| 1. |
- Jansson, Anna M., 1979-
(författare)
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Targeting Infectious Disease : Structural and functional studies of proteins from two RNA viruses and Mycobacterium tuberculosis
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Henriksson, Lena M, 1977-
(författare)
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Structural and Functional Studies of Peptidyl-prolyl cis-trans isomerase A and 1-deoxy-D-xylulose- 5-phosphate reductoisomerase from Mycobacterium tuberculosis
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.
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| 3. |
- Roos, Annette K., 1978-
(författare)
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Structural and Functional Studies of Ribose-5-phosphate isomerase B
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribose 5-phosphate isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view.Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.
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