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  1. Article ; Online: Kinetic Detection of E3:PROTAC:Target Ternary Complexes Using NanoBRET Technology in Live Cells.

    Mahan, Sarah D / Riching, Kristin M / Urh, Marjeta / Daniels, Danette L

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2365, Page(s) 151–171

    Abstract: Heterobifunctional small-molecule degraders known as Proteolysis Targeting Chimeras (PROTACs) serve as a chemical bridge bringing into direct association a target protein with an active E3 ligase complex, called the ternary complex, to facilitate ... ...

    Abstract Heterobifunctional small-molecule degraders known as Proteolysis Targeting Chimeras (PROTACs) serve as a chemical bridge bringing into direct association a target protein with an active E3 ligase complex, called the ternary complex, to facilitate targeted protein degradation. This ternary complex formation is the first key mechanistic step in a cascade of events that results in ubiquitination and subsequent degradation of the target protein via the ubiquitin-proteasome pathway. The ternary complex, however, is a nonnative cellular complex; therefore, PROTAC compound design has many challenges to overcome to ensure successful formation, including achieving structural and electrostatic favorability among target and ligase. Due to these challenges, finding successful PROTACs typically requires testing of extensive libraries of heterobifunctional compounds with varying linkers and E3 handles. As PROTAC ternary complex formation is also critically dependent on cellular context, live cell assays and technologies for rapid and robust screening are highly enabling for triaging of early stage compounds. Here, we present cellular assays utilizing NanoBRET technology for the study of ternary complexes, showing examples with two most popular PROTAC E3 ligase components, VHL (von Hippel-Lindau disease tumor suppressor) and CRBN (Cereblon). These assays can be run in either endpoint or real-time kinetic formats, are compatible with high-throughput workflows, and provide insight into how altering the PROTAC chemical composition affects the formation and stability of the ternary complex in live cells.
    MeSH term(s) Cell Survival ; Proteolysis ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination ; Nanotechnology
    Chemical Substances Ubiquitin-Protein Ligases (EC 2.3.2.27)
    Language English
    Publishing date 2021-08-24
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1665-9_8
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  2. Article ; Online: High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds using HiBiT CRISPR Cell Lines.

    Riching, Kristin M / Mahan, Sarah D / Urh, Marjeta / Daniels, Danette L

    Journal of visualized experiments : JoVE

    2020  , Issue 165

    Abstract: 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 ...

    Abstract 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.
    MeSH term(s) Cell Adhesion ; Cell Line ; Cell Survival ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fluorescence ; HEK293 Cells ; High-Throughput Screening Assays ; Humans ; Kinetics ; Proteolysis ; Ubiquitination
    Language English
    Publishing date 2020-11-09
    Publishing country United States
    Document type Journal Article ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/61787
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  3. Article ; Online: Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.

    Caine, Elizabeth A / Mahan, Sarah D / Johnson, Rebecca L / Nieman, Amanda N / Lam, Ngan / Warren, Curtis R / Riching, Kristin M / Urh, Marjeta / Daniels, Danette L

    Current protocols in pharmacology

    2021  Volume 91, Issue 1, Page(s) e81

    Abstract: To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative ... ...

    Abstract To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.
    MeSH term(s) CRISPR-Cas Systems ; Cell Line ; Electroporation ; Humans ; Proteolysis ; Recombinant Fusion Proteins/chemistry
    Chemical Substances Recombinant Fusion Proteins
    Language English
    Publishing date 2021-07-29
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2179074-7
    ISSN 1934-8290 ; 1934-8282
    ISSN (online) 1934-8290
    ISSN 1934-8282
    DOI 10.1002/cpph.81
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  4. Article ; Online: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity.

    Imaide, Satomi / Riching, Kristin M / Makukhin, Nikolai / Vetma, Vesna / Whitworth, Claire / Hughes, Scott J / Trainor, Nicole / Mahan, Sarah D / Murphy, Nancy / Cowan, Angus D / Chan, Kwok-Ho / Craigon, Conner / Testa, Andrea / Maniaci, Chiara / Urh, Marjeta / Daniels, Danette L / Ciulli, Alessio

    Nature chemical biology

    2021  Volume 17, Issue 11, Page(s) 1157–1167

    Abstract: Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could ... ...

    Abstract Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.
    MeSH term(s) Humans ; Proteolysis ; Ubiquitin-Protein Ligases/metabolism
    Chemical Substances Ubiquitin-Protein Ligases (EC 2.3.2.27)
    Language English
    Publishing date 2021-10-21
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2202962-X
    ISSN 1552-4469 ; 1552-4450
    ISSN (online) 1552-4469
    ISSN 1552-4450
    DOI 10.1038/s41589-021-00878-4
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    In stock of ZB MED Cologne/Königswinter

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  5. Article ; Online: Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

    Mishra, Sweta / Van Rechem, Capucine / Pal, Sangita / Clarke, Thomas L / Chakraborty, Damayanti / Mahan, Sarah D / Black, Joshua C / Murphy, Sedona E / Lawrence, Michael S / Daniels, Danette L / Whetstine, Johnathan R

    Cell

    2018  Volume 175, Issue 6, Page(s) 1716

    Language English
    Publishing date 2018-12-13
    Publishing country United States
    Document type Published Erratum
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2018.11.018
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  6. Article ; Online: Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

    Mishra, Sweta / Van Rechem, Capucine / Pal, Sangita / Clarke, Thomas L / Chakraborty, Damayanti / Mahan, Sarah D / Black, Joshua C / Murphy, Sedona E / Lawrence, Michael S / Daniels, Danette L / Whetstine, Johnathan R

    Cell

    2018  Volume 174, Issue 4, Page(s) 803–817.e16

    Abstract: Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional ... ...

    Abstract Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.
    MeSH term(s) Cell Cycle ; Chromatin/metabolism ; DNA Copy Number Variations ; DNA Methylation ; HEK293 Cells ; Histones/metabolism ; Humans ; Lysine/metabolism ; Retinoblastoma-Binding Protein 2/genetics ; Retinoblastoma-Binding Protein 2/metabolism
    Chemical Substances Chromatin ; Histones ; KDM5A protein, human (EC 1.14.11.-) ; Retinoblastoma-Binding Protein 2 (EC 1.14.11.27) ; Lysine (K3Z4F929H6)
    Language English
    Publishing date 2018-07-26
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2018.06.018
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  7. Article: Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications

    Mishra, Sweta / Van Rechem, Capucine / Pal, Sangita / Clarke, Thomas L / Chakraborty, Damayanti / Mahan, Sarah D / Black, Joshua C / Murphy, Sedona E / Lawrence, Michael S / Daniels, Danette L / Whetstine, Johnathan R

    Cell. 2018 Aug. 09, v. 174, no. 4

    2018  

    Abstract: Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional ... ...

    Abstract Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.[Display omitted]
    Keywords DNA ; chromatin ; epigenetics ; gene overexpression ; genomics ; histones ; loci ; lysine ; methylation ; methyltransferases ; neoplasms
    Language English
    Dates of publication 2018-0809
    Size p. 803-817.e16.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2018.06.018
    Database NAL-Catalogue (AGRICOLA)

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