I04-Macromolecular Crystallography
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Adam M.
Thomas
,
Marta
Serafini
,
Emma K.
Grant
,
Edward A. J.
Coombs
,
Joseph P.
Bluck
,
Matthias
Schiedel
,
Michael A.
Mcdonough
,
Jessica K.
Reynolds
,
Bernadette
Lee
,
Michael
Platt
,
Vassilena
Sharlandjieva
,
Philip C.
Biggin
,
Fernanda
Duarte
,
Thomas A.
Milne
,
Jacob T.
Bush
,
Stuart J.
Conway
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
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Oct 2023
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I03-Macromolecular Crystallography
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Angelina R.
Sekirnik
,
Jessica K.
Reynolds
,
Larissa
See
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Cynthia
Tallant
,
Bernadette
Lee
,
Katarzyna B.
Leszczynska
,
Rachel L.
Grimley
,
R. Ian
Storer
,
Marta
Malattia
,
Sara
Crespillo
,
Sofia
Caria
,
Stephanie
Duclos
,
Ester M.
Hammond
,
Stefan
Knapp
,
Garrett M.
Morris
,
Fernanda
Duarte
,
Philip C.
Biggin
,
Stuart J.
Conway
Open Access
Abstract: 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.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18145]
Open Access
Abstract: A novel crystallographic fragment screening data set was generated and used in the SAMPL7 challenge for protein-ligands. The SAMPL challenges prospectively assess the predictive power of methods involved in computer-aided drug design. Application of various methods to fragment molecules are now widely used in the search for new drugs. However, there is little in the way of systematic validation specifically for fragment-based approaches. We have performed a large crystallographic high-throughput fragment screen against the therapeutically relevant second bromodomain of the Pleckstrin-homology domain interacting protein (PHIP2) that revealed 52 different fragments bound across 4 distinct sites, 47 of which were bound to the pharmacologically relevant acetylated lysine (Kac) binding site. These data were used to assess computational screening, binding pose prediction and follow-up enumeration. All submissions performed randomly for screening. Pose prediction success rates (defined as less than 2 Å root mean squared deviation against heavy atom crystal positions) ranged between 0 and 25% and only a very few follow-up compounds were deemed viable candidates from a medicinal-chemistry perspective based on a common molecular descriptors analysis. The tight deadlines imposed during the challenge led to a small number of submissions suggesting that the accuracy of rapidly responsive workflows remains limited. In addition, the application of these methods to reproduce crystallographic fragment data still appears to be very challenging. The results show that there is room for improvement in the development of computational tools particularly when applied to fragment-based drug design.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Petra
Lukacik
,
C. David
Owen
,
Gemma
Harris
,
Jani Reddy
Bolla
,
Sarah
Picaud
,
Irfan
Alibay
,
Joanne E.
Nettleship
,
Louise E.
Bird
,
Raymond
Owens
,
Philip C.
Biggin
,
Panagis
Filippakopoulos
,
Carol V.
Robinson
,
Martin A.
Walsh
Diamond Proposal Number(s):
[4990, 5073, 4988]
Open Access
Abstract: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.
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Oct 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with 10-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL, and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable −4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.
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Jun 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: Centrioles are key eukaryotic organelles responsible for the formation of cilia and flagella, and for organising the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerisation of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organisation of further organelle components. A dimerisation interaction between SAS-6 N-terminal ‘head’ domains was previously shown to be essential for protein oligomerisation in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low molecular weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerisation. We demonstrate using nuclear magnetic resonance spectroscopy that a ligand from this family binds at the head domain dimerisation site of algae, nematode and human SAS-6 variants, but also that another ligand specifically recognises human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest ligand specificity derives from favourable Van der Waals interactions with a hydrophobic cavity at the dimerisation site.
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Sep 2020
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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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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12305]
Abstract: γ-Aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABAAR function requires high-resolution structures. Here we present the first atomic structures of a GABAAR chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABAARs in neurological disorders.
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Oct 2017
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I03-Macromolecular Crystallography
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Open Access
Abstract: Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.
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Mar 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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