I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402, 33658]
Open Access
Abstract: Environment-sensitive fluorescent probes are indispensable tools for studying biological systems and advancing drug discovery. This study reports the development of 4-sulfamoyl-7-aminobenzoxadiazole (SBD)-based fluorescent probes for the allosteric site of the liver isoform of pyruvate kinase (PKL). By integrating SBD moieties into known activator scaffolds, such as mitapivat and diarylsulfonamide (DASA) ligands, probes for indicator displacement assays were designed to quantify ligand interactions in the allosteric site. Compound 4a displayed dose-dependent fluorescence enhancement in response to PKL binding and was used in a competitive binding assay with unlabelled ligands: mitapivat, TEPP-46, DASA-58 and reported activator 21. Structure–activity relationship (SAR) analysis revealed key structural features influencing activity and fluorescence sensitivity. The probes report selectively on the allosteric site ligands as the binding was not affected by natural ligands, such as ADP, fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and phenylalanine. These findings provide a practical framework for detecting allosteric ligand engagement in PKL and expand the repertoire of molecular tools for advancing PKL-targeted therapies.
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Nov 2025
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I24-Microfocus Macromolecular Crystallography
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Natalia
Venetz-Arenas
,
Tim
Schulte
,
Sandra
Müller
,
Karin
Wallden
,
Stefanie
Fischer
,
Tom
Resink
,
Nadir
Kadri
,
Maria
Paladino
,
Nicole
Pina
,
Filip
Radom
,
Denis
Villemagne
,
Sandra
Bruckmaier
,
Andreas
Cornelius
,
Tanja
Hospodarsch
,
Evren
Alici
,
Hans-Gustaf
Ljunggren
,
Benedict J.
Chambers
,
Xiao
Han
,
Renhua
Sun
,
Marta
Carroni
,
Victor
Levitsky
,
Tatyana
Sandalova
,
Marcel
Walser
,
Adnane
Achour
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.
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Nov 2025
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I03-Macromolecular Crystallography
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Cholpon
Tilegenova
,
Tun
Liu
,
Qian
Zhao
,
Mahati
Are
,
Yu
Zhao
,
Woo Suk
Choi
,
Anusarka
Bhaumik
,
Ruth
Steele
,
Nicholas A.
Manieri
,
Bengi
Turegun
,
Alex
Ni
,
Rosa M. F.
Cardoso
,
Paul
Shaffer
,
Desmond
Clark
,
Robin
Ernst
,
Wenyu
Li
,
Tracy
Taylor
,
Suresh Kumar
Swaminathan
,
Bhargavi
Ramaraju
,
Kevin
Liaw
,
Steven A.
Jacobs
,
Sujata
Sharma
,
Wan Cheung
Cheung
,
Adam
Zwolak
Diamond Proposal Number(s):
[34711]
Abstract: Bispecific antibodies (bsAbs) can enable therapeutic mechanisms, such as dual antigen targeting or receptor agonism, that are impossible using monoclonal antibodies. BsAbs with IgG-like format (bsIgG) are comprised of two unique heavy chains, each having a cognate light chain. Co-expression of these four unique polypeptides often leads to several mispaired species that are difficult to separate from the target bsIgG due to their similar biophysical properties. Here we describe a set of mutations called ProAla that exploit a the unfolded protein response pathway of cells. ProAla heavy chains are engineered with higher folding energy barriers such that only the cognate light and heavy chains can induce folding, chaperone release and secretion. The structures of the ProAla Fab and Fc regions are identical in structure to normal antibodies, enabling maintenance of half-life and function. Mispaired polypeptides fail to secrete from the cell due to enhanced interaction with the endoplasmic reticulum chaperone BiP, resulting in increased purity of secreted bsIgGs.
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Oct 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Anaïs F. M.
Noisier
,
Jenny
Sandmark
,
Fredrik
Edfeldt
,
Anna
Backmark
,
Johan
Broddefalk
,
Joanna
Wandzik
,
Ulrik
Jurva
,
Margareta
Ek
,
Carina A.
Johansson
,
Louise
Barlind
,
Jenny
Gunnarsson
,
Janna M.
Bigalke
,
Yafeng
Xue
,
Andrey I.
Frolov
,
Cecilia
Kankkonen
,
Robert G.
Roth
,
Maria
Fritsch
,
Sophie
Watcham
,
Katerine
Van Rietschoten
,
Gemma E.
Mudd
,
Helen
Harrison
,
Liuhong
Chen
,
Michael J.
Skynner
,
David J.
Craik
,
Sunay V.
Chankeshwara
,
Malin
Lemurell
Abstract: The GDF15–GFRaL–RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15–GFRaL protein–protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.
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Oct 2025
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Krios I-Titan Krios I at Diamond
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Andreas
Schedlbauer
,
Xu
Han
,
Wouter
Van Bakel
,
Tatsuya
Kaminishi
,
Borja
Ochoa-Lizarralde
,
Idoia
Iturrioz
,
Retina
Çapuni
,
Ransford
Parry
,
Ronny
Zegarra
,
David
Gil-Carton
,
Jorge P.
López-Alonso
,
Kristina
Barragan Sanz
,
Letizia
Brandi
,
Claudio O.
Gualerzi
,
Paola
Fucini
,
Sean R.
Connell
Diamond Proposal Number(s):
[17171, 31586]
Open Access
Abstract: The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator fMet–tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B, and B1 with molecular masses of 643–658 Da). Previous biochemical and structural characterization of GE81112 indicates that the primary mechanism of action of this antibiotic is to (i) prevent the initiator fMet–tRNA from binding correctly to the P-site and (ii) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryo-EM, we have determined the binding site of GE81112 in initiation complexes (3.2–3.7 Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel but remote from the binding site of the initiation factors and initiator fMet–tRNA. This suggests that it acts allosterically to prevent the initiator fMet–tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.
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Oct 2025
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B23-Circular Dichroism
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Open Access
Abstract: The emergence of multidrug-resistant (MDR) bacterial pathogens is an alarming global health threat that demands new therapeutic strategies beyond conventional antibiotics. Here, we present a rationally designed antimicrobial peptide (AMP) derived from mammalian cathelicidins and defensins that selectively targets bacterial membranes with low cytotoxicity toward mammalian cells. Circular dichroism spectroscopy revealed that the peptide adopts an α-helical conformation upon membrane interaction, a key feature of its mechanism. Surface plasmon resonance and isothermal titration calorimetry demonstrated high-affinity and selective binding to bacterial lipid membranes. Functionally, the peptide was strongly bactericidal against clinical MDR Escherichia coli (E. coli) and clinically important ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). Compared with the parent peptide LL-37, our AMP exhibited lower minimum inhibitory concentrations (MICs) and faster bactericidal kinetics across both Gram-negative and Gram-positive strains. Calcein leakage assays, showing effective membrane disruption. Importantly, cytotoxicity experiments with human epithelial (Caco-2) and immune (THP-1) cells indicated low cytotoxicity at concentrations exceeding bactericidal levels, supporting a favorable therapeutic window. ELISA quantifications of cytokines (IL-6, TNF-α) further suggested immunomodulatory effects at bactericidal concentrations. Transcriptomic profiling of E. coli treated with sub-lethal concentrations of the peptide exhibited upregulation of bacterial stress response pathways and downregulation of vital metabolic processes, reflecting the complex antimicrobial action of the peptide. Collectively, these findings highlight this LL-37-derived AMP as a promising candidate for treating MDR bacterial infections caused by E. coli and ESKAPE pathogens and for guiding the development of next-generation antimicrobial agents.
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Oct 2025
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I04-Macromolecular Crystallography
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Anastasia
Polycarpou
,
Tara
Wagner-Gamble
,
Roseanna
Greenlaw
,
Lauren
O'Neill
,
Varsha
Kanabar
,
Alanoud
Alrehaili
,
Yusun
Jeon
,
Jonathan
Baker
,
Mona
Bafadhel
,
Hataf
Khan
,
Michael H.
Malim
,
Marco
Romano
,
Conrad A.
Farrar
,
Dorota
Smolarek
,
Rocio
Martinez-Nunez
,
Katie J.
Doores
,
Russell
Wallis
,
Linda S.
Klavinskis
,
Steven H.
Sacks
Open Access
Abstract: SARS-CoV-2 respiratory-tract infection affects both vaccinated and unvaccinated persons suggesting factors besides adaptive immunity are operative. We investigated the role of collectin-11 (CL-11), an epithelial-secreted carbohydrate-binding lectin that drives innate immunity and eliminates pathogens by complement activation. SARS-CoV-2, despite binding CL-11 to activate complement, was resistant to lysis. Remarkably, opsonization by CL-11 enhanced virus production by infected respiratory epithelial cells independently of complement. Furthermore, infected cells expressing SARS-CoV-2 spike protein displayed enhanced vulnerability to CL-11 binding and membrane attack by complement. The mechanism of enhanced infectivity was ablated in the presence of L-fucose, which occupied the extended carbohydrate-binding cleft of CL-11 in a crystallographic analysis of complexes between L-fucose and CL-11. Our study suggests pathogenicity of SARS-CoV-2 is related to complement-resistance together with enhanced infectivity and injury of respiratory epithelial cells mediated by locally released CL-11.
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Oct 2025
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I04-Macromolecular Crystallography
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Zhe
Nie
,
Michael
Trzoss
,
Andrew T.
Placzek
,
Lynnie
Trzoss
,
Goran
Krilov
,
Shulu
Feng
,
Morgan
Lawrenz
,
Min
Ye
,
Netonia
Marshall
,
Karen H.
Dingley
,
Robert D.
Pelletier
,
W. George
Lai
,
Jeffrey A.
Bell
,
Haifeng
Tang
,
Paul
Devine
,
Zhijie
Liu
,
Peter
Skrdla
,
Roman
Shimanovich
,
Matt
Liu
,
Renchao
Wang
,
Xiaoming
Xu
,
Robert
Abel
,
Karen
Akinsanya
,
Wu
Yin
Abstract: MALT1 is a key component of the CARD11-BCL10-MALT1 (CBM) complex downstream from BTK on the B-cell receptor signaling pathway. It is a key mediator of NF-κB signaling and considered a potential therapeutic target for several subtypes of non-Hodgkin’s B-cell lymphomas. By applying advanced physics-based modeling techniques, including combining free energy calculations with machine learning methods and a chemistry-aware compound enumeration workflow, extensive sets of de novo design ideas were explored to quickly identify a novel hit series. Multiparameter optimization allowed efficient prioritization of molecules with good potency and drug-like properties during lead optimization, which led to the discovery of a highly potent MALT1 inhibitor, SGR-1505, with a well-balanced property profile. It demonstrated strong antitumor activity alone and in combination with BTK inhibitor in multiple in vivo B-cell lymphoma xenograft models and progressed to a phase 1 clinical trial in patients with mature B-cell neoplasms.
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Oct 2025
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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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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[39391]
Open Access
Abstract: Mutations in the T-cell receptor signalling pathway have been identified in patients with adult T-cell leukaemia/lymphoma (ATLL) and one of the most frequently observed targets of these mutations is protein kinase C beta (PKCb). Here we have characterised the most frequent mutation in PKCb (D427N) addressing the issue of gain/loss of function, neomorphic change, assessing the impact of mutation in vivo, in cells, biochemically and structurally. It is concluded that this mutation is a gain-of-function, activating mutation that confers an altered substrate specificity on this protein kinase. In a constitutive knock-in mouse model this activated allele induces splenomegaly associated with extramedullary haematopoiesis. Pharmacologically, the D427N mutant protein displays poor sensitivity to established PKCb inhibitors, necessitating development of bespoke therapeutics for any ATLL intervention through this target. Such efforts could be guided by the availability the D427N mutant-ruboxistaurin structure presented here.
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Oct 2025
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