Re-evaluation of Cyclic Peptide Binding to Neurotensin Receptor 1 by Shifting the Peptide Register during Synthesis

The head-to-tail cyclic peptide <i>cyclo</i>[Arg-Lys-Pro-Tyr-Tle-Leu] (peptide <b>1</b>, where Tle is l-<i>tert</i>-Leu) has previously been reported to bind to neurotensin receptor 1 (NTS1) (pKi = 5.97). Upon seeking to reproduce this finding, we found that peptide <b>1</b> did not have a measurable affinity for NTS1. However, a semipurified preparation of peptide <b>1</b> appeared to bind to NTS1 with pKi = 5.83 ± 0.25 SEM. Resynthesis of peptide <b>1</b> using a shifted peptide register gave linear and cyclic forms of peptide <b>1</b> that were both unable to bind to NTS1. We observe that the previously reported activity of peptide <b>1</b> may be due to the presence of high affinity linear contaminants. Approximately 3% contamination with the linear variant would explain the apparent binding of the semipure peptide <b>1</b> sample. From this study, we propose that shifting the peptide register during synthesis as a strategy to minimize the presence of potent precursor contaminants.