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Applications of paramagnetic NMR spectroscopy in drug discovery

Chen, Wan-Na

Description

NMR spectroscopy of proteins with paramagnetic metal ions, first performed with metalloproteins, is a unique technique to obtain long-range distance information for three-dimensional structure determinations. This thesis focuses on developing applications of paramagnetic NMR spectroscopy, particularly pseudocontact shifts, in drug discovery. The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) from serotype 2 is a well-established drug target, but...[Show more]

dc.contributor.authorChen, Wan-Na
dc.date.accessioned2016-06-05T23:36:04Z
dc.identifier.otherb39905299
dc.identifier.urihttp://hdl.handle.net/1885/101990
dc.description.abstractNMR spectroscopy of proteins with paramagnetic metal ions, first performed with metalloproteins, is a unique technique to obtain long-range distance information for three-dimensional structure determinations. This thesis focuses on developing applications of paramagnetic NMR spectroscopy, particularly pseudocontact shifts, in drug discovery. The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) from serotype 2 is a well-established drug target, but drug development has been hampered for many years by lack of structural information. In earlier work, pseudocontact shifts (PCSs) induced by lanthanide binding tags at multiple sites had successfully been used to determine the closed conformation of NS2B in the presence of a small inhibitor molecule. Subsequently, PCSs were used to prove that an unlinked construct of NS2B-NS3pro exists predominately in the closed conformation in solution, showing that the open conformation observed previously is an artefact generated by a covalent link between NS2B and NS3 (Paper 1). Next, PCSs generated for NS2B, NS3pro and bovine pancreatic trypsin inhibitor (BPTI) were used to show that the C-terminal segment of NS2B remains in the closed conformation in the presence of BPTI, correcting a crystallographic artefact (Paper 2). The work described in Papers 1 and 2 confirmed that the closed conformation of dengue virus NS2B-NS3pro is the best model for structure-guided drug design. As the sensitivity of NMR spectra of dynamic proteins, such as the dengue virus protease, is compromised by excessive line broadening, alternative NMR tags were sought. O-tert-butyltyrosine incorporated in proteins proved to be an outstanding NMR probe for conformational studies of high-molecular-weight systems and measurement of submicromolar ligand binding affinities in one-dimensional 1H-NMR spectra without any isotope labelling (Paper 3). A tert-butyl probe was also introduced into a tightly binding lead compound against the dengue virus protease. Measurement of ligand PCSs from intense intramolecular NOESY cross-peaks with the tert-butyl group allowed positioning of the ligand on the protein with respect to the paramagnetic centre, while strong intermolecular NOEs validated the structural model of the complex established with the use of PCSs (Paper 4). In summary, the paramagnetic NMR approach, demonstrated on the dengue virus NS2B-NS3 protease, presents a broadly applicable and elegant way for structure-guided drug design at atomic resolution.
dc.language.isoen
dc.subjectNMR spectroscopy
dc.subjectpseudocontact shifts
dc.subjectdengue virus NS2B-NS3 protease
dc.subjecttert-butyl group
dc.titleApplications of paramagnetic NMR spectroscopy in drug discovery
dc.typeThesis (PhD)
local.contributor.supervisorOtting, Gottfried
local.contributor.supervisorcontactgottfried.otting@anu.edu.au
dcterms.valid2016
local.type.degreeDoctor of Philosophy (PhD)
dc.date.issued2016
local.contributor.affiliationResearch School of Chemistry, College of Physical and Mathematical Sciences, The Australian National University
local.identifier.doi10.25911/5d78d57314dc7
dc.provenance6.2.2020 - Made open access after no response to emails re: extending restriction.
local.mintdoimint
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