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Structural and functional studies of proteins of medical relevance: Protein-ligand complexes in cancer and novel structural folds in bacteria
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-4854-5531
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

X-ray crystallography is a tool for determining the structures of proteins and protein-ligand complexes. In this thesis the method has been employed to study several proteins of medical relevance.

Cancer is a terrible disease, severely impacting those affected, as well as their family and friends. Current cancer treatments involve a combination of cytostatic drugs, surgery and radiation treatment. Unfortunately many cytostatic drugs also kill healthy cells, which gives rise to serious side-effects. The discovery of treatments which selectively inhibit proteins essential for cancer cell survival but which are non-essential in normal cells, could reduce such side-effects.

MTH1 is a protein that degrades oxidised nucleotides, which when incorporated into DNA cause mutations and subsequent cell death. Cancer cells have higher levels of reactive oxygen species, which create oxidised nucleotides.  In Paper I it was discovered that cancer cells are dependent on MTH1 for their survival. Crystal structures of MTH1 in complex with small molecules guided their development into potent MTH1 inhibitors, capable of killing cancer cells. Cells with increased amounts of oxidised nucleotides, or with induced hypoxia, were more susceptible to MTH1 inhibition, as shown in Paper II. In Paper III several MTH1 orthologues from organisms often used in pre-clinical studies were tested for MTH1 inhibition. Leucine 116 of mouse MTH1 was determined to be important for the lower inhibition of the developed inhibitors towards this enzyme. A virtual fragment screening study using commercial chemicals resulted in several potent MTH1 inhibitors, as shown in Paper IV. The crystal structures with the fragments or optimised inhibitors did in most cases agree with the docking pose determined from the virtual screening. In addition to the known function of MTH1 in the degradation of oxidised nucleotides, Paper V showed that MTH1 also degrades methylated nucleotides.

MTHFD2 is responsible for providing one-carbon units for nucleotide synthesis in cancer cells. As MTHFD2 is present in cancer cells but not in healthy cells, targeting the enzyme would make it possible to selectively kill cancer cells. Paper VI presents the first structure of MTHFD2, along with the first inhibitor of the protein. This information provides a starting point for the development of potent and selective MTHFD2 inhibitors.

The botulinum neurotoxin from the bacterium Clostridium Botulinum is the causative agent of the deadly disease botulism. The action of the botulinum neurotoxin on nerve cells results in paralysis, and is life-threatening if the patient is not helped with breathing support. However, low doses of the neurotoxin are used as a successful treatment for several medical conditions, such as involuntary spasms. In Paper VII the structure of two proteins, P47 and OrfX2, encoded in the gene cluster of a botulinum neurotoxin, were determined. The structures resembled tubular lipid-binding proteins, previously only found in eukaryotes. The proteins were also found to be able to bind lipids. This work gives new insight into the structure and function of this group of proteins, which help the deadly botulinum neurotoxins.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018. , p. 79
Keywords [en]
X-ray crystallography, Cancer, MTH1, oxidised nucleotides, MTHFD2, nucleotide metabolism, one-carbon metabolism, Botulism, Botulinum neurotoxin, OrfX, OrfX gene cluster
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-150688ISBN: 978-91-7797-097-2 (print)ISBN: 978-91-7797-098-9 (electronic)OAI: oai:DiVA.org:su-150688DiVA, id: diva2:1170266
Public defence
2018-02-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.

Available from: 2018-01-24 Created: 2018-01-02 Last updated: 2018-05-09Bibliographically approved
List of papers
1. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
Open this publication in new window or tab >>MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
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2014 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 508, no 7495, p. 215-221Article in journal (Refereed) Published
Abstract [en]

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102367 (URN)10.1038/nature13181 (DOI)000333979900038 ()
Available from: 2014-04-02 Created: 2014-04-03 Last updated: 2018-01-08Bibliographically approved
2. Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition
Open this publication in new window or tab >>Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition
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2016 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, no 8, p. 2366-2375Article in journal (Refereed) Published
Abstract [en]

Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1 alpha response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-130645 (URN)10.1158/0008-5472.CAN-15-2380 (DOI)000374170700031 ()
Available from: 2016-06-02 Created: 2016-05-27 Last updated: 2018-01-08Bibliographically approved
3. Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity
Open this publication in new window or tab >>Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 5, p. 593-603Article in journal (Refereed) Published
Abstract [en]

MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to non-malignant cells making cancer cells more dependent on MTH1 for survival. The possibility to specifically target cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. Progression of MTH1 inhibitors into the clinic requires animal studies and knowledge about species differences in potency of inhibitors are of vital importance. We here show that the human MTH1 inhibitor TH588 is approximately twenty fold less potent for inhibition of mouse MTH1 compared to human, rat, pig, and dog MTH1. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant for TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.

Keywords
MTH1, MutT, Cancer biology, NUDT1, Inhibitor, TH588, 8-oxo-dGTP, oxidative stress, hydrolase, nucleoside/nucleotide analogue
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-150682 (URN)10.1021/acs.biochem.7b01163 (DOI)000424723300023 ()
Funder
Swedish Research Council, 2014-5667Wenner-Gren FoundationsSwedish Cancer SocietyÅke Wiberg FoundationKnut and Alice Wallenberg Foundation
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-03-06Bibliographically approved
4. Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space
Open this publication in new window or tab >>Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space
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2017 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 19, p. 8160-8169Article in journal (Refereed) Published
Abstract [en]

Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 mu M. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.

National Category
Biological Sciences
Research subject
Biochemistry; Biochemistry towards Bioinformatics
Identifiers
urn:nbn:se:su:diva-149015 (URN)10.1021/acs.jmedchem.7b01006 (DOI)000413131400015 ()28929756 (PubMedID)
Available from: 2017-11-20 Created: 2017-11-20 Last updated: 2018-04-10Bibliographically approved
5. MutT homologue 1 (MTH1) catalyses the hydrolysis of mutagenic O6-methyl-dGTP
Open this publication in new window or tab >>MutT homologue 1 (MTH1) catalyses the hydrolysis of mutagenic O6-methyl-dGTP
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Structural Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-150683 (URN)
Funder
Swedish Research Council, 2014-5667Knut and Alice Wallenberg FoundationWenner-Gren FoundationsÅke Wiberg FoundationSwedish Cancer Society
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-01-22Bibliographically approved
6. Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor
Open this publication in new window or tab >>Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor
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2017 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 77, no 4, p. 937-948Article in journal (Refereed) Published
Abstract [en]

To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD(+) and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-141414 (URN)10.1158/0008-5472.CAN-16-1476 (DOI)000393887800014 ()27899380 (PubMedID)
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2018-01-08Bibliographically approved
7. Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster
Open this publication in new window or tab >>Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster
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2017 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 22, p. 3781-3792Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

Keywords
botulinum neurotoxin, Crystallography, gene cluster, TULIP, X-ray
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-150675 (URN)10.1002/1873-3468.12889 (DOI)000416726600008 ()
Funder
Swedish Research Council, 2014-5667Wenner-Gren FoundationsSwedish Cancer Society
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-01-08Bibliographically approved

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