Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8–13) analog

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.

placement of a proline in the native NT (8)(9)(10)(11)(12)(13). We first examined the binding affinities of this novel NT (8)(9)(10)(11)(12)(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.

Description of data collection
Radioactivity counts retained on GF/C filters were counted on a γ -counter.
Filtered luminescence readings of BRET experiments were recorded in endpoint readout using a multimode plate reader equipped with a BRET2 filter set. Optical density (absorbance) of the colorimetric reaction for cell-surface ELISA was recorded in endpoint readout using a multimode plate reader using a 450 nm filter. Western blots for phosphorylation of p42/p44 were revealed using an enhanced chemiluminescence detection with high sensitivity films.
( continued on next page ) Remaining intact peptide in plasma stability assay was quantified using an internal standard and UPLC/MS system. Graphs, data normalization, and non-linear regression fits were done using GraphPad Prism v7.0a. Western blots for phosphorylation of p42/p44 were revealed using an enhanced chemiluminescence detection with high sensitivity films. Remaining intact peptide in plasma stability assay was quantified using an internal standard and UPLC/MS system. Graphs, data normalization, and non-linear regression fits were done using GraphPad Prism v7.0a.

Data source location
Institut

Value of the Data
• These data characterize the in vitro and in cellulo behavior of a novel neurotensinergic compound with analgesic properties.
• These data provide insights into different G protein activation and β-arrestin recruitment on the NTS1 receptor and functional assay on the NTS2 receptor including p42/p44 phosphorylation and receptor internalization. • These data provide insights into the molecular mechanisms underlying the action of JMV2009

Data description
This article describes the data that are analysed, interpreted, and discussed in Tétreault et al. [1] . Raw data are made freely available at https://doi.org/10.6084/m9.figshare.11962689 .

JMV2009 synthesis and chemical characterization
The hexapeptide JMV2009 ( Scheme 1 ) was synthesized by solide-phase method using Wang resin preloaded with Leucine residue ( Fig. 1 ) as described in Section 2.2 . below. The 9fluorenylmethyloxycarbonyl (Fmoc) protection was used as temporary protection of the Nterminal amino groups, and N-tert-Butyloxycarbonyl (Boc) and tert -Butyl (tBu) were used as orthogonal side-chain protections. Couplings of protected amino acids were carried out with a solution of HBTU/HOBt reagents. The unnatural amino acid Silaproline (Sip) has been synthetized as previously described [ 2 , 3 ], Fmoc-protected, and incorporated in the automated synthesis as other natural amino acid. The use of Wang resin allowed peptide release from the resin and the deprotection of side chains of the desired protected peptide with TFA in the presence of anisole as scavenger. The resulting peptide JMV 2009 was purified by preparative reverse-phase HPLC on a C 18 column and its purity and structure were confirmed by HPLC-UV and ESI mass spectrometry, respectively ( Fig. 2 ).

JMV2009 binding at NT receptors
We first evaluated the binding affinities of this new analog on both NTS1 and NTS2 receptors. Binding experiments of neurotensin (NT) and JMV2009 were carried out on freshly prepared Scheme 1. Chemical structure of JMV2009.

Table 1
Binding affinities of NT and JMV2009.
Values are expressed as IC 50 ± SEM of at least three independent determinations. membranes of CHO-K1 cells expressing the human NTS1 receptor or 1321N1 cells expressing the human NTS2 receptor as previously described [4] . Concentration-displacement curves ( Fig. 3 ) were used to fit a non-linear regression model in Graphpad Prism and determine the IC 50 values for NT and JMV2009 ( Table 1 ).

JMV2009 plasma stability
Finally, we assessed the plasma stability of this novel neurotensin-like compound bearing a proline substitute. We incubated JMV2009 for various time points in rat plasma, and after protein precipitation and centrifugation, the intact remaining peptide was dosed by HPLC/UV-MS. We observed that JMV2009 possesses a plasma half-life of 6.24 ± 2.9 min, compared to 1.49 ± 0.4 min for the native NT ( Fig. 4 ).

Signalling signature of JMV2009 at NT receptors
We next assessed the signalling signature of this novel neurotensinergic compound, JMV2009, in comparison with the hexapeptide C-terminal fragment of neurotensin, NT(8-13). We used a bioluminescence resonance energy transfer-based assay to monitor the effect of NT (8)(9)(10)(11)(12)(13) and JMV2009 on the activation of four G proteins known to be activated by NTS1 (G α q , G α 13 , G α i1 , and G α oA ) as well as the two β-arrestins ( β-arr), also known to be recruited by NTS1 upon activation [5] . We observed a concentration-dependent response of NT(8-13) and JMV2009 for all G protein and β-arr pathways monitored ( Fig. 5 ). A non-linear regression fit in  Graphpad Prism has been used to determine the potency values (EC 50 ) of NT(8-13) and JMV2009 ( Table 2 ). We further evaluated the ability of JMV2009 to induce an activation of the mitogen-activated protein kinases (MAPK) pathway after incubation at various time points with cells stably expressing either the NTS1 or NTS2 receptor. Thus, we performed western blots to monitor the phosphorylation of p42/p44 proteins (ERK 1/2) after stimulation with 1 μM of NT or JMV2009, as previously described by Gendron, et al. [6] We report here the time-dependent phosphorylation of p42/p44 proteins by the JMV2009 ( Fig. 6 ).
We finally investigated the ability of JMV2009 to trigger the internalization of NT receptors using a cell-surface ELISA assay, after stimulation of CHO-K1 cells transfected with the HA-tagged human NTS1 or NTS2 receptors. We found that JMV2009 was able to promote the internalization of both NT receptors ( Fig. 5 and Table 3 ) after a 1 h-incubation period ( Fig. 7 ).    Values are expressed as mean ± SEM of at least three independent determinations.

JMV2009 synthesis
Leucine residue-preloaded Wang resin was purchased from Novabiochem; amino acids bearing Fmoc-protection were obtained from ISIS Biotech. HBTU, HOBt, DIEA, TEA and piperidine were purchased from Aldrich. Acetonitrile and trifluoroacetic acid (TFA) were from Merck. ESI-MS was performed on a Micromass Platform II quadrupole mass spectrometer (Micromass) coupled with an HPLC.
Reverse phase analytical chromatograms were obtained using a C18 column (3.5 μm, 4.6 × 50 mm), coupled to a UV-Vis detector with a linear gradient of acetonitrile in water from 0 to 100% in 15 min at a flow of 1 mL/min. Retention time ( t R ) are given in minutes.
Waters Delta-Prep 40 0 0 chromatography equipped with a 214 nm UV detector and mounted with a Delta-Pak C18 column (40 × 100 mm, 15 μm, 100 Å ) was used as a preparative set-up with a flow rate of 50 mL min −1 of a binary eluent system of A: H 2 O, TFA 0.1% / B: CH 3 CN, TFA 0.1%.
Automated solid-phase peptide synthesis with a PerkinElmer ABI433A automatic synthesizer was used for the NT hexapeptide on a 0.25 mmol scale starting with Wang resin loaded with a leucine residue (loading 0,84 mmol/g) as previously described [7] . HBTU/HOBt (0.45 M) was used as coupling reagent with a 4-time excess of Fmoc-protected amino acid (1 mmol). Fmoc-Sip-OH has been synthesized according to published procedures [ 2 , 3 ]. piperidine:DMF (20:80) was used for deprotection and deprotection steps were followed using conductimetry. DMF with DIEA (2 M) as base were used during the 30-min coupling steps. Resin was washed between coupling steps using DMF and DCM. TFA:anisole 8:2 mixture was used for the final deprotection and cleavage for 3 h. The resin was washed extensively with DCM and filtered over cotton

Cell culture
Cell lines stably expressing the human NTS1 receptor were cultured in DMEM/F12. NTS2expressing cells were cultured in DMEM. Culture media were supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 2 mM l -glutamine, 20 mM HEPES, and 0.4 mg/mL of G418. CHO-K1 cells were cultured in the same DMEM/F12 as NTS1 cells but without G418 supplementation. Cells were kept at 37 °C under 5% CO 2 . All cell lines were used below passage 25.

Radioligand binding experiments
Binding experiments were carried out on freshly prepared membrane homogenates as previously described [4] . Competition radioligand binding experiments were performed by incubating cell membranes with 125 I-[T yr 3 ]-NT (specific activity of 2200 Ci/mmol) and different concentrations of ligands (ranging from 10 −11 to 10 −5 M) for a hour at room temperature. All binding data were plotted and fitted by using the One site -Fit Log(IC 50 ) of Prism v7.0a (GraphPad, La Jolla, CA, USA) and represent the mean ± SEM of three separate determinations. Blood sampling and preparation of plasma : Plasma was sampled from anesthetized rats by cardiac puncture in 4.5 mL plasma separating tubes coated with lithium heparin (from BD). Tubes were then centrifuged at 2500 rpm for 15 min at 4 °C to separate the plasma from the blood cells. Plasma was stored at −80 °C in 500 μL aliquots until use.
6 μL of 1 mM solution of Fmoc-Gly-was added in each sample as an internal standard for quantification. Ratio between AUC of test compound and AUC of Fmoc-Gly-was used to determine remaining test compound percentage. One-phase decay non-linear regression from Prism v7.0a was used to determine the half-life. Each point represent the mean ± SEM of three independent determinations. Leduc). The assays were performed as previously described [8] . Briefly, 1.5 × 10 6 CHO-K1 cells were seeded into 55 mm 2 cell culture dishes and transfected 24 hours later. The cells were transfected with either of the following biosensor couples: hNTS1, G α q -RlucII, or G α 13 -RlucII, or G α i1 -RlucII, or G α oA -RlucII, together with G β 1 and G γ 1 -GFP 10 as described [5] . On the final day of the experiment, cells were washed with 100 μL of PBS and stimulated with increasing concentrations of NT (8)(9)(10)(11)(12)(13) or JMV2009 prepared in HBSS containing 20 mM HEPES. The cells were then stimulated with 5 μM of coelenterazine 400A (GoldBio, St-Louis, MO, USA), incubated at 37 °C for 5 minutes, and read on a GENios Pro plate reader (Tecan, Durham, NC, USA) using a BRET 2 filter set (410 nm and 515 nm emission filters).

BRET-based assays for the activation of G proteins and recruitment of β-arrestins
For each well, a BRET2 ratio was determined by dividing the GFP10-associated light emission by RlucII-associated light emission. The data was subsequently normalized relative to NT(8-13); values for non-treated cells were set as 0% pathway activation, and those for cells treated with

β-arrestin recruitment
The monitoring of β-arrestin recruitment was done by the transient transfection of CHO-K1 cells with plasmids containing cDNAs encoding hNTS1-GFP 10 and RlucII-β-arrestin 1 or 2. The same protocol as the one used for G protein activation was then used except that incubation time before luminescence reading was increased to 15 min.

Western blot analyses of ERK1/2 activity
Cells stably expressing NTS1 or NTS2 were grown for 48 h in complete culture media before being incubated for 16 h in serum-free media. Cells were then stimulated with either NT or JMV2009. Aspiration of media and addition of ice-cold PBS blocked any further protein phosphorylation. Cells were then lysed in RIPA containing proteases and phosphatases inhibitors before being centrifuged at 80 0 0 g for 15 min.

JMV2009-induced NT receptors internalization
Receptor internalization using CHO-K1 cells transiently transfected with either HA-NTS1 or HA-NTS2 was carried out as previously described in details [9][10][11] . Before ELISA detection, cells were washed with PBS and stimulated using 1 μM of NT or JMV2009 for 60 min at 37 °C in serum-free media. Cells were then washed with PBS and remaining ELISA steps form the detailed protocol were followed and unchanged. Absorbance was read at 450 nm and data were normalized according to the protocol.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Ethics statement
Experimental procedures were approved by the Animal Care and Ethical Committee of the Université de Sherbrooke (protocol 035-18) and were in accordance with policies and directives of the Canadian Council on Animal Care, the ARRIVE recommendations [12] , and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023).