Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7001
>0.1 ppm were considered as significant59 and were used as an
indication of structural perturbation upon amino acid muta-
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Radioligand Binding Studies. For radioligand binding studies,
membranes from transfected COS-7 cells were incubated in
96-well plates with [3H]nisoxetine (4.3 nM) in the absence or
presence of χ-conopeptide (1 nM to 100 μM, in triplicate) in
buffer for 1 h at RT, as previously described.15 Filter-retained
radioactivity was quantified by liquid scintillation counting.
Curve fitting of concentration-response data was performed
by nonlinear regression using individual data points with
GraphPad Prism 3.0. The equation of Cheng and Prusoff60
was used to convert IC50 values for [3H]nisoxetine displacement
to pKi values, since χ-conopeptides act competitively with this
ligand.9 Statistical analyses were undertaken using the Graph-
Pad Prism software package with a statistical significance
criterion of P < 0.05.
In Vivo Studies. The CCI rat model of neuropathic pain was
performed as described in Nielsen et al.10 Three animals were
used for each analogue tested, with dose selected on the basis of
potency to inhibit the NET transport of NE.
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aspects. Diabetes/Metab. Res. Rev. 2008, 24 (S1), S52–S57.
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R. M. Protein folding determinants: structural features determin-
ing alternative disulfide pairing in R- and χ/λ-conotoxins. Biochem-
istry 2007, 46, 3338–3355.
(24) Nilsson, K. P. R.; Lovelace, E. S.; Caesar, C. E.; Tynngard, N.;
Alewood, P. F.; Johansson, H. M.; Sharpe, I. A.; Lewis, R. J.; Daly,
N. L.; Craik, D. J. Solution structure of χ-conopeptide MrIA, a
modulator of the human norepinephrine transporter. Pept. Sci.
2005, 80, 815–823 (PDB 2ew4) .
Acknowledgment. We thank Norelle L. Daly for assistance
in generation of the NMR structure and related graphics. This
work was supported in part by an AusIndustry START Grant
and an NHMRC Program Grant.
Supporting Information Available: HPLC purity data of all
peptides, details of peptide quantification used in the determi-
nation of concentration used in in vitro assays, buffer and
plasma stability data, receptor selectivity data of 3, and in vitro
functional assay results for 3. This material is available free of
(25) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. An efficient meth-
od for anchoring Fmoc-amino acids to hydoxyl-functionalised
solid supports. Tetrahedron Lett. 1990, 31, 1701–1704.
(26) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. In
situ neutralization in Boc-chemistry solid phase peptide synthesis.
Rapid, high yield assembly of difficult sequences. Int. J. Pept.
Protein Res. 1992, 40, 180–193.
(27) Blankenship, J. W.; Balambika, R.; Dawson, P. E. Probing back-
bone hydrogen bonds in the hydrophobic core of GCN4. Biochem-
istry 2002, 41, 15676–15684.
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€
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