Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8–13) analog
Élie Besserer‐OffroyPascal TétreaultRebecca L. BrouilletteAdeline RenéAlexandre MurzaRoberto FanelliKaryn KirbyAlexandre J. ParentIsabelle DubucNicolas BeaudetJérôme CôtéJean‐Michel LongpréJean MartínezFlorine CavelierPhilippe Sarret
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Abstract:
Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8-13) (NT(8-13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8-13). We first examined the binding affinities of this novel NT(8-13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article "Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog" published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.Keywords:
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Neurotensin and several sequence analogues have been synthesized using solid‐phase technology. The purity of the following derivatives: neurotensin, neurotensin‐(10–13), neurotensin‐(9–13). neurotensin‐(8–13), neurotensin‐(6–13), neurotensin‐(4–13), [Cit 8 ]neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Cit 9 ]neurotensin‐(8–13),[Lys 9 ]neurotensin‐(8–13), [Phe 11 ]neurotensin‐(8–13), [Ala 12 ]neurotensin‐(8–13) and [Ala 13 ]‐ neurotensin‐(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse‐phase high‐ performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N‐acetylation of neurotensin‐(6–13), neurotensin‐(8–13), [Cit 8 ]neurotensin‐(8–13), [Cit 9 ]‐ neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Lys 9 ]neurotensin‐(8–13) and [Phe 11 ]neurotensin‐(8–13), as well as native xenopsin, were all tested for binding competition with [ 3 H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C‐terminal region analogues up to the size of neurotensin‐ (8–13). The two peptides neurotensin‐(6‐ 13) and neurotensin‐(4–13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C‐terminal peptide ‐Arg‐Arg‐Pro‐Tyr‐Ile‐Leu fulfills all the structural requirements for mimicking the entire sequence, provided its α‐amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin‐(8–13), acts similarly to acetyl‐neurotensin‐(8–13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C‐terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.
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Abstract: The binding of [ 3 H]neurotensin(8–13) to membranes from human frontal cortex at 0°C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant ( K D ) of 0.52 n M , and the maximal number of binding sites ( B max) was 3.5 pmol/g original wet weight of tissue. Scat‐chard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [ 3 H]‐neurotensin(8–13) bound to single, noncooperative sites. The K D values of several analogs of neurotensin determined in competition with [ 3 H]neurotensin(8–13) were similar to those previously determined in competition with [ 3 H]‐neurotensin. The regional distribution of binding sites for [ 3 H]neurotensin(8–13) was also similar to that for [ 3 H]‐neurotensin. These results suggest that [ 3 H]neurotensin(8–13) binds to the same sites as [ 3 H]neurotensin and that [ 3 H]neurotensin(8–13) has a higher affinity than [ 3 H]‐neurotensin for these sites in human brain.
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SUMMARY 1. Neurotensin is released from the intestine into the portal circulation and to exert a systemic effect it must traverse the liver intact. 2. The role of the liver in neurotensin clearance was examined using the isolated perfused rat liver preparation. Two concentrations of neurotensin were used to determine the extraction capacity of the liver. 3. Approximately 10% of the added neurotensin (with either dose) was extracted in a single pass through the liver. This extraction rate was low when compared to previous studies with cholecystokinin (60% extraction in a single pass) and vasoactive intestinal peptide (100%). 4. It is concluded that there is a small but high capacity for direct extraction of neurotensin. This low direct extraction percentage supports our previous contention that the major influence of the liver on the metabolism of neurotensin is by the release of neurotensin degrading peptidases into the circulation.
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The role of neurotensin in radiation-induced hypothermia was examined. Intracerebroventricular (ICV) administration of neurotensin produced dose-dependent hypothermia. Histamine appears to mediate neurotensin-induced hypothermia because the mast cell stabilizer disodium cromoglycate and antihistamines blocked the hypothermic effects of neurotensin. An ICV pretreatment with neurotensin antibody attenuated neurotensin-induced hypothermia, but did not attenuate radiation-induced hypothermia, suggesting that radiation-induced hypothermia was not mediated by neurotensin.
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Serotonin 5-HT2B receptors are often coexpressed with 5-HT1B receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT1B and 5-HT2B receptor cross-talk have not been elucidated. We hypothesized that 5-HT2B and 5-HT1B receptors each affect the others9 signaling by modulating the others9 trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT2B and 5-HT1B receptors when expressed alone and upon coexpression in LMTK– murine fibroblasts. Time-lapse confocal microscopy and whole-cell radioligand binding analyses revealed that, when expressed alone, 5-HT2B and 5-HT1B receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT2B receptors was accelerated 5-fold and was insensitive to a 5-HT2B receptor antagonist. In this context, 5-HT2B receptors did internalize in response to a 5-HT1B receptor agonist. In contrast, co-expression did not render 5-HT1B receptor internalization sensitive to a 5-HT2B receptor agonist. The altered internalization kinetics of both receptors upon coexpression was probably not due to direct interaction because only low levels of colocalization were observed. Antibody knockdown experiments revealed that internalization of 5-HT1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT2B receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT1B receptor internalization became entirely Caveolin1-independent in a protein kinase Cϵ-dependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT1B and 5-HT2B receptors influences the internalization pathways and kinetics of both receptors.
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Antibody-drug conjugates (ADCs) have promising potential as an effective therapeutic agent for various cancer therapies. Antigen-mediated internalization induces the delivery of ADCs into cancer cells, resulting in activation of the attached cytotoxic agents. The internalization and internalization efficiency of ADC are critical for its anti-cancer efficacy. How to verify the internalization and the internalization efficiency of ADCs could be very useful to identify appropriate antibody candidates with internalization characteristics favorable for ADCs. This chapter describes the experiments for evaluating internalization and strategies to improve internalization efficiency of ADCs, which would benefit for the identification and development of next generation ADCs in the future.
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