I03-Macromolecular Crystallography
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Open Access
Abstract: Design and synthesis of two 3-substituted swainsonine derivatives with the aim to improve the potency and selectivity towards Golgi alpha-mannosidase II.
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May 2025
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I03-Macromolecular Crystallography
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Joanna L.
Chen
,
Joey L.
Methot
,
Matthew J.
Mitcheltree
,
Andrew
Musacchio
,
Emily B.
Corcoran
,
Guo
Feng
,
Alfred
Lammens
,
Klaus
Maskos
,
Rachel L.
Palte
,
Meredith M.
Rickard
,
Karin M.
Otte
,
My S.
Mansueto
,
Sriraman
Venkat
,
Christopher
Sondey
,
Maren
Thomsen
,
Charles A.
Lesburg
,
Xavier
Fradera
,
Matthew J.
Fell
,
Erin F.
Dimauro
,
Phieng
Siliphaivanh
Abstract: Receptor-interacting protein kinase 1 (RIPK1) plays an essential role in necroptosis, a form of inflammatory, caspase-independent, programmed cell death. Allosteric inhibitors of RIPK1 have been shown to block necroptotic cell death and thus may offer potential therapeutic opportunities across a range of infectious, autoimmune, and neurodegenerative diseases. We report the structure-informed discovery of a novel series of bridged benzoazepine amides as part of our efforts to develop a CNS-penetrant small-molecule inhibitor of RIPK1 with a low projected oral human dose.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32544]
Open Access
Abstract: The recent discovery of the PETase enzyme family offers a sustainable solution for depolymerizing poly(ethylene terephthalate) (PET), one of the most widespread plastic compounds, under mild conditions. This enables the environmentally beneficial conversion of plastic waste into value-added products. Among this enzyme family, PETase from Ideonella sakaiensis has been the most extensively studied. Although other similar enzymes have been discovered, our knowledge about the catalytic and structural properties of this class remains limited. In this study, a PETase-like enzyme (PETase SM14) from Streptomyces sp. SM14 was heterologously produced in Escherichia coli, and its activity was tested on post-consumer plastic substrates using high-performance liquid chromatography for product quantification as well as scanning electron microscopy and atomic force microscopy for substrate surface imaging evaluation. PETase SM14 exhibited high salt tolerance (1.5 M), good heat resistance (Tm 56.26 °C), and optimal activity at pH 9.0, highlighting its potential for PET waste bioremediation. Furthermore, its X-ray crystal structure was solved at 1.43 Å resolution, revealing conserved features of the PETase family with potential relevance for future engineering applications.
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May 2025
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I03-Macromolecular Crystallography
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Jeffrey A.
Boerth
,
Marianne
Schimpl
,
Simon C. C.
Lucas
,
Jingwen
Zhang
,
Erin L.
Code
,
Kevin J.
Embrey
,
Philip B.
Rawlins
,
Haixia
Wang
,
R. Ian
Storer
,
Paolo
Di Fruscia
,
Jennifer E.
Nelson
,
Alexander G.
Milbradt
,
Ulf
Börjesson
,
Andrea
Gohlke
,
Victoria
Korboukh
,
Ariamala
Gopalsamy
Diamond Proposal Number(s):
[20015]
Abstract: Suppression of oncogenic gene expression is an effective strategy for the treatment of cancer. The SWI/SNF (SWItch/Sucrose Non-Fermentable) complex plays an important role in regulating gene activation or repression, and its dysregulation has been linked to aberrant transcription activity in many types of cancer. Targeting the subunits of this complex, such as SMARCA2, SMARCA4, and PBRM1, which are part of the bromodomain family VIII, has significant therapeutic potential. Herein we report the discovery of pyrimidoindolones as a novel series of bromodomain family VIII binders identified through an NMR-based fragment screen. These binders have been optimized to achieve sub-μM affinity for the family VIII proteins SMARCA2, SMARCA4, and PRBM1, with promising physicochemical properties.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zihao
Wang
,
Guodong
Cao
,
Miranda P.
Collier
,
Xingyu
Qiu
,
Sophie
Broadway-Stringer
,
Dominik
Šaman
,
Jediael Z. Y.
Ng
,
Navoneel
Sen
,
Amar J.
Azad
,
Charlotte
Hooper
,
Johannes
Zimmermann
,
Michael A.
Mcdonough
,
Jurgen
Brem
,
Patrick
Rabe
,
Haigang
Song
,
T. Reid
Alderson
,
Christopher J.
Schofield
,
Jani R.
Bolla
,
Kristina
Djinovic-Carugo
,
Dieter O.
Fürst
,
Bettina
Warscheid
,
Matteo T.
Degiacomi
,
Timothy M.
Allison
,
Georg K. A.
Hochberg
,
Carol V.
Robinson
,
Katja
Gehmlich
,
Justin L. P.
Benesch
Open Access
Abstract: The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.
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May 2025
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I03-Macromolecular Crystallography
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Rebecca
Crawshaw
,
Ross
Smithson
,
Johannes
Hofer
,
Florence J.
Hardy
,
George W.
Roberts
,
Jonathan S.
Trimble
,
Anna R.
Kohn
,
Colin W.
Levy
,
Deborah A.
Drost
,
Christian
Merten
,
Derren J.
Heyes
,
Richard
Obexer
,
Thorsten
Bach
,
Anthony P.
Green
Diamond Proposal Number(s):
[31850]
Open Access
Abstract: The development of [2 + 2] cyclases containing benzophenone triplet sensitizers highlights the potential of engineered enzymes as a platform for stereocontrolled energy transfer photocatalysis. However, the suboptimal photophysical features of benzophenone necessitates the use of ultraviolet light, limits photochemical efficiency and restricts the range of chemistries accessible. Here we engineer an orthogonal Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA pair for encoding thioxanthone triplet sensitizers into proteins, which can efficiently harness visible light to drive photochemical conversions. Initially, we developed an enantioselective [2 + 2] cyclase that is orders of magnitude more efficient than our previously developed photoenzymes (kcat = 13 s−1, >1,300 turnovers). To demonstrate that thioxanthone-containing enzymes can enable more challenging photochemical conversions, we developed a second oxygen-tolerant enzyme that can steer selective C–H insertions of excited quinolone substrates to afford spirocyclic β-lactams with high selectivity (99% e.e., 22:1 d.r.). This photoenzyme also suppresses a competing substrate decomposition pathway observed with small-molecule sensitizers, underscoring the ability of engineered enzymes to control the fate of excited-state intermediates.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Listeria monocytogenes is a ubiquitous, psychrotrophic human pathogen that can cause listeriosis, a serious illness for vulnerable populations. Some foods, such as Hispanic-style fresh cheeses like queso fresco, pose a specific risk because there are no widely accepted or available methods for L. monocytogenes mitigation that are both effective and able to maintain the properties of the products. Listeria-specific bacteriophages encode endolysins that are able to cleave the peptidoglycan layer of L. monocytogenes cells externally, showing promise as a potential solution to this problem. PlyP100, from the GRAS Listeria phage P100, is one such endolysin that can prevent the growth of L. monocytogenes in both lab culture conditions and a miniaturized queso fresco model. In this work, we aimed to understand the structural and functional properties of PlyP100. An AlphaFold prediction suggested the presence of three separate domains (D1, D2, and D3). By solving a crystal structure of D1 and assessing various domain truncations, we present evidence that D1 is responsible for catalytic activity, D3 is sufficient for cell wall binding, and D2 is necessary for full function of the enzyme against live cells. Additionally, we performed point mutations in D1 and compared PlyP100 to proteins with similar structures, including S. pneumoniae LytA and Listeria endolysin Ply511, in order to understand its specific enzymatic mechanism and target strain specificity. These insights into the structure and function of PlyP100 will aid future work aiming to engineer better endolysins as safe food antimicrobials.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Matthew J.
Beech
,
Edmond C.
Toma
,
Helen G.
Smith
,
Maria M.
Trush
,
Jit H. J.
Ang
,
Mei Y.
Wong
,
Chung H. J.
Wong
,
Hafiz S.
Ali
,
Zakia
Butt
,
Viha
Goel
,
Fernanda
Duarte
,
Alistair J. M.
Farley
,
Timothy R.
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[31353]
Open Access
Abstract: The Tet(X) flavin-dependent monooxygenases enable tetracycline antibiotic resistance by catalysing inactivating hydroxylation, so preventing inhibition of bacterial ribosomes. Tet(X) resistance is growing rapidly, threatening the efficacy of important last-resort tetracyclines such as tigecycline. Tet(X) inhibitors have potential to protect tetracyclines in combination therapies, but their discovery has been hampered by lack of high-throughput assays. We report the development of an efficient fluorescence polarisation Tet(X) binding assay employing a tetramethylrhodamine-glycyl-minocycline conjugate that enables inhibitor discovery. The assay was applied to tetracycline substrates and reported inhibitors, providing insight into their binding modes. Screening of a bioactive molecule library identified novel Tet(X) inhibitors, including psychoactive phenothiazine derivatives and the 5-HT4 agonist tegaserod, the activities of which were validated by turnover assays. Crystallographic studies of Tet(X4)-inhibitor complexes reveal two new inhibitor binding modes, importantly providing evidence for active site binding of Tet(X) inhibitors that do not share structural similarity with tetracycline substrates. In some cases, potentiation of tigecycline activity was observed in bacteria expressing Tet(X4). The combined results provide non-tetracycline scaffolds for development of potent Tet(X) inhibitors and highlight the need to evaluate the impact of non-antibiotics on antimicrobial resistance.
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May 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[30489]
Open Access
Abstract: Crystallographic fragment screening is a powerful methodology that enables the identification of low molecular weight ligands and has shown great promises in drug discovery. In this work we report the results of a fragment screening carried out in an effort to further map the cavities of trypanothione reductase from Trypanosoma brucei (TbTR), a critical target for drug design against human African trypanosomiases (HAT), for which efficient and non-toxic trypanocidal drugs are lacking. Moreover, the conservation of trypanothione reductase among trypanosomatids, including Leishmania, could facilitate the design of a wide-spectrum drug against many parasitic diseases. At the XCHEM facility (Diamond Light Sources, United Kingdom) we performed the soaking of TbTR monoclinic crystals with fragments from DSIpoised and EubOPEN DSIp libraries and we identified eight new hits binding to different cavities of TR including the trypanothione and the NADPH binding cavities. These fragments exhibited affinities ranging from submillimolar to millimolar, as determined by surface plasmon resonance (SPR). While the newly identified fragments did not significantly alter TbTR’s enzymatic activity—consistent with the nature of low-affinity ligands—they provide valuable insights into key interactions of fragments with TR and, together with prior fragment screening campaigns, pave the way towards follow-up chemical optimization into lead compounds.
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May 2025
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I03-Macromolecular Crystallography
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Yue
Wu
,
Zhihong
Li
,
Samanpreet
Kaur
,
Zewei
Zhang
,
Jie
Yue
,
Anthony
Tumber
,
Haoshu
Zhang
,
Zhe
Song
,
Peiyao
Yang
,
Ying
Dong
,
Fulai
Yang
,
Xiang
Li
,
Christopher J.
Schofield
,
Xiaojin
Zhang
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a JmjC domain 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase that catalyzes protein hydroxylations, including of specific asparagines in the C-terminal transcriptional activation domains of hypoxia-inducible factor alpha (HIF-α) isoforms. FIH is of medicinal interest due to its ability to alter metabolism and modulate the course of the HIF-mediated hypoxic response. We report the development of a light-induced, lysine (Lys106)-targeting irreversible covalent inhibitor of FIH. The approach is complementary to optogenetic methods for regulation of transcription. The covalently reacting inhibitor NBA-ZG-2291 was the result of structure-guided modification of the reported active site binding FIH inhibitor ZG-2291 with an appropriately positioned o-nitrobenzyl alcohol (o-NBA) group. The results demonstrate that NBA-ZG-2291 forms a stable covalent bond in a light-dependent process with Lys106 of FIH, inactivating its hydroxylation activity and resulting in sustained upregulation of FIH-dependent HIF target genes. The light-controlled inhibitors targeting a lysine residue enable light and spatiotemporal control of FIH activity in a manner useful for dissecting the context-dependent physiological roles of FIH.
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May 2025
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