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15 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis for uracil removal from DNA by human SMUG1 Julian M. Ludäscher , Emma Scaletti Hutchinson , Guillem Vila-Julià , Ann-Sofie Jemth , Saher Shahid , Elisee Wiita , Israel Cabeza De Vaca , Szymon Pach , Lukas Gajdos , Swati Aggarwal , Ellen Walse , Oliver Mortusewicz , Thomas Helleday , Jens Carlsson , Pal Stenmark Nature Communications 17 DOI: 10.1038/s41467-026-72937-0 Diamond Proposal Number(s): [29948] Open Access Show Abstract ▼ Abstract: Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.	Jun 2026
I03-Macromolecular Crystallography	Uridine cytidine kinases govern molnupiravir bioactivation and anti-SARS-CoV-2 activity Huazhang Shu , Julian M. Ludäscher , Sushma Sharma , Seher Alam , Lilian Frank , Emma Scaletti Hutchinson , Marianna Tampere , Chloé Lévêque , André B. P. Van Kuilenburg , Nicholas C. K. Valerie , Mikael Altun , Andrei Chabes , Pal Stenmark , Sean G. Rudd , Si Min Zhang Plos Pathogens 22 DOI: 10.1371/journal.ppat.1014225 Diamond Proposal Number(s): [36026] Open Access Show Abstract ▼ Abstract: Molnupiravir is a nucleoside analogue antiviral drug against RNA viruses, including its clinical indication SARS-CoV-2. Whilst its mechanism-of-action is well defined, host factors that regulate its therapeutic responses have not been thoroughly deciphered and characterized. Here we show that uridine cytidine kinases (UCKs), key enzymes in pyrimidine salvage, effectively phosphorylate and thereby bioactivate N4-hydroxycytidine (NHC) – the active compound of molnupiravir, thus dictating its anti-SARS-CoV-2 efficacy and furthermore selectivity. In vitro, both isoforms of UCKs (UCK1 and UCK2) effectively phosphorylated NHC, where the structural basis of the catalysis was further deciphered via the first complete substrate bound co-crystal structure of UCK, i.e., UCK1-NHC-AMPPNP. In SARS-CoV-2-infected cells, UCK2 knockdown via siRNA hampered the intracellular accumulation of the tri-phosphorylated antiviral metabolite of NHC, resulting in a 10-fold reduction of the antiviral efficacy, and surprisingly, 2-fold reduction of its selectivity, which were critically recapitulated in a dose-dependent manner using a pan-UCK inhibitor. Altogether, this work underscores UCKs as pivotal players in upholding molnupiravir efficacy and therapeutic window of molnupiravir, and furthermore as pharmacologically tractable targets for tailoring the drug response.	May 2026
I04-Macromolecular Crystallography	A versatile nanobody platform for live and super-resolution imaging of synaptic vesicle dynamics and plasticity in rodent and human neurons Rashi Goel , Kristina Jevdokimenko , Ronja Rehm , Jannik Hentze , Paola Agüi-Gonzalez , Momchil Ninov , Erik Maier , Yin Wu , Felix Lange , Agata Witkowska , Svenja Bolz , Francesca Pennacchietti , Martina Damenti , Natalie Kaempf , Vladimir Khayenko , Carles Calatayud , Viveka Nand Malviya , Natali L. Chanaday , Emma Scaletti Hutchinson , Hao Liu , Kirsten Weyand , Valentin Schwarze , Daniela Ivanova , Tristan P. Wallis , Christopher Small , Hans M. Maric , Merja Joensuu , Michael A. Cousin , Frédéric A. Meunier , Patrik Verstreken , Ilaria Testa , Ege T. Kavalali , Volker Haucke , Stefan Jakobs , Henning Urlaub , Nils Brose , Benjamin H. Cooper , Pal Stenmark , Felipe Opazo , Reinhard Jahn , Silvio O. Rizzoli , Eugenio F. Fornasiero Journal Of Nanobiotechnology 24 DOI: 10.1186/s12951-026-04489-w Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.	May 2026
I03-Macromolecular Crystallography	Nucleobase catalysts for the enzymatic activation of 8-oxoguanine DNA glycosylase 1 Emily C. Hank , Nicholas D. D'Arcy-Evans , Emma R. Scaletti , Carlos Benítez-Buelga , Olov Wallner , Florian Ortis , Kaixin Zhou , Liuzhen Meng , Alicia Del Prado , Patricia Calvo , Ingrid Almlöf , Elisée Wiita , Karen Nierlin , Sara Kosenina , Andreas Krämer , Alice Eddershaw , Mario Kehler , Maeve Long , Ann-Sofie Jemth , Holly Dawson , Josephine Stewart , Adam Dickey , Mikhael E. Astorga , Marek Varga , Evert J. Homan , Martin Scobie , Stefan Knapp , Leandro Sastre , Pal Stenmark , Miguel De Vega , Thomas Helleday , Maurice Michel Rsc Chemical Biology DOI: 10.1039/D4CB00323C Diamond Proposal Number(s): [15806, 21625] Open Access Show Abstract ▼ Abstract: Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.	Oct 2025
I03-Macromolecular Crystallography	Structural and functional insights into Listeria monocytogenes phage endolysin PlyP100 - a promising food safety tool Karson R. Bateman , Emma Scaletti Hutchinson , Göran Widmalm , Michael J. Miller , Pal Stenmark Journal Of Biological Chemistry 12 DOI: 10.1016/j.jbc.2025.110295 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ 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.	May 2025
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Virtual fragment screening for DNA repair inhibitors in vast chemical space Andreas Luttens , Duc Duy Vo , Emma R. Scaletti , Elisée Wiita , Ingrid Almlöf , Olov Wallner , Jonathan Davies , Sara Kosenina , Liuzhen Meng , Maeve Long , Oliver Mortusewicz , Geoffrey Masuyer , Flavio Ballante , Maurice Michel , Evert Homan , Martin Scobie , Christina Kalderén , Ulrika Warpman Berglund , Andrii V. Tarnovskiy , Dmytro S. Radchenko , Yurii S. Moroz , Jan Kihlberg , Pål Stenmark , Thomas Helleday , Jens Carlsson Nature Communications 16 DOI: 10.1038/s41467-025-56893-9 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging optimization process. To expand the explored chemical space, we employ structure-based docking to evaluate orders-of-magnitude larger libraries than those used in traditional fragment screening. We computationally dock a set of 14 million fragments to 8-oxoguanine DNA glycosylase (OGG1), a difficult drug target involved in cancer and inflammation, and evaluate 29 highly ranked compounds experimentally. Four of these bind to OGG1 and X-ray crystallography confirms the binding modes predicted by docking. Furthermore, we show how fragment elaboration using searches among billions of readily synthesizable compounds identifies submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cells. Comparisons of virtual screening strategies to explore a chemical space of 1022 compounds illustrate that fragment-based design enables enumeration of all molecules relevant for inhibitor discovery. Virtual fragment screening is hence a highly efficient strategy for navigating the rapidly growing combinatorial libraries and can serve as a powerful tool to accelerate drug discovery efforts for challenging therapeutic targets.	Feb 2025
I03-Macromolecular Crystallography	Kinetic and structural characterization of NUDT15 and NUDT18 as catalysts of isoprene pyrophosphate hydrolysis Emma Scaletti , Judith E. Unterlass , Ingrid Almlöf , Tobias Koolmeister , Karl S. Vallin , Despina Kapsitidou , Viktoriia Tsuber , Thomas Helleday , Pal Stenmark , Ann-Sofie Jemth The Febs Journal 39 DOI: 10.1111/febs.17202 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.	Jun 2024
I03-Macromolecular Crystallography	Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis Emma R. Scaletti , Pontus Pettersson , Joan Patrick , Patrick J. Shilling , Robert Gustafsson Westergren , Daniel O. Daley , Lena Mäler , Göran Widmalm , Pal Stenmark Journal Of Biological Chemistry 280 DOI: 10.1016/j.jbc.2023.105256 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose and UDP-GalNAc. Structural comparison with the homologue from Escherichia coli (EcWaaG) revealed five key differences in the sugar binding pocket. Solution-state NMR analysis showed that wildtype PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither wildtype PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.	Sep 2023
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Optimization of N-piperidinyl-benzimidazolone derivatives as potent and selective inhibitors of 8-oxo guanine DNA glycosylase 1 Olov Wallner , Armando Cázares-Körner , Emma R. Scaletti , Geoffrey Masuyer , Tove Bekkhus , Torkild Visnes , Kirill Mamonov , Florian Ortis , Thomas Lundbäck , Maria Volkova , Tobias Koolmeister , Elisée Wiita , Olga Loseva , Monica Pandey , Evert Homan , Carlos Benítez-Buelga , Jonathan Davies , Martin Scobie , Ulrika Warpman Berglund , Christina Kalderén , Pal Stenmark , Thomas Helleday , Maurice Michel Chemmedchem DOI: 10.1002/cmdc.202200310 Diamond Proposal Number(s): [15806, 21625] Open Access Show Abstract ▼ Abstract: 8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G:C→T:A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N -piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of different members of the class. Potent members adopt a chair within the N -Piperidinyl-linker, while a boat conformation was found for weaker analogues.	Sep 2022
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function Maurice Michel , Carlos Benítez-Buelga , Patricia A. Calvo , Bishoy M. F. Hanna , Oliver Mortusewicz , Geoffrey Masuyer , Jonathan Davies , Olov Wallner , Sanjiv Kumar , Julian J. Albers , Sergio Castañeda-Zegarra , Ann-Sofie Jemth , Torkild Visnes , Ana Sastre-Perona , Akhilesh N. Danda , Evert J. Homan , Karthick Marimuthu , Zhao Zhenjun , Celestine N. Chi , Antonio Sarno , Elisée Wiita , Catharina Von Nicolai , Anna J. Komor , Varshni Rajagopal , Sarah Müller , Emily C. Hank , Marek Varga , Emma R. Scaletti , Monica Pandey , Stella Karsten , Hanne Haslene-Hox , Simon Loevenich , Petra Marttila , Azita Rasti , Kirill Mamonov , Florian Ortis , Fritz Schömberg , Olga Loseva , Josephine Stewart , Nicholas D’arcy-Evans , Tobias Koolmeister , Martin Henriksson , Dana Michel , Ana De Ory , Lucia Acero , Oriol Calvete , Martin Scobie , Christian Hertweck , Ivan Vilotijevic , Christina Kalderén , Ana Osorio , Rosario Perona , Alexandra Stolz , Pal Stenmark , Ulrika Warpman Berglund , Miguel De Vega , Thomas Helleday Science 376, 1471 - 1476 DOI: 10.1126/science.abf8980 Diamond Proposal Number(s): [15806, 21625] Show Abstract ▼ Abstract: Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.	Jun 2022

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