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Targeting Infectious Disease: Structural and functional studies of proteins from two RNA viruses and Mycobacterium tuberculosis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (T. Alwyn Jones)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections.

Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera.

The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies.

1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1028
Keyword [en]
RNA virus, coronavirus, alphacoronavirus, nsp1, TGEV, flavivirus, Modoc virus, NS5, methyltransferase, mRNA capping, Mycobacterium tuberculosis, tuberculosis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, DXR, fosmidomycin analogues, MEP pathway, drug development, xray-crystallography
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology; Biology with specialization in Molecular Biology; Biochemistry; Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-196623ISBN: 978-91-554-8618-1 (print)OAI: oai:DiVA.org:uu-196623DiVA: diva2:610521
Public defence
2013-04-26, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research
Available from: 2013-04-05 Created: 2013-03-11 Last updated: 2013-08-30Bibliographically approved
List of papers
1. Structure of Alphacoronavirus Transmissible Gastroenteritis Virus nsp1 Has Implications for Coronavirus nsp1 Function and Evolution
Open this publication in new window or tab >>Structure of Alphacoronavirus Transmissible Gastroenteritis Virus nsp1 Has Implications for Coronavirus nsp1 Function and Evolution
2013 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 87, no 5, 2949-2955 p.Article in journal (Refereed) Published
Abstract [en]

Coronavirus nsp1 has been shown to induce suppression of host gene expression and to interfere with the host immune re- sponse. However, the mechanism is currently unknown. The only available structural information on coronavirus nsp1 is the nuclear magnetic resonance (NMR) structure of the N-terminal domain of nsp1 from severe acute respiratory syndrome corona- virus (SARS-CoV) from the betacoronavirus genus. Here we present the first nsp1 structure from an alphacoronavirus, transmis- sible gastroenteritis virus (TGEV) nsp1. It displays a six-stranded -barrel fold with a long alpha helix on the rim of the barrel, a fold shared with SARS-CoV nsp113–128. Contrary to previous speculation, the TGEV nsp1 structure suggests that coronavirus nsp1s have a common origin, despite the lack of sequence homology. However, comparisons of surface electrostatics, shape, and amino acid conservation between the alpha- and betacoronaviruses lead us to speculate that the mechanism for nsp1-induced suppression of host gene expression might be different in these two genera. 

Place, publisher, year, edition, pages
American Society for Microbiology, 2013
Keyword
Coronavirus, Alphacoronavirus, nsp1
National Category
Structural Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-196611 (URN)10.1128/JVI.03163-12 (DOI)000314876900051 ()
Available from: 2013-03-11 Created: 2013-03-11 Last updated: 2017-12-06Bibliographically approved
2. Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector
Open this publication in new window or tab >>Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector
Show others...
2009 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 65, 796-803 p.Article in journal (Refereed) Published
Abstract [en]

The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-l-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-128367 (URN)10.1107/S0907444909017260 (DOI)000268136800008 ()
Available from: 2010-07-22 Created: 2010-07-20 Last updated: 2017-12-12Bibliographically approved
3. DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues
Open this publication in new window or tab >>DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues
Show others...
2013 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 56, no 15, 6190-6199 p.Article in journal (Refereed) Published
Abstract [en]

The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway producing the universally essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the cramped substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.

Keyword
Mycobacterium tuberculosis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, DXR
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology; Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-196616 (URN)10.1021/jm4006498 (DOI)000323082400015 ()
Funder
Swedish Foundation for Strategic Research Swedish Research Council
Note

De tre (3) första författarna delar förstaförfattarskapet.

Available from: 2013-03-11 Created: 2013-03-11 Last updated: 2017-12-06Bibliographically approved

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