DOCSLIB.ORG

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents Against MYC-Driven Pediatric Cancer P

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents Against MYC-Driven Pediatric Cancer P Published OnlineFirst July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934 CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer P. Jake Slavish1, Liying Chi1, Mi-Kyung Yun2, Lyudmila Tsurkan1, Nancy E. Martinez1, Barbara Jonchere3, Sergio C. Chai1, Michele Connelly1, M. Brett Waddell4, Sourav Das1, Geoffrey Neale5, Zhenmei Li2, William R. Shadrick1, Rachelle R. Olsen6, Kevin W. Freeman6, Jonathan A. Low1, Jeanine E. Price1, Brandon M. Young1, Nagakumar Bharatham1, Vincent A. Boyd1,†, Jun Yang7, Richard E. Lee1, Marie Morfouace3, Martine F. Roussel3, Taosheng Chen1, Daniel Savic8, R. Kiplin Guy1, Stephen W. White2, Anang A. Shelat1, and Philip M. Potter1 ABSTRACT ◥ Inhibition of members of the bromodomain and extraterminal MYC proteins and downstream targets. These compounds were (BET) family of proteins has proven a valid strategy for cancer potent cytotoxins toward numerous pediatric cancer cell lines and chemotherapy. All BET identified to date contain two bromodo- were minimally toxic to nontumorigenic cells. In addition, unlike mains (BD; BD1 and BD2) that are necessary for recognition of the pan BETi (þ)-JQ1, these BD2-selective inhibitors demonstrated acetylated lysine residues in the N-terminal regions of histones. no rebound expression effects. Finally, we report a pharmacoki- Chemical matter that targets BET (BETi) also interact via these netic-optimized, metabolically stable derivative that induced domains. Molecular and cellular data indicate that BD1 and BD2 growth delay in a neuroblastoma xenograft model with minimal have different biological roles depending upon their cellular context, toxicity. We conclude that BD2-selective agents are valid candidates with BD2 particularly associated with cancer. We have therefore for antitumor drug design for pediatric malignancies driven by the pursued the development of BD2-selective molecules both as MYC oncogene. chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of Significance: This study presents bromodomain-selective BET tetrahydroquinoline analogs that exhibit >50-fold selectivity for inhibitors that act as antitumor agents and demonstrates that these BD2 versus BD1. This selective targeting resulted in engagement molecules have in vivo activity towards neuroblastoma, with essen- with BD-containing proteins in cells, resulting in modulation of tially no toxicity. 1Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Introduction 2 Hospital, Memphis, Tennessee. Department of Structural Biology, St. Jude The targeting of chromatin-modifying proteins has become an Children’s Research Hospital, Memphis, Tennessee. 3Department of Tumor Cell fi ’ 4 attractive avenue for drug design, speci cally for antitumor Biology, St. Jude Children s Research Hospital, Memphis, Tennessee. Molecular – Interaction Analysis Shared Resource, St. Jude Children’s Research Hospital, agents (1 3). Consequently, there are considerable ongoing efforts Memphis, Tennessee. 5Hartwell Center, St. Jude Children’s Research Hospital, to develop chemical matter that demonstrates specificity for such Memphis, Tennessee. 6Department of Oncology, St. Jude Children’s Research proteins. The bromodomain and extraterminal (BET) family of Hospital, Memphis, Tennessee. 7Department of Surgery, St. Jude Children’s proteins, which includes BRD2, BRD3, BRD4, and BRDT, contains 8 Research Hospital, Memphis, Tennessee. Department of Pharmaceutical two domains (BD1 and BD2) that are required for the recognition of ’ Sciences, St. Jude Children s Research Hospital, Memphis, Tennessee. acetylated lysine residues in histones (4, 5). These bromodomains Note: Supplementary data for this article are available at Cancer Research (BD) demonstrate considerable amino acid and structural homol- Online (http://cancerres.aacrjournals.org/). ogy, and hence, it has been difficult to develop small molecules with Current address for W.R. Shadrick: Vaccine Production Program, VRC, NIAID, significant selectivity toward either BD1 or BD2 (6). Therefore, NIH, Gaithersburg, Maryland; current address for R.R. Olsen, Department of most clinical candidates that target BET inhibit both BD with Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, Utah; current similar efficiencies. This includes the exemplar compound address for K.W. Freeman, Genetics, Genomics & Informatics, The University of þ Tennessee Health Science Center, Memphis, Tennessee; current address for M. ( )-JQ1 (1), based upon a benzodiazepine scaffold, which is Morfouace, EORTC, Brussels, Belgium; and current address for R.K. Guy, College currently the basis for at least four molecules in clinical trials (see of Pharmacy, University of Kentucky, Lexington, Kentucky. http://clinicaltrials.gov). P.J. Slavish, L. Chi, M.-K. Yun, and L. Tsurkan contributed equally to this article. However, biochemical and cellular studies have revealed that independently modulating these domains results in different down- †Deceased. stream sequelae, arguing that the recognition of acetylated lysine Corresponding Authors: Philip M. Potter and Anang A, Shelat, St. Jude Chil- residues by BD1 and BD2 is specific and leads to the regulation of ’ dren s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2729. disparate gene sets (7–11). For example, BRDT-BD1 (but not BRDT- Phone: 901-595-2825; Fax: 901-595-4293; E-mail: [email protected]; and Anang A. Shelat, [email protected] BD2) is required for spermatogenesis (8). Similarly, isothermal titra- tion calorimetry (ITC) studies indicate that BD1 preferentially binds to – Cancer Res 2020;80:3507 18 histone H3 sequences, whereas BD2 has higher affinity for histone H4 doi: 10.1158/0008-5472.CAN-19-3934 and acetylated lysine peptides derived from cyclin T1 (12). BRD3-BD1 Ó2020 American Association for Cancer Research. has been shown to be important for the expression of erythroid and AACRJournals.org | 3507 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934 Slavish et al. megakaryocyte-specific genes through interactions with the acetylated collected to establish a baseline. A single laser pulse lasting 0.09 transcription factor (TF) GATA1 (7). In contrast, BRD4-BD2 recruits seconds was then targeted onto the nucleus and single section images TWIST, and BRD4-BD1 serves to anchor the complex to chroma- were collected at 200 ms intervals. Fluorescence intensity plots were tin (9). Because there are greater sequence homologies among the BET generated using Slidebook 6 Â 64 software (3i Technologies), and BD1 BDs and BD2 BDs, as compared with BD1 versus BD2, this FRAP determined as described previously (17). suggests that the separate bromodomains have different biological functions. We hypothesized therefore, that targeting either BD1 or Microarray BD2 may be exploited in anticancer drug design. Total RNA was hybridized to an Affymetrix Human Gene 2.0 ST We have developed BD-selective BET inhibitors (BETi) chemical Array (Thermo Fisher Scientific) and signals normalized using probes that target BD1 and BD2s, and assessed their activity in the multiarray average algorithm (18). Datasets were subsequently pediatric tumor models. Using the tetrahydroquinoline (THQ) analyzed by a one-factor ANOVA model (Partek Genomics Suite) scaffold (6, 13) and a combination of structure-based iterative drug where drug plus concentration were used as the treatment factor. A design, coupled with biochemical analyses, we have generated FDR threshold of <0.05 was used to identify differentially expressed highly potent, BD2-selective BETi. These molecules: bind BET in transcripts and were analyzed for functional enrichment using the cultured cells; downregulate MYC and associated targets; are cyto- Enrichr (19) and DAVID (20) bioinformatics databases. toxic to tumor, but less so to nontumorigenic cells; and are minimally toxic in vivo and induce antitumor activity. While Chromatin immunoprecipitation sequencing BD-selective probes have been synthesized previously (14–16), our Chromatin immunoprecipitation sequencing (ChIP-seq) was per- studies seek to validate the activity of such compounds toward formed as described previously (21). Differential histone 3, lysine pediatric tumors with dire prognoses. 27 acetylation (H3K27ac) read enrichment was identified using DESeq2 (22) on normalized read depth at the union of all reproducible H3K27ac sites. Principal component analysis was performed using the Materials and Methods prcomp function in R. CentriMo (23) and was used to identify enriched Reagents TF motifs within differentially enriched H3K27ac sites. The list of cell lines, their sources, culture conditions, and antibodies used in this study are reported in the Supplementary Information BRD2-BD1 and BRD2-BD2 expression (Supplementary Tables S1–S3). Three neuroblastoma lines were cDNAs encoding the human BRD2-BD1 (residues 67–200) and used for subsequent studies based upon their MYC expression status: BRD2-BD2 (amino acids 348–455) domains were expressed from SJ-N-AS—Amplified c-MYC, no expression of MYCN; SK-N-SH— pET28a(þ) containing a N-terminal His-tag. Detailed methods are Expresses c-MYC, no expression of MYCN; IMR32—No expression of provided in the Supporting Information. c-MYC, MYNC amplified (Supplementary Fig. S1). Cell lines
Load more

Recommended publications
  • Bromodomain Factors of BET Family Are New Essential Actors Of
    Bromodomain factors of BET family are new essential actors of pericentric heterochromatin transcriptional activation in response to heat shock Edwige Col, Neda Hoghoughi, Solenne Dufour, Jessica Penin, Sivan Koskas, Virginie Faure, Maria Ouzounova, Hector Hernandez-Vargash, Nicolas Reynoird, Sylvain Daujat, et al. To cite this version: Edwige Col, Neda Hoghoughi, Solenne Dufour, Jessica Penin, Sivan Koskas, et al.. Bromodomain factors of BET family are new essential actors of pericentric heterochromatin transcriptional acti- vation in response to heat shock. Scientific Reports, Nature Publishing Group, 2017, 7, pp.5418. 10.1038/s41598-017-05343-8. hal-02989794 HAL Id: hal-02989794 https://hal.archives-ouvertes.fr/hal-02989794 Submitted on 5 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. www.nature.com/scientificreports OPEN Bromodomain factors of BET family are new essential actors of pericentric heterochromatin Received: 5 August 2016 Accepted: 30 May 2017 transcriptional activation in Published: xx xx xxxx response to heat shock Edwige Col1, Neda Hoghoughi1, Solenne Dufour1, Jessica Penin1, Sivan Koskas1, Virginie Faure1, Maria Ouzounova2, Hector Hernandez-Vargash2, Nicolas Reynoird1, Sylvain Daujat4, Eric Folco1, Marc Vigneron3, Robert Schneider4,5, André Verdel1, Saadi Khochbin1, Zdenko Herceg2, Cécile Caron1 & Claire Vourc’h1 The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin.
    [Show full text]
  • Functional Roles of Bromodomain Proteins in Cancer
    cancers Review Functional Roles of Bromodomain Proteins in Cancer Samuel P. Boyson 1,2, Cong Gao 3, Kathleen Quinn 2,3, Joseph Boyd 3, Hana Paculova 3 , Seth Frietze 3,4,* and Karen C. Glass 1,2,4,* 1 Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, VT 05446, USA; [email protected] 2 Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington, VT 05405, USA; [email protected] 3 Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT 05405, USA; [email protected] (C.G.); [email protected] (J.B.); [email protected] (H.P.) 4 University of Vermont Cancer Center, Burlington, VT 05405, USA * Correspondence: [email protected] (S.F.); [email protected] (K.C.G.) Simple Summary: This review provides an in depth analysis of the role of bromodomain-containing proteins in cancer development. As readers of acetylated lysine on nucleosomal histones, bromod- omain proteins are poised to activate gene expression, and often promote cancer progression. We examined changes in gene expression patterns that are observed in bromodomain-containing proteins and associated with specific cancer types. We also mapped the protein–protein interaction network for the human bromodomain-containing proteins, discuss the cellular roles of these epigenetic regu- lators as part of nine different functional groups, and identify bromodomain-specific mechanisms in cancer development. Lastly, we summarize emerging strategies to target bromodomain proteins in cancer therapy, including those that may be essential for overcoming resistance. Overall, this review provides a timely discussion of the different mechanisms of bromodomain-containing pro- Citation: Boyson, S.P.; Gao, C.; teins in cancer, and an updated assessment of their utility as a therapeutic target for a variety of Quinn, K.; Boyd, J.; Paculova, H.; cancer subtypes.
    [Show full text]
  • Histone-Binding of DPF2 Mediates Its Repressive Role in Myeloid Differentiation
    Histone-binding of DPF2 mediates its repressive role in myeloid differentiation Ferdinand M. Hubera,1, Sarah M. Greenblattb,1, Andrew M. Davenporta,1, Concepcion Martinezb,YeXub,LyP.Vuc, Stephen D. Nimerb,2, and André Hoelza,2 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; bSylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136; and cMolecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065 Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved April 26, 2017 (received for review January 6, 2017) Double plant homeodomain finger 2 (DPF2) is a highly evolution- RUNX1 form a methylation-dependent repressive complex in arily conserved member of the d4 protein family that is ubiqui- AML, although it remains unclear whether the two proteins bind tously expressed in human tissues and was recently shown to each other directly or act concertedly as part of a larger complex. inhibit the myeloid differentiation of hematopoietic stem/progen- Here, we present the crystal structure of the human DPF2 itor and acute myelogenous leukemia cells. Here, we present the tandem PHD finger domain at a 1.6-Å resolution. We demon- crystal structure of the tandem plant homeodomain finger domain strate that the DPF2 tandem PHD finger domain binds acetylated of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts H3 and H4 histone tails, identify the primary determinants of with the acetylated tails of both histones 3 and 4 via bipartite histone recognition, and confirm these interactions in vivo.
    [Show full text]
  • Function of Bromodomain and Extra-Terminal Motif Proteins (Bets) in Gata1-Mediated Transcription
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription Aaron James Stonestrom University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Stonestrom, Aaron James, "Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription" (2015). Publicly Accessible Penn Dissertations. 1148. https://repository.upenn.edu/edissertations/1148 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1148 For more information, please contact [email protected]. Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription Abstract Bromodomain and Extra-Terminal motif proteins (BETs) associate with acetylated histones and transcription factors. While pharmacologic inhibition of this ubiquitous protein family is an emerging therapeutic approach for neoplastic and inflammatory disease, the mechanisms through which BETs act remain largely uncharacterized. Here we explore the role of BETs in the physiologically relevant context of erythropoiesis driven by the transcription factor GATA1. First, we characterize functions of the BET family as a whole using a pharmacologic approach. We find that BETs are broadly required for GATA1-mediated transcriptional activation, but that repression is largely BET-independent. BETs support activation by facilitating both GATA1 occupancy and transcription downstream of its binding. Second, we test the specific olesr of BETs BRD2, BRD3, and BRD4 in GATA1-activated transcription. BRD2 and BRD4 are required for efficient anscriptionaltr activation by GATA1. Despite co-localizing with the great majority of GATA1 binding sites, we find that BRD3 is not equirr ed for GATA1-mediated transcriptional activation.
    [Show full text]
  • Download Product Insert (PDF)
    PRODUCT INFORMATION BRD3 bromodomains 1 and 2 (human, recombinant) Item No. 14864 Overview and Properties Synonyms: Bromodomain containing protein 3, ORFX, RING3L, RING3-like protein Source: Recombinant N-terminal GST-tagged protein expressed in E. coli Amino Acids: 2-434 Uniprot No.: Q15059 Molecular Weight: 75.0 kDa Storage: -80°C (as supplied) Stability: ≥2 years Purity: batch specific (≥85% estimated by SDS-PAGE) Supplied in: 50 mM Tris, pH 8.0, with 150 mM sodium chloride and 20% glycerol Protein Concentration: batch specific mg/ml Activity: batch specific U/ml Specific Activity: batch specific U/mg Information represents the product specifications. Batch specific analytical results are provided on each certificate of analysis. Image 1 2 3 4 · · · · · · ·250 kDa · · · · · · ·150 kDa · · · · · · ·100 kDa · · · · · · ·75 kDa · · · · · · ·50 kDa · · · · · · ·37 kDa · · · · · · ·25 kDa · · · · · · ·20 kDa · · · · · · ·15 kDa Lane 1: BRD3 (4 µg) Lane 2: BRD3 (6 µg) Lane 3: BRD3 (8 µg) Lane 4: MW Markers WARNING CAYMAN CHEMICAL THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE. 1180 EAST ELLSWORTH RD SAFETY DATA ANN ARBOR, MI 48108 · USA This material should be considered hazardous until further information becomes available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash thoroughly after handling. Before use, the user must review the complete Safety Data Sheet, which has been sent via email to your institution. PHONE: [800] 364-9897 WARRANTY AND LIMITATION OF REMEDY [734] 971-3335 Buyer agrees to purchase the material subject to Cayman’s Terms and Conditions. Complete Terms and Conditions including Warranty and Limitation of Liability information can be found on our website.
    [Show full text]
  • Discovery of a Hidden Transient State in All Bromodomain Families
    Discovery of a hidden transient state in all bromodomain families Lluís Raicha, Katharina Meierb, Judith Güntherc, Clara D. Christc, Frank Noéa,d,1, and Simon Olssona,1,2 aDepartment of Mathematics and Computer Science, Freie Universität Berlin, 14195 Berlin, Germany; bComputational Molecular Design, Pharmaceuticals, R&D, Bayer AG, 42096 Wuppertal, Germany; cComputational Molecular Design, Pharmaceuticals, R&D, Bayer AG, 13342 Berlin, Germany; and dDepartment of Chemistry, Rice University, Houston, TX 77005 Edited by David Baker, University of Washington, Seattle, WA, and approved December 1, 2020 (received for review August 17, 2020) Bromodomains (BDs) are small protein modules that interact with comprised by a four-helix bundle (αZ, αA, αB,andαC) with two acetylated marks in histones. These posttranslational modifica- principal loops that connect them (ZA- and BC-loops), con- tions are pivotal to regulate gene expression, making BDs prom- forming a hydrophobic pocket that is suited for acetyl-lysine ising targets to treat several diseases. While the general structure binding (Fig. 1) (24). The BC-loop contains a well-conserved hy- of BDs is well known, their dynamical features and their interplay drogen bond donor or “reader”—usually an asparagine—that is with other macromolecules are poorly understood, hampering the key for recognition. The ZA-loop is the longest loop and the one rational design of potent and selective inhibitors. Here, we com- that displays more variability in terms of sequence and structure, bine extensive molecular dynamics simulations, Markov state presenting short helices, turns, and even hairpin insertions in BD modeling, and available structural data to reveal a transiently family VIII (Fig.
    [Show full text]
  • Acetyltransferase Machinery Conserved in P300/CBP-Family Proteins
    Oncogene (2002) 21, 2253 ± 2260 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc SHORT REPORT Acetyltransferase machinery conserved in p300/CBP-family proteins L Wuchao Yuan1 and Antonio Giordano*,2 1Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts, MA 02118, USA; 2Department of Pathology, Anatomy and Cell Biology, Jeerson Medical College, Philadelphia, Pennsylvania, PA 19107, USA CREB-binding protein (CBP) and p300 are highly supports this proposal. Later, this protein family was conserved and functionally related transcription coacti- expanded after the discovery of Drosophila CBP (dCBP), vators and histone/protein acetyltransferases. They are which was also able to bind to E1A and coactivate tumor suppressors, participate in a wide variety of CREB-dependent transactivation (Akimaru et al., physiological events, and serve as integrators among 1997a). Recently, p300/CBP-like proteins of the plant dierent signal transduction pathways. In this study, 11 Arabidopsis thaliana have been reported (Bordoli et al., distinct proteins that have a high degree of homology 2001), suggesting that they are possible members of the with the amino acid sequence of p300 have been p300/CBP family. It is interesting to know how large the identi®ed in current protein databases. All of these 11 p300/CBP protein family could be and how this family of proteins belong to either animal or plant multicellular proteins is conserved evolutionarily. organisms (higher eucaryotes). Conservation of p300/ Taking advantage of recent progress in genomic CBP domains among these proteins was examined sequencing of dierent organisms, we performed a blast further by sequence alignment and pattern search.
    [Show full text]
  • The Role of Bromodomain Proteins in Regulating Gene Expression
    Genes 2012, 3, 320-343; doi:10.3390/genes3020320 OPEN ACCESS genes ISSN 2073-4425 www.mdpi.com/journal/genes Review The Role of Bromodomain Proteins in Regulating Gene Expression Gabrielle A. Josling, Shamista A. Selvarajah, Michaela Petter and Michael F. Duffy * Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Australia; E-Mails: [email protected] (G.A.J.); [email protected] (S.A.S.); [email protected] (M.P.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +61-38344-3262; Fax: +61-39347-1863. Received: 30 April 2012; in revised form: 11 May 2012 / Accepted: 17 May 2012 / Published: 29 May 2012 Abstract: Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription. Keywords: bromodomain; histone acetylation; histone modifications; histone code; epigenetics 1. Introduction The post-translational modification of histones plays an important role in regulating gene expression in eukaryotes.
    [Show full text]
  • BRD2-2 (His) (Bromodomain Containing Protein 2 (RING3), Bromodomain 2)
    BRD2-2 (His) (Bromodomain containing protein 2 (RING3), bromodomain 2) CATALOG NO.: RD-11-145 LOT NO.: DESCRIPTION: Human recombinant BRD2, bromodomain-2 (residues 344-454; Genbank Accession # NM_005104; MW = 15.7 kDa) expressed in E. coli with an N-terminal His-tag. BRD2, like other human members of the BET family of chromatin-binding proteins (BRD3, BRD4, BRDT), comprises two bromodomains (see reviews 1,2 ), protein modules that bind ε-N-acetyllysine residues 3,4 . When overexpressed in 293 cells, BRD2, along with BRD3, binds the hyperacetylated chromatin of transcribed genes, regions enriched in acetylated histone H4 lysine-5 (H4K5Ac), H4K12Ac, H3K14Ac, but deficient in H4K16Ac and H3K9me 5. A single H4K5AcK12Ac peptide can bind two copies of BRD2-2 (BRD2, bromodomain 2), each interacting with one of the two acetylated lysines 6. In an in vitro RNA polymerase II transcription system, binding of either BRD2 or BRD3 to a chromatin template assembled with hyperacetylated histones enabled transcription through the nucleosomes 5. Further, BRD2 displayed histone chaperone activity, catalyzing the transfer of histone octamers from hyperacetylated oligonucleosomes to a labeled 190 bp 5s rDNA fragment 5. Like BRD4, BRD2 is a ubiquitously expressed 7 transcriptional regulator 8 and atypical protein kinase 9, with functions in cell cycle progression 8 and embryogenesis 10,11 . BRD2 binds preferentially to hyperacetylated histone H4 in H2A.Z-containing nucleosomes and this interaction is required for activation of androgen receptor (AR)-regulated genes in prostate cancer cells 12 . In addition to prostate cancer, leukemia is a potential indication for specific BRD2 inhibition9,13 . BRD2 suppresses HIV transcription in latently infected cells and may therefore represent a target in therapeutic strategies involving viral reactivation 14 .
    [Show full text]
  • Do Protein Motifs Read the Histone Code? Xavier De La Cruz,2,3 Sergio Lois,3 Sara Sa´ Nchez-Molina,1,3 and Marian A
    Review articles Do protein motifs read the histone code? Xavier de la Cruz,2,3 Sergio Lois,3 Sara Sa´ nchez-Molina,1,3 and Marian A. Martı´nez-Balba´ s1,3* Summary the interaction between the SANT domain and histones is The existence of different patterns of chemical modifica- also well documented. Overall, experimental evidence tions (acetylation, methylation, phosphorylation, ubiqui- suggests that these domains could be involved in tination and ADP-ribosylation) of the histone tails led, the recruitment of histone-modifying enzymes to discrete some years ago, to the histone code hypothesis. Accord- chromosomal locations, and/or in the regulation their ing to this hypothesis, these modifications would provide enzymatic activity. Within this context, we review the dis- binding sites for proteins that can change the chromatin tribution of bromodomains, chromodomains and SANT state to either active or repressed. Interestingly, some domains among chromatin-modifying enzymes and dis- protein domains present in histone-modifying enzymes cuss how they can contribute to the translation of the are known to interact with these covalent marks in the histone code. BioEssays 27:164–175, 2005. histone tails. This was first shown for the bromodomain, ß 2005 Wiley Periodicals, Inc. which was found to interact selectively with acetylated lysines at the histone tails. More recently, it has been The histone code hypothesis described that the chromodomain can be targeted to The packing of the eukaryotic genome into chromatin provides methylation marks in histone N-terminal domains. Finally, the means for compaction of the entire genome inside the nucleus. However, this packing restricts the access to DNA of the many regulatory proteins essential for biological pro- 1 Instituto de Biologı´a Molecular de Barcelona.
    [Show full text]
  • CBP30, a Selective CBP/P300 Bromodomain Inhibitor, Suppresses Human Th17 Responses
    CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses Ariane Hammitzscha, Cynthia Tallantb,c, Oleg Fedorovb,c, Alison O’Mahonyd, Paul E. Brennanb,c, Duncan A. Hayb,1, Fernando O. Martineza, M. Hussein Al-Mossawia, Jelle de Wita, Matteo Vecellioa, Christopher Wellsb,2, Paul Wordswortha, Susanne Müllerb,c, Stefan Knappb,c,e,3, and Paul Bownessa,3 aBotnar Research Institute, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, United Kingdom; bStructural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom; cTarget Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom; dBioSeek Division of DiscoveRx Corporation, South San Francisco, CA 94080; and eInstitute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, 60438 Frankfurt am Main, Germany Edited by Saadi Khochbin, Université Joseph Fourier, INSERM U823, Grenoble, France, and accepted by the Editorial Board July 13, 2015 (received for review January 29, 2015) Th17 responses are critical to a variety of human autoimmune BET bromodomain inhibitors exhibit antiinflammatory activity by diseases, and therapeutic targeting with monoclonal antibodies inhibiting expression of inflammatory genes (11). The pan-BET in- against IL-17 and IL-23 has shown considerable promise. Here, we hibitor JQ1 ameliorated collagen-induced arthritis and experimental report data to support
    [Show full text]
  • Structural Basis of Molecular Recognition of Helical Histone H3 Tail by PHD Finger Domains
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Crossref Biochemical Journal (2017) 474 1633–1651 DOI: 10.1042/BCJ20161053 Research Article Structural basis of molecular recognition of helical histone H3 tail by PHD finger domains Alessio Bortoluzzi*, Anastasia Amato*, Xavier Lucas, Manuel Blank and Alessio Ciulli Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee DD1 5EH, U.K. Correspondence: Alessio Ciulli ([email protected]) The plant homeodomain (PHD) fingers are among the largest family of epigenetic domains, first characterized as readers of methylated H3K4. Readout of histone post- translational modifications by PHDs has been the subject of intense investigation; however, less is known about the recognition of secondary structure features within the histone tail itself. We solved the crystal structure of the PHD finger of the bromodomain adjacent to zinc finger 2A [BAZ2A, also known as TIP5 (TTF-I/interacting protein 5)] in complex with unmodified N-terminal histone H3 tail. The peptide is bound in a helical folded-back conformation after K4, induced by an acidic patch on the protein surface that prevents peptide binding in an extended conformation. Structural bioinformatics ana- lyses identify a conserved Asp/Glu residue that we name ‘acidic wall’, found to be mutu- ally exclusive with the conserved Trp for K4Me recognition. Neutralization or inversion of the charges at the acidic wall patch in BAZ2A, and homologous BAZ2B, weakened H3 binding. We identify simple mutations on H3 that strikingly enhance or reduce binding, as a result of their stabilization or destabilization of H3 helicity.
    [Show full text]