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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Diamond Proposal Number(s):
[15806]
Open Access
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.
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Mar 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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.
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May 2019
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