B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jean-Philippe
Lambert
,
Sarah
Picaud
,
Takao
Fujisawa
,
Huayun
Hou
,
Pavel
Savitsky
,
Liis
Uusküla-Reimand
,
Gagan D.
Gupta
,
Hala
Abdouni
,
Zhen-Yuan
Lin
,
Monika
Tucholska
,
James D. R.
Knight
,
Beatriz
Gonzalez-Badillo
,
Nicole
St-Denis
,
Joseph A.
Newman
,
Manuel
Stucki
,
Laurence
Pelletier
,
Nuno
Bandeira
,
Michael D.
Wilson
,
Panagis
Filippakopoulos
,
Anne-Claude
Gingras
Diamond Proposal Number(s):
[6391, 10619, 15433]
Open Access
Abstract: Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
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Dec 2018
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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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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sarah
Picaud
,
Katharina
Leonards
,
Jean-Philippe
Lambert
,
Oliver
Dovey
,
Christopher
Wells
,
Oleg
Fedorov
,
Octovia
Monteiro
,
Takao
Fujisawa
,
Chen-Yi
Wang
,
Hannah
Lingard
,
Cynthia
Tallant
,
Nikzad
Nikbin
,
Lucie
Guetzoyan
,
Richard
Ingham
,
Steven V.
Ley
,
Paul
Brennan
,
Susanne
Muller
,
Anastasia
Samsonova
,
A.-C.
Gingras
,
Juerg
Schwaller
,
George
Vassiliou
,
Stefan
Knapp
,
Panagis
Filippakopoulos
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.
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Oct 2016
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.
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Jun 2020
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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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I03-Macromolecular Crystallography
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Martin M.
Matzuk
,
Michael R.
Mckeown
,
Panagis
Filippakopoulos
,
Qinglei
Li
,
Lang
Ma
,
Julio E.
Agno
,
Madeleine E.
Lemieux
,
Sarah
Picaud
,
Richard N.
Yu
,
Jun
Qi
,
Stefan
Knapp
,
James E.
Bradner
Diamond Proposal Number(s):
[443]
Open Access
Abstract: A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.
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Aug 2012
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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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I03-Macromolecular Crystallography
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Abstract: The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains. High-resolution crystal structures of the complexes of key fragments binding to BRD4(1), CREBBP, and PCAF were determined to provide binding mode data to aid the development of potent and selective inhibitors of PCAF, CREBBP, and BRD4.
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Dec 2016
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6391]
Open Access
Abstract: PGAM5 is a mitochondrial membrane protein that functions as an atypical Ser/Thr phosphatase and is a regulator of oxidative stress response, necroptosis, and autophagy. Here we present several crystal structures of PGAM5 including the activating N-terminal regulatory sequences, providing a model for structural plasticity, dimerization of the catalytic domain, and the assembly into an enzymatically active dodecameric form. Oligomeric states observed in structures were supported by hydrogen exchange mass spectrometry, size-exclusion chromatography, and analytical ultracentrifugation experiments in solution. We report that the catalytically important N-terminal WDPNWD motif acts as a structural integrator assembling PGAM5 into a dodecamer, allosterically activating the phosphatase by promoting an ordering of the catalytic loop. Additionally the observed active site plasticity enabled visualization of essential conformational rearrangements of catalytic elements. The comprehensive biophysical characterization offers detailed structural models of this key mitochondrial phosphatase that has been associated with the development of diverse diseases.
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Jun 2017
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I03-Macromolecular Crystallography
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John
Spencer
,
Storm
Hassell
,
Sarah
Picaud
,
Ralph
Lengacher
,
Joshua
Csuker
,
Regis
Millet
,
Gilles
Gasser
,
Roger
Alberto
,
Hannah
Maple
,
Robert
Felix
,
Zbigniew
Leśnikowski
,
Helen
Stewart
,
Timothy
Chevassut
,
Simon
Morley
,
Panagis
Filippakopoulos
Diamond Proposal Number(s):
[19301]
Abstract: A series of bulky organometallic and organic analogues of the bromodomain (BRD) inhibitor (+)‐JQ1 have been prepared. The most potent, N‐[(adamantan‐1‐yl)methyl]‐2‐[(9S)‐7‐(4‐chlorophenyl)‐4,5,13‐trimethyl‐3‐thia‐1,8,11,12‐tetraazatricyclo[8.3.0.02,6]trideca‐2(6),4,7,10,12‐pentaen‐9‐yl]acetamide, 2e , showed excellent potency with an K D = ca. 130 nM vs BRD4(1) and a ca. 2‐fold selectivity over BRD4(2) (K D = ca. 260 nM). Its binding to the first bromodomain of BRD4 was determined by a protein cocrystal structure.
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Jan 2021
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