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23 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
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress Nadilly Bonagas , Nina M. S. Gustafsson , Martin Henriksson , Petra Marttila , Robert Gustafsson , Elisée Wiita , Sanjay Borhade , Alanna C. Green , Karl S. A. Vallin , Antonio Sarno , Richard Svensson , Camilla Göktürk , Therese Pham , Ann-Sofie Jemth , Olga Loseva , Victoria Cookson , Nicole Kiweler , Lars Sandberg , Azita Rasti , Judith E. Unterlass , Martin Haraldsson , Yasmin Andersson , Emma R. Scaletti , Christoffer Bengtsson , Cynthia B. J. Paulin , Kumar Sanjiv , Eldar Abdurakhmanov , Linda Pudelko , Ben Kunz , Matthieu Desroses , Petar Iliev , Katarina Färnegårdh , Andreas Krämer , Neeraj Garg , Maurice Michel , Sara Häggblad , Malin Jarvius , Christina Kalderén , Amanda Bögedahl Jensen , Ingrid Almlöf , Stella Karsten , Si Min Zhang , Maria Häggblad , Anders Eriksson , Jianping Liu , Björn Glinghammar , Natalia Nekhotiaeva , Fredrik Klingegård , Tobias Koolmeister , Ulf Martens , Sabin Llona-Minguez , Ruth Moulson , Helena Nordström , Vendela Parrow , Leif Dahllund , Birger Sjöberg , Irene L. Vargas , Duy Duc Vo , Johan Wannberg , Stefan Knapp , Hans E. Krokan , Per I. Arvidsson , Martin Scobie , Johannes Meiser , Pal Stenmark , Ulrika Warpman Berglund , Evert J. Homan , Thomas Helleday Nature Cancer 3, 156 - 172 DOI: 10.1038/s43018-022-00331-y Open Access Show Abstract ▼ Abstract: The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.	Feb 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural and biochemical investigation of class I ribonucleotide reductase from the hyperthermophile Aquifex aeolicus Daniel Rehling , Emma Rose Scaletti , Inna Rozman Grinberg , Daniel Lundin , Margareta Sahlin , Anders Hofer , Britt-Marie Sjöberg , Pal Stenmark Biochemistry DOI: 10.1021/acs.biochem.1c00503 Open Access Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.	Dec 2021
I24-Microfocus Macromolecular Crystallography	Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity Daniel Rehling , Si Min Zhang , Ann-Sofie Jemth , Tobias Koolmeister , Adam Throup , Olov Wallner , Emma Scaletti , Takaya Moriyama , Rina Nishii , Jonathan Davies , Matthieu Desroses , Sean G. Rudd , Martin Scobie , Evert Homan , Ulrika Warpman Berglund , Jun J. Yang , Thomas Helleday , Pal Stenmark Journal Of Biological Chemistry 271 DOI: 10.1016/j.jbc.2021.100568 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: The enzyme NUDT15 efficiently hydrolyses the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and pre-emptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants due to their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared to TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.	Mar 2021
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
I24-Microfocus Macromolecular Crystallography	Structural and functional analysis of the inhibition of equine glutathione transferase A3-3 by organotin endocrine disrupting pollutants Jana Skerlova , Aram Ismail , Helena Lindström , Birgitta Sjödin , Bengt Mannervik , Pal Stenmark Environmental Pollution DOI: 10.1016/j.envpol.2020.115960 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrine-disrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects. Our enzyme kinetics and structural data on EcaGST A3-3 explain on a molecular level the neurotoxic and endocrine disrupting effects of organotin pollutants.	Oct 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site Markel Martinez-Carranza , Venkateswara Rao Jonna , Daniel Lundin , Margareta Sahlin , Lars-Anders Carlson , Newal Jemal , Martin Högbom , Britt-Marie Sjöberg , Pal Stenmark , Anders Hofer Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.014895 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.	Sep 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
I24-Microfocus Macromolecular Crystallography	Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target Jana Skerlova , Judith Unterlass , Mona Göttmann , Petra Marttila , Evert Homan , Thomas Helleday , Ann-Sofie Jemth , Pal Stenmark Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.013695 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and L-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.	Jun 2020

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