Neurotensin receptor 1 agonist provides neuroprotection in pre-diabetic rats.

Exogenous treatment of a neurotensin receptor 1 (NTR1) agonist exerted the neuroprotection in an obese and Alzheimer's model. However, the effects of NTR1 modulation on peripheral/hippocampal impairment and cognitive deficit following sustained HFD consumption are poorly understood. Forty rats received a normal diet (ND) or HFD for 16 weeks. At week 13, the ND group received a vehicle (n = 8). Thirty-two HFD-fed group were randomized into four subgroups (n = 8/subgroup) with a vehicle, 1 mg/kg of NTR1 agonist, 1 mg/kg of NTR antagonist, and combined treatment (NTR1 agonist-NTR antagonist) for 2 weeks, s.c. injection. Then, the cognitive tests and peripheral/hippocampal parameters were determined. Our findings demonstrated that NTR1 activator reversed obesity and attenuated metabolic impairment in pre-diabetic rats. It also alleviated hippocampal pathologies and synaptic dysplasticity, leading to deceleration or prevention of cognitive impairment progression. Therefore, NTR1 activation would be a possible novel therapy to decelerate or prevent progression of neuropathology and cognitive impairment in the pre-diabetes.

Virtual screening of a natural compound library at orthosteric and allosteric binding sites of the neurotensin receptor.

Molecular dynamics (MD) simulation using the AMBER force field has been performed on the neurotensin (NT) receptor, a class A type G-protein-coupled receptor in its activated conformation co-crystallized with the non-peptide agonists. For structure-based hit molecule identification via natural chemical compound library, orthosteric sites on NT receptor have been mapped by docking using AutoDock4.0 and Vina with the known agonists and antagonists SR48692, SR142948, ML301 and ML314 of the receptor. Furthermore, clustering analysis on the MD trajectories by SIMULAID has been performed to filter receptor conformations for the allosteric binders from the Otava natural compound library. Comparative mappings of contrasting binding region patterns have been done between the crystal structure orthosteric sites as well as the binding regions in the SIMULAID-based cluster center conformations from MD trajectories with the FTmap server using the small organic molecule fragments as the probes. The distinct binding region in the cluster-based conformations in the extracellular region of the receptor has been identified for targeted docking by Otava natural chemical compound library using AutoDock4.0 and Vina docking suites to obtain putative allosteric binders. A group of compounds from the Otava library has been identified as showing high free energy in both AutoDock4.0 and Vina docking suites. Biophysical assessments on the natural compound computational hit molecules will be done to identify lead structures from the hit molecules. Communicated by Ramaswamy H. Sarma.

SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands.

The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments.

Discovery of novel antagonists of human neurotensin receptor 1 on the basis of ligand and protein structure.

Neurotensin receptor 1 (NTR1) is a cell surface receptor belonging to the G protein-coupled receptor (GPCR) A superfamily. NTR1 plays an important role in neuronal and non-neuronal systems. Using the previously identified crystal structure of rat NTR1 (rNTR1), we screened for potential candidates of human NTR1 (hNTR1) ligand. Approximately 10,000 compounds were selected using the docking score, followed by pharmacophore-based virtual screening and a two-dimensional (2D)-fingerprint structural similarity search. The identified molecules were tested by in vitro calcium flux assay. Four compounds showed micromolar level affinity, of which, one compound can inhibit hNTR1/CHO cells' proliferation by cell viability assays. To improve the affinity of these positive hit compounds, a homology model of hNTR1 was built on the basis of the crystal structure of rNTR1. The hit compounds will be further optimized on the basis of the structure of the hNTR1 receptor to be the targets for drugs directed against diseases associated with hNTR1. The results demonstrate that the method we used is valid, which will be treated as a useful tool to search for the agonists or antagonists of our interested target protein. Moreover, the compound we tested may provide a hopeful clue for treating the diseases related with hNTR1.

Label-free cell phenotypic profiling and pathway deconvolution of neurotensin receptor-1.

Neurotensin (NT), an endogenous peptide found in the central nervous system and in peripheral tissues, contributes to the pathophysiology of neurodegenerative and psychiatric diseases, cancer, inflammation, and immunomodulatory disease. NT exerts its physiological effects predominantly through its cognate high-affinity neurotensin receptor-1 (NTS1). NTS1 emerges as a druggable target; however, there are limited numbers of NTS1 active compounds reported to date. Here we reported a label-free cell phenotypic profiling model for screening NTS1 ligands and differentiating their biased agonism. Resonant waveguide grating enabled dynamic mass redistribution (DMR) assay was first optimized against cell confluency and then used to characterize the endogenous NTS1 in HT-29 cell using known agonists and antagonists. Pathway modulators were also used to deconvolute the signaling pathways of endogenous NTS1. Results showed that the NTS1 DMR assay is robust for screening and can differentiate biased agonism; and the activation of NTS1 in HT-29 triggers multiple pathways including Gq signaling and epidermal growth factor receptor transactivation. This study highlighted the power of label-free DMR assay to characterize receptor signaling and pharmacology of distinct classes of ligands for NTS1, G protein-coupled receptors in general.

Hypothalamic neurotensin projections promote reward by enhancing glutamate transmission in the VTA.

The lateral hypothalamus (LH) sends a dense glutamatergic and peptidergic projection to dopamine neurons in the ventral tegmental area (VTA), a cell group known to promote reinforcement and aspects of reward. The role of the LH to VTA projection in reward-seeking behavior can be informed by using optogenetic techniques to dissociate the actions of LH neurons from those of other descending forebrain inputs to the VTA. In the present study, we identify the effect of neurotensin (NT), one of the most abundant peptides in the LH to VTA projection, on excitatory synaptic transmission in the VTA and reward-seeking behavior. Mice displayed robust intracranial self-stimulation of LH to VTA fibers, an operant behavior mediated by NT 1 receptors (Nts1) and NMDA receptors. Whole-cell patch-clamp recordings of VTA dopamine neurons demonstrated that NT (10 nm) potentiated NMDA-mediated EPSCs via Nts1. Results suggest that NT release from the LH into the VTA activates Nts1, thereby potentiating NMDA-mediated EPSCs and promoting reward. The striking behavioral and electrophysiological effects of NT and glutamate highlight the LH to VTA pathway as an important component of reward.

The role of endogenous neurotensin in psychostimulant-induced disruption of prepulse inhibition and locomotion.

The neuropeptide neurotensin (NT) is closely associated with dopaminergic and glutamatergic systems in the rat brain. Central injection of NT into the nucleus accumbens (NAcc) or peripheral administration of NT receptor agonists, reduces many of the behavioral effects of psychostimulants. However, the role of endogenous NT in the behavioral effects of psychostimulants (e.g. DA agonists and NMDA receptor antagonists) remains unclear. Using a NTR antagonist, SR142948A, the current studies were designed to examine the role of endogenous NT in DA receptor agonist- and NMDA receptor antagonist-induced disruption of prepulse inhibition of the acoustic startle response (PPI), locomotor hyperactivity and brain-region specific c-fos mRNA expression. Adult male rats received a single i.p. injection of SR142948A or vehicle followed by D-amphetamine, apomorphine or dizocilpine challenge. SR142948A had no effect on baseline PPI, but dose-dependently attenuated d-amphetamine- and dizocilpine-induced PPI disruption and enhanced apomorphine-induced PPI disruption. SR142948A did not significantly affect either baseline locomotor activity or stimulant-induced hyperlocomotion. Systemic SR142948A administration prevented c-fos mRNA induction in mesolimbic terminal fields (prefrontal cortex, lateral septum, NAcc, ventral subiculum) induced by all three psychostimulants implicating the VTA as the site for NT modulation of stimulant-induced PPI disruption. Further characterization of the NT system may be valuable to find clinical useful compounds for schizophrenia and drug addiction.

Design and in vitro evaluation of branched peptide conjugates: turning nonspecific cytotoxic drugs into tumor-selective agents.

The use of peptide receptors as targets for tumor-selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo-branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor-specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug-conjugated branched NT peptides. We present results obtained with oligo-branched neurotensin peptides conjugated to 6-mercaptopurin (6-MP), combretastain A-4 (CA4) and monastrol (MON). Drugs were conjugated to oligo-branched neurotensin through different linkers, and the mode-of-release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug-armed branched peptides, NT4-CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide-drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.

Endogenous neurotensin is involved in estrous cycle related alterations in prepulse inhibition of the acoustic startle reflex in female rats.

Ovarian hormones regulate prepulse inhibition (PPI) of the acoustic startle reflex. Results from studies in intact female rodents investigating sex, estrous cycle and ovarian hormone regulation of PPI are inconsistent. In experiment #1, we investigated whether PPI in female rats is influenced by the time of day of testing and the estrous cycle stage of the rat. PPI was examined across the day of proestrus (P) and diestrus 1 (D1) in female rats and compared to males. PPI in males and P females was significantly higher than in D1 females. PPI in males and D1 females was significantly affected by the time of day of testing with PPI being reduced in the afternoon and evening compared to morning. PPI in P females was not significantly affected by the time of day of testing. Previous studies have demonstrated estrous cycle regulation of central nervous system neurotensin (NT) neurons and peripherally administered NT receptor agonists regulate PPI in a manner similar to antipsychotic drugs. Experiment #2 of this study was designed to examine whether endogenous NT is involved in estrous cycle regulation of PPI. The NT receptor antagonist SR 142948A reduced the high levels of PPI during D1 and P. In contrast, when tested at a time of day in which PPI was low in D1 females, administration of both the typical antipsychotic drug haloperidol and the NT receptor antagonist significantly increased PPI. These data support an effect of time of day and estrous cycle stage on PPI in female rats. The estrous cycle variations in PPI are mediated in part by endogenous NT.

Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells.

The binding and signaling properties of neuronal NTS2 neurotensin (NT) receptors were examined in cultured rat cerebellar granule cells. As shown by reverse transcription-PCR, receptor autoradiography, and confocal microscopic localization of fluorescent NT, these cells selectively express the NTS2 receptor subtype. Accordingly, a single apparent class of (125)I-NT-binding sites, with an affinity of 3.1 nm, was detected in cerebellar granule cell cultures. This binding was competed for with high affinity (IC(50) = 5.7 nm) by the NTS2 ligand levocabastine and with low affinity (IC(50) = 203 nm) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound (125)I-NT was internalized at equilibrium via a clathrin-dependent pathway. In cells loaded with the Ca(2+)-sensitive fluorescent dye Fluo4, SR48692, but neither NT nor levocabastine, triggered a marked increase in cytosolic [Ca(2+)](i). By contrast, both NT and levocabastine, but not SR48692, induced a sustained (>60 min) activation of the mitogen-activated protein kinases, p42/p44, indicating functional coupling of NTS2 receptors. Complementary experiments carried out on synaptosomes from adult rat cerebellum demonstrated the presence of presynaptic NTS2 receptors. However, in contrast to perikaryal NTS2 sites, these presynaptic receptors did not internalize in response to NT stimulation. Taken together, the present results demonstrate that NTS2 receptors are present both presynaptically and postsynaptically in central neurons and that NT and levocabastine act as agonists on these receptors.

Enhanced neurotensin neurotransmission is involved in the clinically relevant behavioral effects of antipsychotic drugs: evidence from animal models of sensorimotor gating.

To date, none of the available antipsychotic drugs are curative, all have significant side-effect potential, and a receptor-binding profile predictive of superior therapeutic ability has not been determined. It has become increasingly clear that schizophrenia does not result from the dysfunction of a single neurotransmitter system, but rather from an imbalance between several interacting systems. Targeting neuropeptide neuromodulator systems that concertedly regulate all affected neurotransmitter systems could be a promising novel therapeutic approach for schizophrenia. A considerable database is concordant with the hypothesis that antipsychotic drugs act, at least in part, by increasing the synthesis and release of the neuropeptide neurotensin (NT). In this report, we demonstrate that NT neurotransmission is critically involved in the behavioral effects of antipsychotic drugs in two models of antipsychotic drug activity: disrupted prepulse inhibition of the acoustic startle response (PPI) and the latent inhibition (LI) paradigm. Blockade of NT neurotransmission using the NT receptor antagonist 2-[[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methylcarbamoyl)-2-isopropylphenyl)-1H- pyrazole-3-carbonyl]-amino]-adamantane-2-carboxylic acid, hydrochloride (SR 142948A) prevented the normal acquisition of LI and haloperidol-induced enhancement of LI. In addition, SR 142948A blocked the PPI-restoring effects of haloperidol and the atypical antipsychotic drug quetiapine in isolation-reared animals deficient in PPI. We also provide evidence of deficient NT neurotransmission as well as a left-shifted antipsychotic drug dose-response curve in isolation-reared rats. These novel findings, together with previous observations, suggest that neurotensin receptor agonists may represent a novel class of antipsychotic drugs.

Ionic mechanisms of action of neurotensin in acutely dissociated neurons from the diagonal band of Broca of the rat.

Whole cell recordings were performed on acutely dissociated neurons from the horizontal limb of the diagonal band of Broca (hDBB) from rats to elucidate the ionic mechanisms of action of neurotensin. Neurotensin caused a decrease in whole cell voltage-activated outward currents and failed to elicit a response when Ca2+ influx was blocked by changing the external solution to the one containing 0 mM Ca2+ and 50 microM Cd2+, suggesting the involvement of Ca2+-dependent conductances. Charybdotoxin, a specific blocker of voltage-sensitive calcium-activated K+ channels (IC), caused a decrease in outward currents comparable with that caused by blocking calcium influx and occluded the neurotensin-induced decrease in outward currents. Similarly, 50 microM tetraethylammonium ions also blocked the neurotensin response. Also neurotensin reduced whole cell barium currents (IBa) and calcium currents (ICa). Amiloride and omega-conotoxin GVIA, but not nimodipine, were able to eliminate the neurotensin-induced decrease in IBa. Thus T- and N- but not L-type calcium channels are subject to modulation by neurotensin, and this may account for its effects on IC. The predicted changes in action potential as a result of the blockade of currents through calcium channels culminating into changes in IC were confirmed in the bridge current-clamp recordings. Specifically, neurotensin application led to depolarization of the resting membrane potential, broadening of spike and a decrease in afterhyperpolarization and accommodation. These alterations in action potential characteristics that resulted in increased firing rate and excitability of the hDBB neurons also were produced by application of charybdotoxin. Neurotensin effects on these properties were occluded by 2 - [(1 - 7 - chloro - 4 - quinolinyl) - 5 - (2, 6 - di - methoxyphenyl) pyrazol-3-yl) carbonylamino] tricyclo (3.3.1.1.)decan-2-carboxylic acid, a nonpeptide high-affinity neurotensin receptor antagonist. Neurotensin blockade of IC, possibly through ICa, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Stimulation by neurotensin of (3H)5-hydroxytryptamine (5HT) release from rat frontal cortex slices.

The effect of neurotensin (NT) on the K+-evoked (3H)5HT release from brain frontal cortex slices was studied in rats. NT(1-13) and NT(8-13) increased (3H)5HT release with EC50 values in the nanomolar range and Emax values in the range of 100% of control, whereas D-tyr11-NT was inactive. Concerning NT receptor antagonists, SR 48692 and SR 142948A antagonized with IC50 values of 4.8+/-1.8 nM and 4.5+/-1.8 nM respectively, the NT stimulated K+-evoked (3H)5HT release. SR 48527 also antagonized NT induced (3H)5HT release with an IC50 value of 0.95+/-0.06 nM whereas the inactive R(-) enantiomer SR 49711 only inhibited this effect with IC50 value close to 10(-6)M. The 5HT-releasing effect of NT was completely inhibited by tetrodotoxin suggesting that NT receptors involved in the control of 5-HT release are not located on 5-HT terminals. After a first NT (10(-7)M) application, the NT (10(-7)M, 10(-6)M) effect under K+ depolarization was drastically decreased, indicating that the NT receptor could be desensitized. No potentiating effect of NT on K+-evoked (3H)5HT release was observed in striatal and hippocampal slices. These results suggest that, in the rat frontal cortex, NT regulates 5HT release through a high affinity NT receptor not associated with 5HT terminals.

Blockade of neurotensin binding in the rat hypothalamus and of the central action of neurotensin on the hypothalamic-pituitary-adrenal axis with non-peptide receptor antagonists.

Central administration of neurotensin (NT) has been shown to activate the hypothalamic-pituitary-adrenal axis, an effect which seems dependent upon the release of corticotropin-releasing factor. In this study, we describe the distribution of NT binding sites in the hypothalamus using film and emulsion receptor autoradiography. Among the 125I-NT-labelled hypothalamic nuclei, relatively high densities of neurotensin binding sites were detected over the paraventricular nucleus. Silver grains on emulsion-coated slides overlaid indiscriminately cell bodies and surrounding processes of magnocellular and parvocellular parts of the nucleus. Two newly developed NT receptor antagonists, SR 48692 and its analog SR 48450, competed for 125I-NT binding to hypothalamic tissue sections and membrane preparations with Ki values in the nanomolar range. Moreover, intracerebrally injected SR 48450 was able to block the NT-induced hypothalamic-pituitary-adrenal axis activation in freely moving rats, whereas its administration alone did not significantly affect basal plasma levels of adrenocorticotropin and corticosterone. These data provide anatomical substrate for a potential neurotensin action at the hypothalamus in the hypothalamic-pituitary-adrenal axis activation and highlight the use of new non-peptide NT receptor antagonists to characterize the effects of NT on neuroendrocrine functions.