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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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Chiara
Marabelli
,
Biagina
Marrocco
,
Simona
Pilotto
,
Sagar
Chittori
,
Sarah
Picaud
,
Sara
Marchese
,
Giuseppe
Ciossani
,
Federico
Forneris
,
Panagis
Filippakopoulos
,
Guy
Schoehn
,
Daniela
Rhodes
,
Sriram
Subramaniam
,
Andrea
Mattevi
Diamond Proposal Number(s):
[16082]
Open Access
Abstract: LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
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Apr 2019
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure–activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors.
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Mar 2012
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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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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
I04-1-Macromolecular Crystallography (fixed wavelength)
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Serena G.
Piticchio
,
Miriam
Martinez-Cartro
,
Salvatore
Scaffidi
,
Moira
Rachman
,
Sergio
Rodriguez-Arevalo
,
Ainoa
Sanchez-Arfelis
,
Carmen
Escolano
,
Sarah
Picaud
,
Tobias
Krojer
,
Panagis
Filippakopoulos
,
Frank
Von Delft
,
Carles
Galdeano
,
Xavier
Barril
Diamond Proposal Number(s):
[15433, 19301]
Abstract: Fragment-based drug discovery (FBDD) is a very effective hit identification method. However, the evolution of fragment hits into suitable leads remains challenging and largely artisanal. Fragment evolution is often scaffold-centric, meaning that its outcome depends crucially on the chemical structure of the starting fragment. Considering that fragment screening libraries cover only a small proportion of the corresponding chemical space, hits should be seen as probes highlighting privileged areas of the chemical space rather than actual starting points. We have developed an automated computational pipeline to mine the chemical space around any specific fragment hit, rapidly finding analogues that share a common interaction motif but are structurally novel and diverse. On a prospective application on the bromodomain-containing protein 4 (BRD4), starting from a known fragment, the platform yields active molecules with nonobvious scaffold changes. The procedure is fast and inexpensive and has the potential to uncover many hidden opportunities in FBDD.
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Dec 2021
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I04-Macromolecular Crystallography
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S.
Picaud
,
O.
Fedorov
,
A.
Thanasopoulou
,
K.
Leonards
,
K.
Jones
,
J.
Meier
,
H.
Olzscha
,
O.
Monteiro
,
S.
Martin
,
M.
Philpott
,
A.
Tumber
,
Panagis
Filippakopoulos
,
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
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
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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