I03-Macromolecular Crystallography
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Open Access
Abstract: TRIM24 is an epigenetic transcriptional coregulator that “reads” KMe3 and KAc histone modifications via its tandem plant homeodomain (PHD) and bromodomain (BRD), respectively. The PHD and BRD are potential therapeutic targets due to the roles of TRIM24 in breast cancer progression. However, there are currently no small-molecule ligands for the PHD, and existing TRIM24 BRD inhibitors lack selectivity over the main off-target, BRPF1. Here, we report the development of the first bivalent tool molecules capable of simultaneously engaging both the TRIM24 PHD and BRD. Key to this strategy was the identification of effective KMe3 bioisosteres that enhance H3 peptide binding to the TRIM24 PHD. The most promising of these was incorporated into a nine amino acid H3-mimicking peptide, and linked to a TRIM24 BRD ligand. The resulting peptide-drug conjugates (PDCs) bind to TRIM24 with picomolar affinity and a slow dissociation rate (koff), which is driven by an in cis bivalent binding mode. Although the PDCs showed limited effects on breast cancer cell proliferation in vitro, this work underscores their potential as tools for studying previously unliganded reader domains and consequently advancing our understanding of multivalent epigenetic regulation in disease.
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Nov 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited, and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains a pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarization assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarization, and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC–CBP30, bromosporine, and I-BRD9; moreover, SGC–CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a possible drug target in Leishmania and provide a foundation for the future development of optimized antileishmanial compounds targeting this epigenetic reader protein.
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Oct 2023
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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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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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I24-Microfocus Macromolecular Crystallography
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Christos
Pliotas
,
Samuel C.
Grayer
,
Silvia
Ekkerman
,
Anthony K. N.
Chan
,
Jess
Healy
,
Phedra
Marius
,
Wendy
Bartlett
,
Amjad
Khan
,
Wilian A.
Cortopassi
,
Shane A.
Chandler
,
Tim
Rasmussen
,
Justin L. P.
Benesch
,
Robert S.
Paton
,
Timothy D. W.
Claridge
,
Samantha
Miller
,
Ian R.
Booth
,
James
Naismith
,
Stuart J.
Conway
Open Access
Abstract: Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.
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Aug 2017
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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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