Topography of the neurotensin (NT)(8−9) binding site of human NT receptor-1 probed with NT(8-13) analogs

• Published in: The journal of peptide research Vol. 59; no. 2; pp. 55 - 61
• Main Authors: Lundquist IV, J.T., Büllesbach, E.E., Golden, P.L., Dix, T.A.
• Format: Journal Article
• Language: English
• Published: Copenhagen, Denmark Munksgaard 01-02-2002
• Subjects: Amino Acid Substitution; Amino Acids; Animals; Arginine; Binding Sites; CHO Cells; Cricetinae; electrostatics; homolysine; Humans; Kinetics; Neurotensin - metabolism; neurotensin receptor; neurotensin(8-13); non-natural amino acids; Peptide Fragments - chemical synthesis; Peptide Fragments - metabolism; Radioligand Assay; Receptors, Neurotensin - chemistry; Receptors, Neurotensin - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; sterics; Transfection
• ISSN: 1397-002X; 1399-3011
• DOI: 10.1046/j.1397-002x.2001.10946.x

:  A series of neurotensin (NT)(8–13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non‐natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor‐1 (hNTR‐1). The modifications were designed to probe specific steric and electrostatic requirements in the N‐terminal cationic region of NT(8–13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non‐natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the α–amino acid carbon. Substitution of Arg8 with NG‐alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8–13) to the hNTR‐1. Position 8 incorporation of Hlys produced the most favorable primary amine side‐chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8–13), in which DAB is 2,4‐diaminobutyric acid, was also prepared and tested as the shorter side‐chain of DAB is known to be favored in position 9 of NT(8–13). This analog maintained 60% of NT(8–13) binding affinity making it the most favored des‐guanidinium‐containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR‐1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8–13) and related analogs.