I04-1-Macromolecular Crystallography (fixed wavelength)
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Xiaomin
Ni
,
R. Blake
Richardson
,
Andre
Schutzer Godoy
,
Matteo P.
Ferla
,
Caroline
Kikawa
,
Jenke
Scheen
,
William W.
Hannon
,
Eda
Capkin
,
Noa
Lahav
,
Blake H.
Balcomb
,
Peter G.
Marples
,
Michael
Fairhead
,
Siyi
Wang
,
Eleanor P.
Williams
,
Charles W. E.
Tomlinson
,
Jasmin C.
Aschenbrenner
,
Ryan
Lithgo
,
Max
Winokan
,
Charline
Giroud
,
Isabela
Dolci
,
Rafaela Sachetto
Fernandes
,
Glaucius
Oliva
,
Anu V.
Chandran
,
Mary-Ann
Xavier
,
Martin A.
Walsh
,
Warren
Thompson
,
Jesse D.
Bloom
,
Nathaniel T.
Kenton
,
Alpha A.
Lee
,
Annette
Von Delft
,
Haim
Barr
,
Karla
Kirkegaard
,
Lizbe
Koekemoer
,
Daren
Fearon
,
Matthew J.
Evans
,
Frank
Von Delft
Diamond Proposal Number(s):
[32627]
Open Access
Abstract: The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 46 fragments with diverse scaffolds are identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we perform deep mutational scanning of the NS2B-NS3 protease. Merging fragment hits yields an extensive set of ‘mergers’, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points. In this work, we maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.
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Oct 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Geoffrey M. T.
Smith
,
Laksh
Aithani
,
Charlotte E.
Barrett
,
Alwin O.
Bucher
,
Christopher D. O.
Cooper
,
Sébastien L.
Degorce
,
Andrew
Dore
,
Catherine T.
Fletcher
,
Sophie
Huber
,
Rosemary
Huckvale
,
Amanda J.
Kennedy
,
Abigail A.
Mornement
,
Mark
Pickworth
,
Prakash
Rucktooa
,
Conor C. G.
Scully
,
Sarah E.
Skerratt
Abstract: Werner (WRN) helicase, has emerged as a promising therapeutic target for cancers associated with microsatellite instability (MSI). This letter describes the discovery of small molecule inhibitors from a fragment screen that occupy a cryptic, allosteric site of WRN helicase. Key findings include the identification of benzimidazole and amino-indazole scaffolds, exploiting their proximity to Cys727 via covalent modification. The use of our proprietary co-folding model DragonFold assisted the identification of novel WRN helicase inhibitors. These, together with near-neighbor profiling, offer tools for furthering the understanding of WRN and BLM helicase function, and potential therapeutic avenues for MSI-associated cancers.
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Oct 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Mingda
Ye
,
Mpho
Makola
,
Mark W.
Richards
,
Joseph A.
Newman
,
Michael
Fairhead
,
Selena G.
Burgess
,
Zhihuang
Wu
,
Elizabeth
Maclean
,
Nathan D.
Wright
,
Lizbe
Koekemoer
,
Andrew
Thompson
,
Gustavo
Arruda Bezerra
,
Gangshun
Yi
,
Huanyu
Li
,
Victor
Rangel
,
Dimitrios
Mamalis
,
Hazel
Aitkenhead
,
Benjamin G.
Davis
,
Robert J. C.
Gilbert
,
Katharina L.
Duerr
,
Richard
Bayliss
,
Opher
Gileadi
,
Frank
Von Delft
Diamond Proposal Number(s):
[26998]
Open Access
Abstract: Design of modular, transferable protein assemblies has broad applicability and in structural biology could help with the ever-troublesome crystallization bottleneck, including finding robustly behaved protein crystals for rapidly characterizing ligands or drug candidates or generating multiple polymorphs to illuminate diverse conformations. Nanobodies as crystallization chaperones are well-established but still unreliable, as we show here. Instead, we show an exemplar of how robust crystallization behavior can be engineered by exploring many combinations (>200) of nanobody surface mutations over several iterations. Critically, what needed testing was crystallization and diffraction quality, since target–nanobody binding affinity is decoupled from crystallizability enhancement. Our study yielded multiple polymorphs, all mediated by the same interface, with dramatically improved resolution and diffraction reliability for some mutants; we thus name them ‘Gluebodies’ (Gbs). We further demonstrate that these Gb mutations do transfer to some other targets, both for achieving robust crystallization in alternative packing forms and for establishing the ability to crystallize a key early stage readout. Since the Gb interface is evidently a favored interaction, it may be broadly applicable for modular assembly; more specifically, this work suggests that Gbs should be routinely attempted for crystallization whenever nanobodies are available.
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Oct 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043, 18548, 25402]
Open Access
Abstract: Casein kinase 2α (CK2α) is an oncology drug target that acts as a positive regulator of many tumorigenic signaling pathways. We previously reported that CK2α has a unique cryptic binding site, the αD pocket, that offers the potential for inhibitors with improved kinase selectivity. The prototype bivalent molecule CAM4066 (6) confirmed that improved selectivity could be achieved while binding in both the ATP-binding site and the αD pocket. A drug discovery project to develop a new series of bivalent CK2α inhibitors with increased cell potency and selectivity identified 61f (APL-5125), a highly potent, ATP-competitive CK2α inhibitor with exquisite kinase selectivity and cellular potency. Compound 61f demonstrates in vivo inhibition of p-AKT S129 in tumors (HCT116) following once-daily oral administration and shows a clear PK–PD relationship with unbound drug exposure. 61f has a superior preclinical profile to existing CK2α inhibitors and is currently under evaluation in patients with advanced solid tumors.
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Oct 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[32633]
Open Access
Abstract: Dengue viruses (DENVs) infect approximately 400 million people each year, and currently, there are no effective therapeutics available. To explore potential starting points for antiviral drug development, we conducted a large-scale crystallographic fragment screen targeting the RNA-dependent RNA polymerase (RdRp) domain of the nonstructural protein 5 (NS5) from DENV serotype 2. Our screening, which involved 1108 fragments, identified 60 hit compounds across various known binding sites, including the active site, N pocket, and RNA tunnel. Additionally, we discovered a novel binding site and a fragment-binding hot spot in thumb site II. These structural findings open amenable avenues for developing non-nucleoside inhibitors and offer valuable insights for future structure-based drug design aimed at DENV and other flaviviral RdRps.
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Sep 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Heparanase is the only human enzyme responsible for heparan sulfate (HS) breakdown, an activity that remodels the extracellular matrix (ECM) and strongly drives cancer metastasis and angiogenesis. Compelling evidence implies that heparanase promotes essentially all aspects of the tumorigenic process, namely, tumor initiation, vascularization, growth, metastasis, and chemoresistance. A key mechanism by which heparanase accelerates cancer progression is by enabling the release and bioavailability of HS-bound growth factors, chemokines, and cytokines, residing in the tumor microenvironment and supporting tumor growth and metastasis. The currently available heparanase inhibitors are mostly HS/heparin-like compounds that lack specificity and exert multiple off-target side effects. To date, only four such compounds have progressed to clinical trials, and none have been approved for clinical use. We have generated and characterized an anti-heparanase monoclonal antibody (A54 mAb) that specifically inhibits heparanase enzymatic activity (ECM degradation assay) and cellular uptake. Importantly, A54 mAb attenuates xenograft tumor growth and metastasis (myeloma, glioma, pancreatic, and breast carcinomas) primarily when administered (syngeneic or immunocompromised mice) in combination with conventional anti-cancer drugs. Co-crystallization of the A54 Fab fragment and the heparanase enzyme revealed that the interaction between the two proteins takes place adjacent to the enzyme HS/heparin binding domain II (HBDII; Pro271-Ala276), likely hindering heparanase from interacting with HS substrates via steric occlusion of the active site cleft. Collectively, we have generated and characterized a novel mAb that specifically neutralizes heparanase enzymatic activity and attenuates its pro-tumorigenic effects in preclinical models, paving the way for its clinical examination against cancer, inflammation, and other diseases.
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Sep 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Ekaterina
Kot
,
Matteo P.
Ferla
,
Patricia H.
Hollinshead
,
Charles W. E.
Tomlinson
,
Daren
Fearon
,
Jasmin C.
Aschenbrenner
,
Lizbe
Koekemoer
,
Max
Winokan
,
Michael
Fairhead
,
Xiaomin
Ni
,
Rod
Chalk
,
Katherine S.
England
,
Laura
Ortega Varga
,
Mark
Greer Montgomery
,
Nicholas P.
Mulholland
,
Frank
Von Delft
Diamond Proposal Number(s):
[28172, 34598, 30602, 36049]
Open Access
Abstract: BACKGROUND: In order to alleviate the growing issue of herbicide resistance, diversification of the herbicide portfolio is necessary. A promising yet underutilized mode-of-action is the inhibition of fatty acid thioesterases (FATs), which terminate de novo fatty acid (FA) biosynthesis by releasing FAs from acyl carrier protein (ACP) cofactors. These enzymes impact plant growth and sterility by determining the amount and length of FAs present. In this study we report a crystallographic fragment screening approach for the identification of novel chemical matter targeting FATs. RESULTS: We have solved the crystal structure of Arabidopsis thaliana FatA to 1.5 Å and conducted a crystallographic fragment screen which identified 129 unique fragments bound in 141 different poses. Ten fragments demonstrated on-scale potency, two of these exploiting different interactions to known herbicides. Elaboration of one of the fragments resulted in an improvement of affinity from ~20 μm to ~90 nm KD. Finally, superposition of our crystal structures revealed that some fragments exploit large conformational changes in the substrate binding site. CONCLUSION: We have fully enabled FatA as a target for rapid, rational hit-to-lead development, with robust structural, biophysical and biochemical assays. We provide a set of fragment hits which represent diverse, novel scaffolds that both recapitulate interactions made by current herbicides, and also target novel regions within the active and dimer sites. Our fragments can be readily merged and allow for effective catalogue-based structure–activity relationship (SAR) exploration. Together these data will accelerate the development of novel, alternative herbicides to combat herbicide resistance.
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Sep 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Hepatoma-derived growth factor-related protein 2 (HRP-2) is a member of the HDGF-related protein family, which has been linked to multiple malignancies. A defining feature of this protein family is the presence of an N-terminal PWWP domain, which enables binding to nucleosomes carrying a dimethylation or trimethylation marker on residue Lys36 of histone H3. To support the rational design of small-molecule drugs that bind to the PWWP domain, crystallographic fragment screening was chosen. A critical requirement for such screening is the ability to reliably produce large batches of high-quality crystals, ideally grown under low-salt conditions to prevent the precipitation of drug-like fragments during crystal soaking. Initial crystallization of the wild-type (WT) HRP-2 PWWP domain only produced crystals under high-salt conditions and these significantly lost diffraction quality over two weeks. It was hypothesized that these complications were caused by oxidation of the solvent-exposed Cys64 residue. To overcome these difficulties, a Cys64Ser mutant was produced. This mutation revealed a substantially improved crystallization propensity, as eight crystal forms could be obtained and resolved versus two forms for the WT. Moreover, the mutant crystals could be grown in PEG-based low ionic strength conditions which are optimal for fragment soaking. Finally, the crystals did not lose their diffraction quality for up to six months. Importantly, systematic analysis of all obtained X-ray structures revealed that the Cys64/Ser64 residue lies at a key lattice interface which is conserved across all crystal forms. This suggests that even minor chemical changes at this position could disrupt important intermolecular contacts, explaining the demonstrated major benefit of the introduced mutation. The presented data underpin the substitution of surface-exposed cysteines as a general strategy to enhance protein crystallization and diffraction quality. Ultimately, the results presented here were pivotal to the successful execution of the crystallographic fragment-screening campaign with the HRP-2 PWWP domain.
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Aug 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Chunyu
Lin
,
Dilip
Narayanan
,
Marilia
Barreca
,
Cecilie
Poulsen
,
Leandro Silva
Da Costa
,
Xiangrong
Chen
,
Kendall G.
Wichman
,
Cherisse A.
Charley
,
Jaslin L.
Lindsay
,
Mahya
Dezfouli
,
Dimitra
Vlissari
,
Thomas S.
Mortensen
,
Camilla B.
Chan
,
Jingyi
Wang
,
William
Richardson
,
Charlotte E.
Manning
,
Zhuoyao
Chen
,
Jie
Zang
,
Helena
Käck
,
Michael
Gajhede
,
Alex N.
Bullock
,
David J.
Blake
,
David
Olagnier
,
Anders
Bach
Diamond Proposal Number(s):
[21630]
Open Access
Abstract: Activating the cytoprotective response of nuclear factor erythroid 2-related factor 2 (Nrf2) can reduce oxidative stress and inflammation. A promising strategy is to inhibit the protein-protein interaction between Kelch-like ECH-associated protein 1 (Keap1) and Nrf2 using noncovalent compounds that target the Keap1 Kelch domain. These compounds may be more specific than covalent Keap1-reacting Nrf2 activators. However, the development of drug-like noncovalent Keap1-Nrf2 inhibitors faces challenges due to the size and polarity of the Kelch binding pocket. Here, we present a new series of noncovalent Keap1-Nrf2 inhibitors developed from a weak fragment hit identified by crystallographic screening. A two-step growing strategy and optimization guided by several X-ray cocrystal structures led to compounds with low nanomolar affinities and complete selectivity for Keap1 in a panel of homologous Kelch domains. In cells, compounds 24 and 28 potently activated the expression of Nrf2-controlled genes and showed anti-inflammatory effects by downregulating NLRP3 inflammasome and STING signalling activation. RNA sequencing revealed activation of cytoprotective pathways and a different profile from typical covalent Nrf2 activators. This work highlights the potential of fragment-based drug discovery for challenging targets like Keap1 and introduces novel Keap1-Nrf2 inhibitors as chemical probes and drug leads.
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Aug 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16258]
Open Access
Abstract: RING family ubiquitin ligases (E3s) employ the RING domain to recruit the E2 thioester ubiquitin (E2∼Ub) intermediate to catalyze the transfer of ubiquitin (Ub) to substrates. A cationic Arg linchpin (LP) residue in the RING domain plays a key role in stabilizing the interface with E2∼Ub, but the identity of the LP residue varies across E3s. Here, we investigate how the LP residue contributes to ubiquitination. Using the model RNF38 system, we demonstrate that substitution of LPArg to the other 19 available amino acids modulates ubiquitination, ranging from minor reduction to complete abolition. The identity of the LP residue influences E2∼Ub binding but does not correlate with E3 activity. NMR and X-ray crystallography analyses reveal that RNF38 LPArg variants stabilize E2∼Ub in a catalytically competent conformation to varying degrees. By altering the LP residue in XIAP, we show that the XIAPY485R variant promotes E2∼Ub stabilization and enhances substrate ubiquitination in cells. Our work demonstrates the importance of the LP residue in modulating E2∼Ub conformation to control ubiquitination.
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Aug 2025
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