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Instruments / Technical Area	Publication Details	Published
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
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
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	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
I04-Macromolecular Crystallography	MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool Emma Rose Scaletti , Karl S. Vallin , Lars Bräutigam , Antonio Sarno , Ulrika Warpman Berglund , Thomas Helleday , Pal Stenmark , Ann-Sofie Jemth Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.012636 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site sub-pocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1 catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.	Mar 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural basis of inhibition of the human serine hydroxymethyltransferase SHMT 2 by antifolate drugs Emma Scaletti , Ann‐sofie Jemth , Thomas Helleday , Pal Stenmark Febs Letters DOI: 10.1002/1873-3468.13455 Show Abstract ▼ Abstract: Serine hydroxymethyltransferase (SHMT) is the major source of 1‐carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2‐antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti‐cancer drug target.	May 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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