I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39189]
Open Access
Abstract: The enzymes Cannabichromenic Acid Synthase (CBCAS), Cannabidiolic Acid Synthase (CBDAS) and Tetrahydrocannabinolic Acid Synthase (THCAS) are together the major cannabinoid synthase enzymes responsible for the biosynthesis of their respective metabolites from a common precursor Cannabigerolic Acid (CBGA). As the catalysts responsible for generating biological molecules of significant pharmaceutical value, there has been considerable interest in the enzymes with respect to heterologous production, mechanism, and incorporation into synthetic biology pathways for the facile industrial production of these molecules. The enzymes share high degrees of homology, and therefore their distinct specificities are governed by very subtle differences in sequence and therefore structure, although, until now, only a structure for THCAS has been reported. In this report, we present structures of CBCAS, CBDAS and a structure of THCAS at a higher resolution than the known structure, each in complex with their flavin coenzyme FAD. The structures reveal active site differences that may be responsible for the complementary activities observed, in terms of both first-shell amino acid substitutions, but also in more remote residues that influence active site topology through referred effects, or that have effects on substrate access. The structures provide a useful and informative platform for the rational engineering of improved or altered chemoselectivity in these enzymes.
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Dec 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34182]
Open Access
Abstract: Chikungunya virus (CHIKV) outbreaks impose significant burdens on healthcare systems and raise an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV infection. CHIKV-encoded macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer based assay, exhibiting an IC50 of 8.9 μM, and its inhibitory activity was validated through multiple orthogonal assays. The compound features a thiobarbiturate-indole scaffold and shows high selectivity over a panel of human and viral ADP-ribose binding and hydrolyzing proteins. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Guided by structure-activity relationship studies, compound 11 (MDOLL-0591) of the developed series with similar IC50 of 10 μM but with increased lipophilicity was discovered to have antiviral activity against CHIKV in cell culture, demonstrating that macrodomain could be targeted in virus infections.
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Nov 2026
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B23-Circular Dichroism
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Xi
Lei
,
Sibo
Peng
,
Nijas
Aliu
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Tiberiu-Marius
Gianga
,
Xin
Jin
,
Sabrina
Ruggiero
,
Shilpa
Chokshi
,
Luca
Urbani
,
Bernhard F.
Gibbs
,
Elena
Klenova
,
Gurprit S.
Lall
,
Gabriela
Dekany
,
Dietmar
Cholewa
,
Steffen M.
Berger
,
Maryam
Abooali
,
Inna M.
Yasinska
,
Song
Xu
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[36941, 38189]
Abstract: Human cancer cells and malignant tumours operate immune evasion machinery, which consists of a set of cross-linked biochemical pathways that suppress the activities of immune cells, such as cytotoxic T cells and natural killer cells. Recent evidence demonstrated that the immune evasion machinery operated by human cancer cells is self-sustaining. However, the mechanisms underlying self-regulation of cancer immune evasion networks remain to be understood. Here we report for the very first time that the cell surface receptor CD44 acts as an enhancer of immune evasion activity in human cancer cells rather than an immune checkpoint per se. Furthermore, upon interaction with galectin-9, CD44 was found to alter cell membrane potential and induce the secretion of TGF-β. Secreted TGF-β displays autocrine and paracrine activities following TGF-β receptor (TGFBR) engagement, which was shown to then activate the Smad3 transcriptional pathway. CD44 complexed to galectin-9 allosterically enhances activity of the TGFBR complex and this was shown to substantially amplify the expression of immune checkpoint proteins crucially associated with suppressing the anti-cancer activities of cytotoxic lymphoid cells. We also demonstrated that this is a fundamental pathway used by human embryos to prevent immune rejection and by innate immune cells to facilitate anti-bacterial innate immune response. Cancer cells and malignant tumours hijack and successfully adapt this fundamental pathway to escape immune surveillance and suppress anti-cancer immune reactions. As such, this autocrine loop allows for the self-regulation of immune evasion networks operated by cancer cells and can be considered as a promising target for highly efficient immunotherapy of cancer.
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Nov 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34263]
Open Access
Abstract: Deoxyuridine triphosphatase (dUTPase) is essential for DNA replication fidelity and embryonic development in metazoa, yet whether a protein inhibitor can acutely disrupt dUTPase function in a living vertebrate has not been tested. The Staphylococcus aureus Stl protein inhibits diverse dUTPases in vitro, but its efficacy in a eukaryotic organism was unknown. We determined the crystal structure of zebrafish dUTPase in complex with the N-terminal Stl fragment to 2.26 Å resolution, revealing conserved active-site engagement and displacement of the C-terminal arm. Biolayer interferometry confirmed low-nanomolar binding affinity with KD = 0.7 nM, and enzyme assays demonstrated ~50% inhibition of zebrafish dUTPase activity at saturating Stl concentrations. These structural and biophysical data establish that Stl forms a tight inhibitory complex with zebrafish dUTPase, prompting us to test whether this interaction can perturb enzyme function in vivo. Microinjection of Stl or its point mutant, retaining dUTPase-inhibitory activity but unable to bind DNA, into fertilized zebrafish oocytes reduces embryo survival to ~60% within 48 h compared to ~90% in controls, demonstrating that lethality depends on dUTPase targeting rather than DNA binding. Accelerated mortality relative to genetic dUTPase knockout indicates that maternally deposited dUTPase supports viability during early cleavage. Stl thus acts as an immediate, proteinaceous dUTPase inhibitor in a living vertebrate, providing a tool to dissect dUTPase function and regulation during embryonic development.
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Oct 2026
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I04-Macromolecular Crystallography
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Open Access
Abstract: Motivated by the need for a convenient way to demonstrate the growth of crystals in glass plates by the lipid cubic phase method at crystallization workshops and courses, we developed a lightweight, portable, compact and inexpensive digital microscope device. The device is built around hand-held Wi-Fi and hardwired digital microscopes that are available commercially at low cost and that can use cell phones and personal computers for imaging without the need for unwieldy eyepieces. The device provides illumination from above and below the crystallization plate over a range of brightness levels. It can be used with regular white light and with polarized light to detect birefringent crystals. While not the focus of this study, the new device has been shown to also work with batch and vapour-diffusion plates. Remote monitoring of crystallization plates in a cold cabinet or walk-in refrigerator at 4 °C is possible with the Wi-Fi microscope. Crystals grown in lipid cubic phase and vapour-diffusion crystallization plates were harvested with the aid of the new device and used for diffraction data collection, leading to structure determination at high resolution. Most parts of the device were 3D printed in polylactic acid plastic. The corresponding STL files, included as part of this publication, can be used to make replicates of the current microscope and to produce bespoke devices suited to the specific needs of the user. We envision using the new device for outreach activities at primary, secondary and third-level schools, and at science fairs in support of the citizen scientist. Relatedly, in this study examples are included of recorded images of plant and animal tissue sections. One reveals birefringent materials in a tomato leaf. Another shows optically active crystals of calcium oxalate in onion tunic tissue.
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Oct 2026
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I04-Macromolecular Crystallography
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Stéphane
Bourg
,
Matthieu
Place
,
Chloé
Copin
,
Apirat
Chaikuad
,
Thomas
Robert
,
Hanna
Holzmann
,
Susanne
Müller
,
Stéphane
Bach
,
Sandrine
Ruchaud
,
Stefan
Knapp
,
Frédéric
Buron
,
Sylvain
Routier
,
Pascal
Bonnet
Open Access
Abstract: CLK1 is one of the four human isoforms of the cdc2-like (CLK) kinases that has been suggested as a therapeutic target in diverse diseases based on its important role regulating mRNA splicing. For example, CLKs and closely related kinases such as DYRK1A have been targeted in Alzheimer’s disease and other diseases in which splice site selection contributes to the disease development. Here we have developed an efficient in silico fragment-based ligand design approach to identify novel CLK1 inhibitors with excellent ligand efficiency based on an imidazo[2,1-b][1,3,4]thiadiazole fragment. More than one million docking poses were generated from 26,225 unique virtual compounds, and after applying several filtering steps, 11 compounds were selected, synthesized and their CLK1 inhibition and cellular potency were evaluated. Gratifyingly, inhibitor potencies were in excellent agreement with predicted values and crystallographic data of an inhibitor bound to CLK1 confirmed the unusual binding mode of the compounds.
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Oct 2026
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Sven
Falke
,
Patrick Y. A.
Reinke
,
Daniel
Rosenberg
,
Pontus
Fischer
,
Jan
Meyer
,
Marina
Galchenkova
,
Alexandra
Tolstikova
,
Valerio
Mariani
,
Johanna M.
Senst
,
Philipp
Lewe
,
Susanne
Witt
,
Armin
Wagner
,
Henry N.
Chapman
,
Mark
Hunter
,
Sebastian
Gunther
,
Alke
Meents
Open Access
Abstract: Emerging human pathogenic RNA viruses such as Lassa virus (LASV) and closely related viruses such as Hantavirus and Andes virus continue to be a major health threat globally. Treatment options for infections are severely limited. The widely conserved cap-snatching endonuclease of LASV (LASVendoN) is therefore used as an interesting drug target for our X-ray compound-screening experiments. Considering recent improvements in instrumentation and serial crystallography method development, different approaches may be used to facilitate pharmaceutical compound screening. Current-generation X-ray free-electron laser (XFEL) sources hold promise for obtaining higher quality diffraction data and achieving a high sample throughput using fixed-target serial femtosecond X-ray crystallography. We collected serial fixed-target X-ray diffraction data at Linac Coherent Light Source and at the synchrotron source PETRA III for comparison and solved the crystal structures of 2,4-dioxo-4-phenylbutanoic acid (DPBA) and baloxavir acid (BXA) binding to the active site of LASVendoN. These solved structures represent a reasonable starting point for drug development. In addition to room-temperature diffraction data, conventional single-crystal diffraction data were recorded at 100 K for comparison. To analyse the sample throughput in a compound-screening experiment at an XFEL, we further present data for the bacterial fosfomycin-resistance protein A with six ligands previously identified in a synchrotron screening experiment. In summary, the XFEL and synchrotron data-collection approaches have proven to be useful for compound screening with minor differences in achievable data quality and resolution. The individual experimental setup, detector and potentially the use of beam sweeping at XFELs at close-to-physiological temperatures combined with reasonable sample consumption and minimized radiation damage are considered to be advantageous for high-throughput drug development.
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Oct 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[36570]
Open Access
Abstract: Leishmaniasis is a neglected disease that affects around two million people every year. Current treatments are often highly toxic or prone to resistance, underscoring the urgent need for new therapeutic strategies. PROTACs may offer a promising alternative, as they can potentially mitigate both toxicity and resistance. However, very little is known about the ubiquitin–proteasome system (UPS) in Leishmania. Notably, only two E3 ligases containing a CULT domain have been identified so far, and none carrying a von Hippel–Lindau (VHL) domain─the classical E3 ligase used by clinically advanced PROTACs. In this work, we take an important step toward understanding the UPS in Leishmania, and we propose that in this organism the UbC4 E2 enzyme, rather than an E3, may be directly exploited to develop a PROTAC able to engage a protein of interest. Here, we report the biochemical and structural characterization of the Leishmania major ubiquitin-conjugating enzyme 4 (UbC4). Through a fragment screening campaign, we identified 10 fragments binding to distinct cavities on UbC4. Among these, five interact with the same noncatalytic pocket that is poorly conserved in humans, while one fragment binds near the catalytic cysteine. Using DeepFrag predictions and molecular docking, we explored fragment elongation strategies to enhance affinity for their respective binding sites, with the goal of guiding the development of E2-recruiting PROTACs or UPS inhibitors for the treatment of leishmaniasis.
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Sep 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: Carbapenem-resistant Enterobacterales (CRE), including Escherichia coli and Klebsiella pneumoniae are increasingly common pathogens. Resistance in CRE is often associated with production of K. pneumoniae (KPC) carbapenemases. We report a structurally modified meropenem derivative, JDB-1-200 (i.e., 8-epi-meropenem, in which the stereochemistry of the C8 hydroxyl is inverted), displaying enhanced activity relative to meropenem against KPC-producing CRE. JDB-1-200 alone displayed superior activity (MICs of 0.5 to 1 mg/L, compared with >8 mg/L for meropenem) against several clinically relevant Enterobacterales, including KPC-producing K. pneumoniae and Citrobacter freundii. Crystallographic analysis of JDB-1-200 complexes with the E166Q deacylation-deficient KPC-2 mutant, compared with an analogous meropenem (8R-hydroxyl) complex, indicates that the improvement arises from the JDB 8S-hydroxyl group acting as a hydrogen-bond donor to the hydrolytic water, slowing acyl-enzyme hydrolysis, with similar interactions observed in a CTX-M-15:JDB-1-200 complex. Kinetic inhibition data for KPC-2 and CTX-M-15 indicate that JDB-1-200 could be used alone or alongside a β-lactamase inhibitor for effective CRE treatment.
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Sep 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[30374]
Open Access
Abstract: Non-tailed phages remain underexplored in marine environments, as tailed phages have long dominated sequence and culture collections. Yet recent surveys suggest that non-tailed phages may be more abundant and have distinct impacts on microbial mortality and gene transfer. Here, we solve the structure of Vibrio anguillarum bacteriophage NO16, one of the simplest members of the Varidnaviria realm. Mass spectrometry detected at least nine different proteins in the virion, which has a pseudoT = 21 capsid similar to that of related double jelly roll (DJR) phages, but differs in the organization of minor capsid proteins, particularly those mediating membrane-capsid contacts. The DJR major capsid protein GP19 is stabilized by strong electrostatic interactions between monomers, and possibly by a cation at its base, as seen in corticovirus PM2. Localized reconstruction revealed a symmetry mismatch at the vertex, where two trimeric GP13 spikes attach to the pentameric GP14 penton base. GP13 carbohydrate-binding sites and predicted glycosylase activity point to a role in host entry. Using structural and functional predictions for its entire proteome, we propose a complete atlas of the NO16 infectious cycle.
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Sep 2026
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