I24-Microfocus Macromolecular Crystallography
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Laura E.
Jennings
,
Matthias
Schiedel
,
David S.
Hewings
,
Sarah
Picaud
,
Corentine M. C.
Laurin
,
Paul A.
Bruno
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Larissa
See
,
Jessica K.
Reynolds
,
Mustafa
Moroglu
,
Ishna N.
Mistry
,
Amy
Hicks
,
Pavel
Guzanov
,
James
Clayton
,
Charles N. G.
Evans
,
Giulia
Stazi
,
Philip C.
Biggin
,
Anna K.
Mapp
,
Ester M.
Hammond
,
Philip G.
Humphreys
,
Panagis
Filippakopoulos
,
Stuart J.
Conway
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½ = 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½ = 388 min).
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May 2018
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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
I04-Macromolecular Crystallography
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Pavel
Savitsky
,
Tobias
Krojer
,
Takao
Fujisawa
,
Jean-Philippe
Lambert
,
Sarah
Picaud
,
Chen-Yi
Wang
,
Erin K.
Shanle
,
Krzysztof
Krajewski
,
Hans
Friedrichsen
,
Alexander
Kanapin
,
Colin
Goding
,
Matthieu
Schapira
,
Anastasia
Samsonova
,
Brian D.
Strahl
,
Anne-Claude
Gingras
,
Panagis
Filippakopoulos
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.
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Dec 2016
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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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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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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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I04-Macromolecular Crystallography
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S.
Picaud
,
O.
Fedorov
,
A.
Thanasopoulou
,
K.
Leonards
,
K.
Jones
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J.
Meier
,
H.
Olzscha
,
O.
Monteiro
,
S.
Martin
,
M.
Philpott
,
A.
Tumber
,
Panagis
Filippakopoulos
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C.
Yapp
,
C.
Wells
,
K. H.
Che
,
A.
Bannister
,
S.
Robson
,
U.
Kumar
,
N.
Parr
,
K.
Lee
,
D.
Lugo
,
P.
Jeffrey
,
S.
Taylor
,
M. L.
Vecellio
,
C.
Bountra
,
P. E.
Brennan
,
A.
O' Mahony
,
S.
Velichko
,
S.
Muller
,
D.
Hay
,
D. L.
Daniels
,
M.
Urh
,
N. B.
La Thangue
,
T.
Kouzarides
,
R.
Prinjha
,
J.
Schwaller
,
S.
Knapp
Diamond Proposal Number(s):
[6391]
Open Access
Abstract: The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein–protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9+ acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. Cancer Res; 75(23); 5106–19. ©2015 AACR.
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Nov 2015
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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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I04-1-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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I04-Macromolecular Crystallography
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S.
Picaud
,
D.
Da Costa
,
A.
Thanasopoulou
,
P.
Filippakopoulos
,
P. V.
Fish
,
M.
Philpott
,
O.
Fedorov
,
P.
Brennan
,
M. E.
Bunnage
,
D. R.
Owen
,
J. E.
Bradner
,
P.
Taniere
,
B.
O'Sullivan
,
S.
Muller
,
J.
Schwaller
,
T.
Stankovic
,
S.
Knapp
Diamond Proposal Number(s):
[8421]
Abstract: Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression.
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May 2013
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