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29 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
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
I04-Macromolecular Crystallography	Targeting OGG1 arrests cancer cell proliferation by inducing replication stress Torkild Visnes , Carlos Benítez-Buelga , Armando Cázares-Körner , Kumar Sanjiv , Bishoy M. F. Hanna , Oliver Mortusewicz , Varshni Rajagopal , Julian J. Albers , Daniel W Hagey , Tove Bekkhus , Saeed Eshtad , Juan Miguel Baquero , Geoffrey Masuyer , Olov Wallner , Sarah Müller , Therese Pham , Camilla Göktürk , Azita Rasti , Sharda Suman , Raúl Torres-Ruiz , Antonio Sarno , Elisée Wiita , Evert J. Homan , Stella Karsten , Karthick Marimuthu , Maurice Michel , Tobias Koolmeister , Martin Scobie , Olga Loseva , Ingrid Almlöf , Judith Edda Unterlass , Aleksandra Pettke , Johan Boström , Monica Pandey , Helge Gad , Patrick Herr , Ann-Sofie Jemth , Samir El andaloussi , Christina Kalderén , Sandra Rodriguez-Perales , Javier Benítez , Hans E Krokan , Mikael Altun , Pal Stenmark , Ulrika Warpman Berglund , Thomas Helleday Nucleic Acids Research 434 DOI: 10.1093/nar/gkaa1048 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.	Nov 2020
I04-Macromolecular Crystallography	Structural and biochemical characterization of botulinum neurotoxin subtype B2 binding to its receptors Jonathan R. Davies , Geoffrey Masuyer , Pal Stenmark Toxins 12 DOI: 10.3390/toxins12090603 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.	Sep 2020
I03-Macromolecular Crystallography	Crystal structure of Exotoxin A from Aeromonas pathogenic species Geoffrey Masuyer Toxins 12 DOI: 10.3390/toxins12060397 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Aeromonas exotoxin A (AE) is a bacterial virulence factor recently discovered in a clinical case of necrotising fasciitis caused by the flesh-eating Aeromonas hydrophila. Here, database mining shows that AE is present in the genome of several emerging Aeromonas pathogenic species. The X-ray crystal structure of AE was solved at 2.3 Å and presents all the hallmarks common to diphthamide-specific mono-ADP-ribosylating toxins, suggesting AE is a fourth member of this family alongside the diphtheria toxin, Pseudomonas exotoxin A and cholix. Structural homology indicates AE may use a similar mechanism of cytotoxicity that targets eukaryotic elongation factor 2 and thus inhibition of protein synthesis. The structure of AE also highlights unique features including a metal binding site, and a negatively charged cleft that could play a role in interdomain interactions and may affect toxicity. This study raises new opportunities to engineer alternative toxin-based molecules with pharmaceutical potential.	Jun 2020
I04-Macromolecular Crystallography	Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy Linxiang Yin , Geoffrey Masuyer , Sicai Zhang , Jie Zhang , Shin-Ichiro Miyashita , David Burgin , Laura Lovelock , Shu-Fen Coker , Tian-Min Fu , Pal Stenmark , Min Dong Plos Biology 18 DOI: 10.1371/journal.pbio.3000618 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.	Mar 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for the interaction of the chaperone Cbp3 with newly synthesized cytochrome b during mitochondrial respiratory chain assembly Mama Ndi , Geoffrey Masuyer , Hannah Dawitz , Andreas Carlström , Mirco Michel , Arne Elofsson , Mikaela Rapp , Pal Stenmark , Martin Ott Journal Of Biological Chemistry DOI: 10.1074/jbc.RA119.010483 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.	Sep 2019
I03-Macromolecular Crystallography	Crystal structure of the catalytic domain of the Weissela oryzae botulinum‐like toxin Sara Kosenina , Geoffrey Masuyer , Sicai Zhang , Min Dong , Pal Stenmark Febs Letters DOI: 10.1002/1873-3468.13446 Diamond Proposal Number(s): [15806] Show Abstract ▼ Abstract: Botulinum neurotoxins (BoNTs) are the most potent toxins known. So far, eight serotypes have been identified that all act as zinc‐dependent endopeptidases targeting SNARE proteins and inhibiting the release of neurotransmitters. Recently, the first botulinum toxin‐like protein was identified outside the Clostridial genus, designated BoNT/Wo in the genome of Weissella oryzae. Here, we report the 1.6 Å X‐ray crystal structure of the light chain of BoNT/Wo (LC/Wo). LC/Wo presents the core fold common to BoNTs but has an unusually wide, open, and negatively charged catalytic pocket, with an additional Ca2+ ion besides the zinc ion and a unique ß‐hairpin motif. The structural information will help establish the substrate profile of BoNT/Wo and help our understanding of how BoNT evolved.	May 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models Mark Elliott , Christine Favre-Guilmard , Sai Man Liu , Jacquie Maignel , Geoffrey Masuyer , Matthew Beard , Christopher Boone , Denis Carré , Mikhail Kalinichev , Stephane Lezmi , Imran Mir , Camille Nicoleau , Shilpa Palan , Cindy Perier , Elsa Raban , Sicai Zhang , Min Dong , Pal Stenmark , Johannes Krupp Science Advances 5 DOI: 10.1126/sciadv.aau7196 Diamond Proposal Number(s): [11265, 15806] Open Access Show Abstract ▼ Abstract: Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.	Jan 2019
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation Torkild Visnes , Armando Cázares-Körner , Wenjing Hao , Olov Wallner , Geoffrey Masuyer , Olga Loseva , Oliver Mortusewicz , Elisée Wiita , Antonio Sarno , Aleksandr Manoilov , Juan Astorga-Wells , Ann-Sofie Jemth , Lang Pan , Kumar Sanjiv , Stella Karsten , Camilla Gokturk , Maurice Grube , Evert J. Homan , Bishoy M. F. Hanna , Cynthia B. J. Paulin , Therese Pham , Azita Rasti , Ulrika Warpman Berglund , Catharina Von Nicolai , Carlos Benitez-Buelga , Tobias Koolmeister , Dag Ivanic , Petar Iliev , Martin Scobie , Hans E. Krokan , Pawel Baranczewski , Per Artursson , Mikael Altun , Annika Jenmalm Jensen , Christina Kalderén , Xueqing Ba , Roman A. Zubarev , Pal Stenmark , Istvan Boldogh , Thomas Helleday Science 362, 834 - 839 DOI: 10.1126/science.aar8048 Diamond Proposal Number(s): [15806] Show Abstract ▼ Abstract: The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.	Nov 2018

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