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

The head-to-tail cyclic peptide cyclo[Arg-Lys-Pro-Tyr-Tle-Leu] (peptide 1, where Tle is l-tert-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 1 did not have a measurable affinity for NTS1. However, a semipurified preparation of peptide 1 appeared to bind to NTS1 with pKi = 5.83 ± 0.25 SEM. Resynthesis of peptide 1 using a shifted peptide register gave linear and cyclic forms of peptide 1 that were both unable to bind to NTS1. We observe that the previously reported activity of peptide 1 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 1 sample. From this study, we propose that shifting the peptide register during synthesis as a strategy to minimize the presence of potent precursor contaminants.