Identification of Histone Peptide Binding Specificity and Small-Molecule Ligands for the TRIM33α and TRIM33β Bromodomains

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.

[1]  H. Ovaa,et al.  Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class , 2021, ChemBioChem.

[2]  S. Conway,et al.  Chemische Epigenetik: der Einfluss chemischer und chemo‐biologischer Techniken auf die Zielstruktur‐Validierung von Bromodomänen , 2019, Angewandte Chemie.

[3]  Huifang Liang,et al.  The Roles of TIF1γ in Cancer , 2019, Front. Oncol..

[4]  C. Arrowsmith,et al.  Targeting non-bromodomain chromatin readers , 2019, Nature Structural & Molecular Biology.

[5]  Jiuhong Kang,et al.  TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells , 2019, Nature Communications.

[6]  R. Sims,et al.  Bromodomains: a new target class for drug development , 2019, Nature Reviews Drug Discovery.

[7]  R. Prinjha,et al.  Advancements in the Development of non‐BET Bromodomain Chemical Probes , 2019, ChemMedChem.

[8]  Gregory A Ross,et al.  Large-scale analysis of water stability in bromodomain binding pockets with grand canonical Monte Carlo , 2018, Communications Chemistry.

[9]  A. Ciulli,et al.  Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach , 2018, ACS chemical biology.

[10]  David J Huggins,et al.  Quantitative metrics for drug-target ligandability. , 2018, Drug discovery today.

[11]  Christopher J. Ott,et al.  Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands , 2018, Nature Chemical Biology.

[12]  Azah Kamilah Muda,et al.  Similarity Measure for Molecular Structure: A Brief Review , 2017 .

[13]  S. Hatakeyama,et al.  TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis. , 2017, Trends in biochemical sciences.

[14]  Jun Chen,et al.  TRIM24 promotes the aggression of gastric cancer via the Wnt/β-catenin signaling pathway. , 2017, Oncology letters.

[15]  R. Prinjha,et al.  Clinical progress and pharmacology of small molecule bromodomain inhibitors. , 2016, Current opinion in chemical biology.

[16]  R. Aebersold,et al.  TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer. , 2016, Cancer cell.

[17]  T. Heightman,et al.  Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3 , 2016, Angewandte Chemie.

[18]  Andrew C Good,et al.  Diving into the Water: Inducible Binding Conformations for BRD4, TAF1(2), BRD9, and CECR2 Bromodomains. , 2016, Journal of medicinal chemistry.

[19]  C. Petosa,et al.  Bromodomains: Structure, function and pharmacology of inhibition. , 2016, Biochemical pharmacology.

[20]  R. Prinjha,et al.  Progress in the Development of non‐BET Bromodomain Chemical Probes , 2016, ChemMedChem.

[21]  M. Barton,et al.  Regulation of gene expression in human cancers by TRIM24. , 2016, Drug discovery today. Technologies.

[22]  W. Palmer Development of small molecule inhibitors of BRPF1 and TRIM24 bromodomains. , 2016, Drug discovery today. Technologies.

[23]  G. Poncet-Montange,et al.  Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. , 2016, Journal of medicinal chemistry.

[24]  H. Yang,et al.  Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial–mesenchymal transition in non-small-cell lung cancer , 2016, Oncogene.

[25]  S. Knapp,et al.  Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF , 2015, Journal of medicinal chemistry.

[26]  Michael Schroeder,et al.  PLIP: fully automated protein–ligand interaction profiler , 2015, Nucleic Acids Res..

[27]  J. Kinney,et al.  The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer , 2015, eLife.

[28]  M. Höss,et al.  Small molecule inhibitors of bromodomain-acetyl-lysine interactions. , 2015, ACS chemical biology.

[29]  K. Aldape,et al.  Tumour suppressor TRIM33 targets nuclear β-catenin degradation , 2014, Nature Communications.

[30]  Hiroshi Egawa,et al.  miR-629 Targets TRIM33 to Promote TGFβ/Smad Signaling and Metastatic Phenotypes in ccRCC , 2014, Molecular Cancer Research.

[31]  Hua Yu,et al.  Knockdown of Tripartite Motif Containing 24 by Lentivirus Suppresses Cell Growth and Induces Apoptosis in Human Colorectal Cancer Cells , 2014, Oncology research.

[32]  A. Gingras,et al.  Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching , 2014, Epigenetics & Chromatin.

[33]  R. Medema,et al.  Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-β-dependent cancer metastasis. , 2014, The Journal of clinical investigation.

[34]  Yu Huang,et al.  Overexpression of TRIM24 Is Associated with the Onset and Progress of Human Hepatocellular Carcinoma , 2014, PloS one.

[35]  S. Ganesan,et al.  Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein* , 2013, The Journal of Biological Chemistry.

[36]  Matthias Mann,et al.  A map of general and specialized chromatin readers in mouse tissues generated by label-free interaction proteomics. , 2013, Molecular cell.

[37]  Andrea Spitaleri,et al.  Exploring PHD Fingers and H3K4me0 Interactions with Molecular Dynamics Simulations and Binding Free Energy Calculations: AIRE-PHD1, a Comparative Study , 2012, PloS one.

[38]  S. Knapp,et al.  Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions. , 2012, Journal of medicinal chemistry.

[39]  S. Conway Bromodomains: are readers right for epigenetic therapy? , 2012, ACS medicinal chemistry letters.

[40]  Nathan Brown,et al.  Druggability Analysis and Structural Classification of Bromodomain Acetyl-lysine Binding Sites , 2012, Journal of medicinal chemistry.

[41]  Ying Xu,et al.  Overexpression of TRIM24 Correlates with Tumor Progression in Non-Small Cell Lung Cancer , 2012, PloS one.

[42]  A. Gingras,et al.  Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family , 2012, Cell.

[43]  D. Patel,et al.  A Poised Chromatin Platform for TGF-β Access to Master Regulators , 2011, Cell.

[44]  S. Knapp,et al.  Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery. , 2011, Molecular bioSystems.

[45]  S. Knapp,et al.  3,5-Dimethylisoxazoles Act As Acetyl-lysine-mimetic Bromodomain Ligands , 2011, Journal of medicinal chemistry.

[46]  S. Dupont,et al.  Recruitment of TIF1γ to chromatin via its PHD finger-bromodomain activates its ubiquitin ligase and transcriptional repressor activities. , 2011, Molecular cell.

[47]  Ming-Ming Zhou,et al.  The PHD finger: a versatile epigenome reader. , 2011, Trends in biochemical sciences.

[48]  Thomas A. Milne,et al.  Recognition of a Mononucleosomal Histone Modification Pattern by BPTF via Multivalent Interactions , 2011, Cell.

[49]  D. Geman,et al.  Identification of family-determining residues in PHD fingers , 2010, Nucleic acids research.

[50]  D. Patel,et al.  TRIM24 links a noncanonical histone signature to breast cancer , 2010, Nature.

[51]  A. Hill,et al.  Getting physical in drug discovery: a contemporary perspective on solubility and hydrophobicity. , 2010, Drug discovery today.

[52]  S. West,et al.  Poly(ADP-ribose)–Dependent Regulation of DNA Repair by the Chromatin Remodeling Enzyme ALC1 , 2009, Science.

[53]  C. Allis,et al.  PHD fingers in human diseases: disorders arising from misinterpreting epigenetic marks. , 2008, Mutation research.

[54]  Ming-Ming Zhou,et al.  Bromodomain: an acetyl‐lysine binding domain , 2002, FEBS letters.

[55]  Alessandro Guffanti,et al.  The tripartite motif family identifies cell compartments , 2001, The EMBO journal.

[56]  P. Wright,et al.  Zinc finger proteins: new insights into structural and functional diversity. , 2001, Current opinion in structural biology.

[57]  P. Evans,et al.  The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p , 2000, The EMBO journal.

[58]  Lei Zeng,et al.  Structure and ligand of a histone acetyltransferase bromodomain , 1999, Nature.

[59]  G. Sbardella Methyl-Readers and Inhibitors , 2019, Topics in Medicinal Chemistry.

[60]  Anton Simeonov,et al.  AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions. , 2016, Methods in molecular biology.