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 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