| 1. |
- Andaloussi, Mounir, et al.
(författare)
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Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:14, s. 4964-4976
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Tidskriftsartikel (refereegranskat)abstract
- The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.
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| 2. |
- Björkelid, Christofer, 1980-
(författare)
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Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria.The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined.The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods.The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.
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| 3. |
- Björkelid, Christofer, et al.
(författare)
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Structural and functional studies of mycobacterial IspD enzymes
- 2011
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 67, s. 403-414
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.
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| 4. |
- Björkelid, Christofer, et al.
(författare)
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Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
- 2012
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68, s. 134-143
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.
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| 5. |
- Bollati, Michela, et al.
(författare)
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Structure and functionality in flavivirus NS-proteins : perspectives for drug design
- 2010
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Ingår i: Antiviral Research. - : Elsevier BV. - 0166-3542 .- 1872-9096. ; 87:2, s. 125-148
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Forskningsöversikt (refereegranskat)abstract
- Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.
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| 6. |
- Castell, Alina, et al.
(författare)
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Structural analysis of mycobacterial branched-chain aminotransferase : implications for inhibitor design
- 2010
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 66, s. 549-557
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Tidskriftsartikel (refereegranskat)abstract
- The branched-chain aminotransferase (BCAT) of Mycobacterium tuberculosis has been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5'-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features of M. tuberculosis BCAT, X-ray structural and functional analyses of the closely related BCAT from M. smegmatis were carried out. The crystal structures include the apo form at 2.2 angstrom resolution and a 1.9 angstrom structure of the holo form cocrystallized with the inhibitor O-benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor.
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| 7. |
- Ericsson, Daniel J., et al.
(författare)
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Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
- 2010
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 16:3, s. 159-164
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.
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| 8. |
- Ingvarsson, Henrik, 1975-, et al.
(författare)
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Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:19, s. 3947-3962
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Tidskriftsartikel (refereegranskat)abstract
- Two structures of monomeric methionyl-tRNA synthetase, from Mycobacterium smegmatis, in complex with the ligands methionine/adenosine and methionine, were analyzed by X-ray crystallography at 2.3 Å and at 2.8 Å, respectively. The structures demonstrated the flexibility of the multidomain enzyme. A new conformation of the structure was identified in which the connective peptide domain bound more closely to the catalytic domain than described previously. The KMSKS(301-305) loop in our structures was in an open and inactive conformation that differed from previous structures by a rotation of the loop of about 90° around hinges located at Asn297 and Val310. The binding of adenosine to the methionyl-tRNA synthetase methionine complex caused a shift in the KMSKS domain that brought it closer to the catalytic domain. The potential use of the adenosine-binding site for inhibitor binding was evaluated and a potential binding site for a specific allosteric inhibitor was identified.
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| 9. |
- Mahalingam, A. Kannan, et al.
(författare)
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HIV-1 Protease Inhibitors with a Transition-State Mimic Comprising a Tertiary Alcohol : Improved Antiviral Activity in Cells
- 2010
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 53:2, s. 607-615
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Tidskriftsartikel (refereegranskat)abstract
- By a small modification in the core structure of the previously reported series of HIV-1 protease inhibitors that encompasses a tertiary alcohol as part of the transition-state mimicking scaffold, up to 56 times more potent compounds were obtained exhibiting EC50 values down to 3 nM. Three of the inhibitors also displayed excellent activity against selected resistant isolates of HIV-1. The synthesis of 25 new and optically pure HIV-1 protease inhibitors is reported, along with methods for elongation of the inhibitor Pl' side chain using microwave-accelerated, palladium-catalyzed cross-coupling reactions, the biological evaluation, and X-ray data obtained from one of the most potent analogues cocrystallized with both the wild type and the L63P, V82T, 184 V mutant of the HIV-1 protease.
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| 10. |
- Nurbo, Johanna, et al.
(författare)
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Novel pseudopeptides incorporating a benzodiazepine-based turn mimetic – targeting Mycobacterium tuberculosis ribonucleotide reductase
- 2013
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 21:7, s. 1992-2000
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Tidskriftsartikel (refereegranskat)abstract
- Peptides mimicking the C-terminus of the small subunit (R2) of Mycobacterium tuberculosis ribonucleotide reductase (RNR) can compete for binding to the large subunit (R1) and thus inhibit RNR activity. Moreover, it has been suggested that the binding of the R2 C-terminus is very similar in M. tuberculosis and Salmonella typhimurium. Based on modeling studies of a crystal structure of the holocomplex of the S. typhimurium enzyme, a benzodiazepine-based turn mimetic was identified and a set of novel compounds incorporating the benzodiazepine scaffold was synthesized. The compounds were evaluated in a competitive fluorescence polarization assay and in an RNR activity assay. These studies revealed that the compounds incorporating the benzodiazepine scaffold have the ability to compete for the M. tuberculosis R2 binding site with low-micromolar affinity.
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