I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Panagis
Filippakopoulos
,
Sarah
Picaud
,
Maria
Mangos
,
Tracy
Keates
,
Jean-Philippe
Lambert
,
Dalia
Barsyte-Lovejoy
,
Ildiko
Felletar
,
Rudolf
Volkmer
,
Susanne
Müller
,
Tony
Pawson
,
Anne-Claude
Gingras
,
Cheryl h.
Arrowsmith
,
Stefan
Knapp
Open Access
Abstract: Bromodomains (BRDs) are protein interaction modules that specifically recognize ?-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.
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Mar 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nathan
Rose
,
Esther C. Y.
Woon
,
Anthony
Tumber
,
Louise J.
Walport
,
Rasheduzzaman
Chowdhury
,
Xuan Shirley
Li
,
Oliver N. F.
King
,
Clarisse
Lejeune
,
Stanley
Ng
,
Tobias
Krojer
,
Mun Chiang
Chan
,
Anna M.
Rydzik
,
Richard J.
Hopkinson
,
Ka Hing
Che
,
Michelle
Daniel
,
Claire
Strain-Damerell
,
Carina
Gileadi
,
Grazyna
Kochan
,
Ivanhoe K. H.
Leung
,
James
Dunford
,
Kar Kheng
Yeo
,
Peter J.
Ratcliffe
,
Nicola
Burgess-Brown
,
Frank
Von Delft
,
Susanne
Muller
,
Brian
Marsden
,
Paul. E.
Brennan
,
Michael A.
Mcdonough
,
Udo
Oppermann
,
Robert J.
Klose
,
Christopher J.
Schofield
,
Akane
Kawamura
Diamond Proposal Number(s):
[7495]
Abstract: The JmjC oxygenases catalyze the N-demethylation of Nε-methyl lysine residues in histones and are current therapeutic targets. A
SET of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.
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Jul 2012
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Apirat
Chaikuad
,
Tracy
Keates
,
Cécile
Vincke
,
Melanie
Kaufholz
,
Michael
Zenn
,
Bastian
Zimmermann
,
Carlos
Gutiérrez
,
Rong‑guang
Zhang
,
Catherine
Hatzos‑skintges
,
Andrzej
Joachimiak
,
Serge
Muyldermans
,
Friedrich w.
Herberg
,
Stefan
Knapp
,
Susanne
Müller
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent ‘off-target’ of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.
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Apr 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Timothy P. C.
Rooney
,
Panagis
Filippakopoulos
,
Oleg
Fedorov
,
Sarah
Picaud
,
Wilian A.
Cortopassi
,
Duncan A.
Hay
,
Sarah
Martin
,
Anthony
Tumber
,
Catherine M.
Rogers
,
Martin
Philpott
,
Minghua
Wang
,
Amber L.
Thompson
,
Tom D.
Heightman
,
David C.
Pryde
,
Andrew
Cook
,
Robert S.
Paton
,
Susanne
Müller
,
Stefan
Knapp
,
Paul E.
Brennan
,
Stuart J.
Conway
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation–? interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.
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Jun 2014
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan A.
Hay
,
Oleg
Fedorov
,
Sarah
Martin
,
Dean C.
Singleton
,
Cynthia
Tallant Blanco
,
Christopher
Wells
,
Sarah
Picaud
,
Martin
Philpott
,
Octovia P.
Monteiro
,
Catherine M.
Rogers
,
Stuart J.
Conway
,
Timothy P. C.
Rooney
,
Anthony
Tumber
,
Clarence
Yapp
,
Panagis
Filippakopoulos
,
Mark E.
Bunnage
,
Susanne
Müller
,
S
Knapp
,
Christopher J.
Schofield
,
Paul E.
Brennan
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.
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Jul 2014
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I04-Macromolecular Crystallography
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Raquel C.
Montenegro
,
Peter G. K.
Clark
,
Alison
Howarth
,
Xiao
Wan
,
Alessandro
Ceroni
,
Paulina
Siejka
,
Graciela A.
Nunez-Alonso
,
Octovia
Monteiro
,
Catherine
Rogers
,
Vicki
Gamble
,
Rommel
Burbano
,
Paul E.
Brennan
,
Cynthia
Tallant
,
Daniel
Ebner
,
Oleg
Fedorov
,
Eric
O'Neill
,
Stefan
Knapp
,
Darren
Dixon
,
Susanne
Müller
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.
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Nov 2014
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I02-Macromolecular Crystallography
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James
Bennett
,
Oleg
Fedorov
,
Cynthia
Tallant
,
Octovia
Monteiro
,
Julia
Meier
,
Vicky
Gamble
,
Pavel
Savitsky
,
Graciela A
Nunez-Alonso
,
Bernard
Haendler
,
Catherine
Rogers
,
Paul E.
Brennan
,
Susanne
Müller
,
Stefan
Knapp
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/ TRIM24 dual inhibitor that bound with a KD of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data.
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May 2015
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Peter G. K.
Clark
,
Lucas C. C.
Vieira
,
Cynthia
Tallant
,
Oleg
Fedorov
,
Dean C.
Singleton
,
Catherine M.
Rogers
,
Octovia P.
Monteiro
,
James M.
Bennett
,
Roberta
Baronio
,
Susanne
Müller
,
Danette L.
Daniels
,
Jacqui
Méndez
,
Stefan
Knapp
,
Paul E.
Brennan
,
Darren J.
Dixon
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure–activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones in vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.
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May 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Ariane
Hammitzsch
,
Cynthia
Tallant Blanco
,
Oleg
Fedorov
,
Alison
O’mahony
,
Paul E.
Brennan
,
Duncan A.
Hay
,
Fernando O.
Martinez
,
M. Hussein
Al-Mossawi
,
Jelle
De Wit
,
Matteo
Vecellio
,
Christopher
Wells
,
Paul
Wordsworth
,
Susanne
Müller
,
Stefan
Knapp
,
Paul
Bowness
Diamond Proposal Number(s):
[8421]
Abstract: Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.
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Aug 2015
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I03-Macromolecular Crystallography
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O.
Fedorov
,
J.
Castex
,
C.
Tallant Blanco
,
D. R.
Owen
,
S.
Martin
,
M.
Aldeghi
,
O.
Monteiro
,
P.
Filippakopoulos
,
S.
Picaud
,
J. D.
Trzupek
,
B. S.
Gerstenberger
,
C.
Bountra
,
D.
Willmann
,
C.
Wells
,
M.
Philpott
,
C.
Rogers
,
P. C.
Biggin
,
P. E.
Brennan
,
M. E.
Bunnage
,
R.
Schule
,
Thomas
Gunther
,
Stefan
Knapp
,
Susanne
Muller
Open Access
Abstract: Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5′-triphosphate)–driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine–dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.
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Nov 2015
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