title: Topography of the neurotensin (NT)(8-9) binding site of human NT receptor-1 probed with NT(8-13) analogs.
abstract: 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 alpha-amino acid carbon. Substitution of Arg8 with N(G)-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.
authors.affiliation: Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston 29425-2303, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Lundquist
authors.forename: E E
authors.initials: EE
authors.lastname: Büllesbach
authors.forename: P L
authors.initials: PL
authors.lastname: Golden
authors.forename: T A
authors.initials: TA
authors.lastname: Dix
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D009496:Neurotensin / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011869:Radioligand Assay; D018028:Receptors, Neurotensin / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; C114036:neurotensin type 1 receptor; D009496:Neurotensin; C054381:neurotensin (8-13); D001120:Arginine