I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15806, 21625]
Open Access
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.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15806, 21625]
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.
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Jun 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Open Access
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.
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Feb 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
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.
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Dec 2021
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I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[21625]
Open Access
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.
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Mar 2021
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15806]
Open Access
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.
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Nov 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
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.
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Oct 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11265]
Open Access
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.
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Sep 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15806]
Open Access
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.
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Sep 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15806]
Open Access
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.
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Jun 2020
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