neurotensin and neurotensin-(8-13)

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( K

Topics: Analgesics, Non-Narcotic; Animals; Binding, Competitive; Blood Pressure; Body Temperature; CHO Cells; Cricetulus; Cyclization; Drug Evaluation, Preclinical; Drug Stability; Male; Molecular Docking Simulation; Neurotensin; Peptide Fragments; Peptides, Cyclic; Rats, Sprague-Dawley; Receptors, Neurotensin; Structure-Activity Relationship; Tyrosine

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Topics: Angiotensin II; Arginine; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Ligands; Molecular Structure; Neuropeptide Y; Neurotensin; Oligopeptides; Peptide Fragments; Receptors, Angiotensin; Receptors, Neuropeptide; Receptors, Neurotensin; Structure-Activity Relationship

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Topics: Aminopeptidases; Animals; Binding, Competitive; Body Temperature; Calcium; Cell Line; Humans; In Vitro Techniques; Intracellular Space; Male; Neurotensin; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, Neurotensin; Structure-Activity Relationship

The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 [3,1'-naphthyl-l-Ala(11)]NT(8-13) has greater than 60-fold higher affinity for rNTR1 (46 and 60 pM for transiently- and stably-transfected cells, respectively) than for hNTR1 (2.8 and 5.8 nM for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of rNTR1 and hNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr(339) in the pocket center, as compared with the corresponding residue Phe(344) in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of rNTR1(F344Y) and hNTR1(Y339F). Examination of the mutant receptors from membranal preparations of transfected cells in radioligand binding assays and with intact cells in functional assays (phosphatidyl-4,5-bisphosphate turnover) showed that the human-like rat receptor and the rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century.

Topics: Amino Acid Substitution; Animals; Cells, Cultured; CHO Cells; Cricetinae; Humans; Mutagenesis, Site-Directed; Neurotensin; Peptide Fragments; Radioligand Assay; Rats; Receptors, Neurotensin; Transfection

Ejections of 10(-5)-10(-3)M neurotensin into the ventral tegmental area increased dopamine efflux measured by electrochemical approaches in the prefrontal cortex of anaesthetized rats. In the same conditions, the effects evoked on dopamine efflux by 10(-5)M neurotensin(8-13) and [D-Tyr(11)]neurotensin were different from each other and depended on the explored area: the prefrontal cortex and the caudal and rostral nucleus accumbens. In the prefrontal cortex, neurotensin(8-13) was as potent as neurotensin, whereas [D-Tyr(11)]neurotensin was ineffective. In the caudal nucleus accumbens, when considering the initial intensity of the effect, neurotensin(8-13) and neurotensin appeared more potent than [D-Tyr(11)]neurotensin. In contrast, in the rostral nucleus accumbens, neurotensin(8-13) was less potent than [D-Tyr(11)]neurotensin and neurotensin. These results support the differential involvement of two pharmacologically distinct neurotensin receptor entities on ventral tegmental area neurons in the modulation of mesolimbic and mesocortical dopaminergic activity.

Topics: Animals; Dopamine; Electrochemistry; Extracellular Space; Male; Microinjections; Neurotensin; Nucleus Accumbens; Peptide Fragments; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Ventral Tegmental Area

Microinjection of neurotensin(1-13) or neurotensin(8-13) into the ventral tegmental area (VTA) of anaesthetized rats produced dose-dependent (1-100 pg) dopamine release in the nucleus accumbens as measured by differential pulse amperometry (DPA). Higher doses (100 pg-10 ng) of [D-Tyr11]neurotensin were required to produce an identical effect. In addition, the 3 peptides enhanced the K(+)-evoked [3H]DA release from nucleus accumbens slices. The stimulatory actions produced by 10(-8) M neurotensin(1-13) and neurotensin(8-13) were respectively of 96% and 72% while the effect of [D-Tyr11]neurotensin was only of 79% at 10(-6) M. Unilateral application of the 3 peptides in the VTA of cannulated rats produced contralateral circling. [D-Tyr11]neurotensin was effective in a dose-dependent manner, between 40 and 320 ng. Similar effects were observed with 80 ng of neurotensin(1-13) and neurotensin(8-13) in presence of the protease inhibitor thiorphan. In view of the higher potency of neurotensin(1-13) and neurotensin(8-13) versus [D-Tyr11]neurotensin to stimulate DA release both in vivo and in vitro and the higher efficacy of [D-Tyr11]neurotensin to induce circling, this study further strengthens the concept of neurotensin receptor heterogeneity.

Topics: Animals; Behavior, Animal; Dopamine; Dose-Response Relationship, Drug; Male; Neurotensin; Nucleus Accumbens; Peptide Fragments; Rats; Rats, Sprague-Dawley; Stereoisomerism; Ventral Tegmental Area