy will have widespread applicability considering the importance of VHL E3 ligase in the targeted protein degradation field.This study was supported by the Engineering and Physical Sciences Research Council (EPSRC) grants EP/R512540/1 and EPSRC Centre for Doctoral Training in Chemical Biology (EP/F500416/1), the Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/S001565/1 and BB/N016947/1, and GlaxoSmithKline Research and Development Ltd.Conflicts of interestM.M. and J.D.H are employees and shareholders of?GlaxoSmithKline (GSK). E.W.T. is a Director and shareholder of Myricx Pharma Ltd. M.D.R is an employee of Merck Sharp & Dohme.ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY 1A. C. Lai and C. M. Crews, Nat. Rev. Drug Discov., 2017, 16, 101–114.2I. Churcher, J. Med. Chem., 2018, 61, 444–452.3Y. Zhang, C. Loh, J. Chen and N. Mainolfi, Drug Discov. Today Technol., 2019, 31, 53–60.4S. R?th, L. J. Fulcher and G. P. 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