Peptides 105 (2018) 51–57

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Peptides

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In vitro and in vivo activity of cyclopeptide Dmt-c[D-Lys-Phe-Asp]NH2,amu T receptor biased toward β-arrestin

Katarzyna Gach-Janczaka,1, Justyna Piekielna-Ciesielskaa,1, Anna Adamska-Bartłomiejczyka, Karol Wtoreka, Federica Ferrarib, Girolamo Calo’b, Agata Szymaszkiewiczc, ⁎ Joanna Piasecka-Zelgad, Anna Janeckaa, a Department of Biomolecular Chemistry, Medical University, Lodz, Poland b Department of Medical Sciences, Section of Pharmacology and Italian Institute of Neuroscience, University of Ferrara, 44121 Ferrara, Italy c Department of Biochemistry, Faculty of Medicine, Medical University of Lodz, Poland d Institute of Occupational Medicine, Research Laboratory for Medicine and Veterinary Products in the GMP Head of Research Laboratory for Medicine and Veterinary Products, Lodz, Poland

ARTICLE INFO ABSTRACT

Keywords: and related drugs, which are the most effective for the relief of severe pain, act through Cyclic opioid peptides activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used Binding assay as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid Calcium mobilization assay research is to understand the mechanism of action of in order to improve therapeutic Bioluminescence resonance energy transfer utility of . effects of morphine are mediated mostly through activation of the mu opioid re- assay ceptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor Hot-plate test fi ff Whole gastrointestinal transit test pro le gained a lot of interest. Recently, the concept of biased agonists able to di erentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while β-arrestin recruitment may be re- sponsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-

selective ligand Tyr-c[D-Lys-Phe-Asp]NH2 with 2′,6′-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[D-

Lys-Phe-Asp]NH2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward β-arrestin. β-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide.

1. Introduction interest [3,4]. Several opioid analogs with peptide or alkaloid structure that represent this new strategy have already been synthesized and Centrally acting opioid agonists, such as morphine, are the most evaluated in vitro and in vivo. widely used analgesics for the treatment of severe pain [1]. Among the Kappa selective agonists produce analgesia accompanied by some three types of classic opioid receptors, mu, delta and kappa, the mu dysphoric effects [5] and this property limited their therapeutic de- receptor was identified as the one responsible primarily for the pain- velopment. However, mixed mu/kappa agonists (such as ethylk- relieving effects but also for a number of undesired side effects, in- etazocine) display fewer side-effects than their kappa-selective coun- cluding sedation, respiratory depression, inhibition of gastrointestinal terparts such as and [6,7] and have been used to transit and also development of tolerance and [2]. treat addiction [8]. Presumably, the effects produced by mu- In the previous decades, extensive structure-activity relationship agonists help attenuate the dysphoric action associated with kappa studies of opioid receptor ligands concentrated on the obtaining analogs agonism. with high selectivity for one opioid receptor type. More recently, the A combination of (a partial mu agonist/kappa an- development of compounds with mixed opioid profile is gaining a lot of tagonist) and (a non-selective antagonist) produced

⁎ Corresponding author at: Department of Biomolecular Chemistry, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland. E-mail address: [email protected] (A. Janecka). 1 Equal contribution. https://doi.org/10.1016/j.peptides.2018.04.014 Received 5 January 2018; Received in revised form 16 April 2018; Accepted 18 April 2018 Available online 22 April 2018 0196-9781/ © 2018 Published by Elsevier Inc. K. Gach-Janczak et al. Peptides 105 (2018) 51–57 antidepressant-like activity in mice and may represent a novel approach very high potency for these two receptors. The pharmacological profile in the treatment of depression [9]. The synthesis of mixed mu/kappa of this cyclopeptide was investigated in detail, both in vitro and in vivo. receptor antagonists, combining these two activities in one compound, would overcome the abuse liability issue and also the issue of a proper 2. Materials and methods dosing ratio of two separate entities. A new orvinol analog BU10119 with the mu/kappa antagonist affinity profile has recently been ob- 2.1. Materials tained by Cueva et al. [10]. In vitro BU 10119 showed high affinity for kappa and mu receptors with little efficacy at both these receptors, Peptide synthesis reagents (amino acids, MBHA Rink-Amide resin, indicating an antagonist-like profile. The initial characterization of this TBTU) were purchased from Bachem AG (Bubendorf, Switzerland). analog in vivo in mouse models of depression showed the therapeutic Opioid radioligands, [3H]DAMGO, [3H]-2 and [3H]U-69593, potential of BU 10119 for the treatment of depression and other stress- and cell membranes expressing human recombinant opioid receptors induced conditions [11]. were purchased from PerkinElmer (Krakow, Poland). hy- In 1991 Abdelhamid et al. [12] demonstrated for the first time that drochloride, β-funaltrexamine hydrochloride (β-FNA) and selective delta opioid receptor blockade with naltrindole (delta opioid hydrochloride (NLT) were obtained from Tocris Bioscience (Ellisville, antagonist) greatly reduced the development of morphine tolerance and MO, USA). dependence in mice. Opioid ligands combining mu agonist/delta an- tagonist activity in one compound were therefore sought for the de- 2.2. Peptide synthesis velopment of analgesics with lower propensity to produce tolerance and physical dependence [13]. Peptides were synthesized on solid support, using Fmoc chemistry The first known compound with mixed mu agonist/delta antagonist as described elsewhere [36]. Crude peptides were purified using a profile was the tetrapeptide amide Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2, Waters semipreparative HPLC (Waters Breeze instrument, Milford, MA, were Tic represents 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) USA) with a Vydac C18 column (10 μm, 22 × 250 mm), using the sol- [14]. TIPP-NH2 showed quite high delta antagonist but modest mu vent system of 0.1% TFA in water (A)/80% acetonitrile in water con- agonist potency. Further modifications including replacement of Tyr1 taining 0.1% TFA (B). A linear gradient of 0–100% B over 50 min at a residue by Dmt (Dmt = 2′,6′-dimethyltyrosine) and a reduction of the flow rate of 1 mL/min was used. The molecular weight of peptides was peptide bond between Tic2 and Phe3 led to a pseudopeptide Dmt- confirmed by ESI–MS recorded on Bruker micrOTOF-Q mass spectro-

Ticψ[CH2NH]-Phe-Phe-NH2 (DIPP-NH2[ψ]) with greatly improved mu meter (Bruker Daltonics, Bremen, Germany). The purity of the cyclo- potency. As expected, this analog exhibited reduced tolerance com- peptides was verified by analytical HPLC employing a Vydac C18 pared to morphine and no dependence, when administered in- column (5 μm, 4.6 × 250 mm) and the solvent system of 0.1% TFA in tracerebroventricularly [15]. However, the therapeutic potential of water (A)/80% acetonitrile in water containing 0.1% TFA (B). All

DIPP-NH2[ψ] was compromised by its poor blood-brain barrier (BBB) peptides were at least 96% pure as determined by HPLC monitored at permeability [16]. 230 nm. Purington et al. [17] reported the synthesis of a cyclic tetrapeptide

KSK-103, containing a S‐CH2‐CH2‐S- bridge that showed equal and high 2.3. Radioligand binding assays affinity for mu and delta receptors but, similar to the previously re- ported ligands, had poor bioavailability. Glycosylation of this cyclo- Radioligand binding assays were performed according to the pre- peptide produced an analog that retained the desirable in vitro profile, viously described method [37] using commercial membranes of Chi- while displaying centrally mediated antinociception after in- nese Hamster Ovary (CHO) cells transfected with human opioid re- traperitoneal (i.p.) injection with a potency similar to morphine [18]. ceptors. The binding affinities for mu, delta and kappa opioid receptors It was also shown that potency and efficacy of the mu agonists can were determined by radioligand competition analysis using [3H] be enhanced by delta agonists. Synergistic antinociceptive effects in DAMGO, [3H]deltorphin-2 and [3H]U-69593, respectively, as specific response to the mu and delta receptor activation were observed in radioligands. Three independent experiments for each assay were car- several in vivo studies. For example, enkephalins, DPDPE or deltorphine ried out in duplicate. The data were analyzed by a nonlinear least potentiated antinociceptive effect of morphine [19–22]. These results square regression analysis computer program Graph Pad PRISM 6.0 pointed at a functional interaction between mu and delta opioid re- (Graph Pad Software Inc., San Diego, USA). The IC50 values were de- ceptors and a potential regulatory role of the endogenous delta ligands termined from the logarithmic concentration-displacement curves, and in controlling pain. the values of the inhibitory constants (Ki) were calculated according to Therefore, the search for single compounds combining mixed mu/ the equation of Cheng and Prusoff [38]. delta agonist activities followed. The best know and the most studied peptide ligand with the dual mu/delta agonist profile is [Tyr-D- 2.4. Calcium mobilization assay Ala-Gly-Phe-NH-NH-Phe-Gly-D-Ala-Tyr], whose structure is based on two enkephalin-like fragments connected “tail-to-tail” by a hydrazine Calcium mobilization assay was performed as reported in detail bridge [23]. Biphalin showed high affinity for the mu and delta re- elsewhere [39], using CHO cells stably co-expressing human re- ceptors (with Ki about 1–4 nM), very good antinociceptive activity and combinant mu or kappa opioid receptors and the C-terminally modified duration of action after intrathecal (i.t.) administration [24], induced Gαqi5 and CHO cells co-expressing the human recombinant delta opioid less physical dependence than morphine [25] and could penetrate the receptor and the GαqG66Di5 chimeric protein. BBB [26]. Several linear analogs of biphalin with amino acid substitu- Agonist potencies of peptides are given as pEC50 that is the negative tions in positions 2,2′ and 4,4′ were also reported [27,28]. logarithm of the molar concentration of an agonist that produces 50% To overcome moderate stability of biphalin in human plasma [29] of the maximal possible effect. Ligand efficacy was expressed as in- the group of Mollica developed several cyclic analogs of biphalin with a trinsic activity (α). disulfide linkage [30,31] or a xylene bridge [32]. Such analogs showed increased in vitro affinity for the mu and delta receptors and prolonged 2.5. Bioluminescence resonance energy transfer (BRET) assay in vivo antinociceptive effect [33,34]. In our earlier paper we described a cyclic tetrapeptide, Tyr-c[D-Lys- BRET assay was described in our earlier paper [40]. SH-SY5Y cell

Phe-Asp]NH2 [35], which was highly mu selective. Here, replacement lines permanently co-expressing mu-RLuc and one of the transduction of Tyr by Dmt resulted in obtaining an analog with mu/delta profile and protein (Gβ1-RGFP or β-arrestin 2-RGFP) were used to determine the

52 K. Gach-Janczak et al. Peptides 105 (2018) 51–57 interaction of the mu receptor with G-protein and β-arrestin 2. Methods plate test in mice as described earlier [45]. The intracerebroventricular for cell culturing, retroviral transduction, and BRET assay have been (i.c.v.) injections (10 μL/animal) of peptides or vehicle were performed described previously [41,42]. Agonist responses were quantified as under inhalational isoflurane anesthesia into the left brain ventricle stimulated BRET ratio obtained by subtracting the vehicle value from with a Hamilton microsyringe (50 μL) connected to a needle (diameter that measured in the presence of the ligand. 0.5 mm). Antagonists were administered 15 min prior to the peptide or For calculation of bias factors EM-2 was used as a standard unbiased vehicle injection. A transparent plastic cylinder (14 cm diameter, 20 cm ligand. height) was used to confine a mouse on the heated (55 ± 0.5 °C) sur- The concentration response curves of each compound were fitted to face of the plate. The animals were placed on the hot-plate 10 min after the Black-Leff operational model described by Nagi et al. [43]: i.c.v. injection. The latencies to hind paw licking were measured. A cut- off time of 120 s was used to avoid tissue injury. The percentage of the []AτEnn response m ff = nn n maximal possible e ect (%MPE) was calculated as: %MPE = (t1-t0)/(t2- []Aτ++ ([] A KA ) t0) × 100, where t0–control latency, t1- test latency and t2 – cut-off time where [A] is the agonist concentration, the maximal response of the (120 s). The median antinociceptive dose (ED50) was calculated ac- fi system is given by Em, n is a fitting parameter for the slope, the affinity cording to the method of Litch eld and Wilcox [46]. The data were of the agonist is represented by the equilibrium dissociation constant of analyzed by a nonlinear least square regression analysis computer the agonist-receptor complex (KA), and the efficacy of the agonist is program Graph Pad PRISM 6.0 (GraphPad Software Inc., San Diego, defined by τ.KA and τ are descriptive parameters of intrinsic efficacy USA). and binding affinity and may be directly obtained by fitting experi- mental data to the operational equation and can be expressed as 2.7.4. Evaluation of whole gastrointestinal transit

“transduction coefficients” log(τ/KA). The relative efficiency of an Whole gastrointestinal transit (WGT) test in mice was performed as agonist producing activation of any pathway can thus be quantified described [47]. A tested compound was injected i.p. (in the final vo- with a “normalized” transduction coefficient, namely Δlog(τ/KA). Fi- lume of 0.1 mL) 15 min before intragastric (i.g.) administration of a nally, the bias factor was calculated as a difference between Δlog(τ/KA) colored marker (0.15 mL of glutinous liquid consisting of 5% Evans blue values for a given agonist between the pathways (G protein and β-ar- and 5% Arabic gum). The colored dye was administered with 18-gauge restin 2): animal feeding tube. Subsequently, mice were placed in the individual cages on a white sheet of paper (in order to facilitate recognition of bias factor = Δlog(τ/K ) − Δlog(τ/K )β A Gprotein A -arrestin2 colored boluses). The whole gastrointestinal transit is calculated as time Bias factors are expressed as the mean ± SEM of at least 5 in- between i.g. administration of the marker and excretion of the first dependent experiments. colored bolus.

2.6. In vitro activity in isolated smooth muscle strips 2.8. Statistical analysis

Organ bath studies were performed as described previously [44]. Statistical analysis was performed using GraphPad Prism 6.0 Mice were sacrificed by cervical dislocation. The colon was rapidly (GraphPad Software Inc., San Diego, CA, USA). All data are expressed removed and 0.5 cm long full-thickness fragments were distincted. as mean ± SEM Analysis of multiple treatment was performed using – Preparations were kept in cold oxygenated Krebs–Ringer Solution. one way analysis of variance (ANOVA) followed by Newman Keuls fi Electrical field stimulation (EFS; 4 Hz, 24 V, stimulus duration 0.5 ms, post-hoc test. P values < 0.05 were considered statistically signi cant. train duration 10 s) was applied using a S88X stimulator (Grass Tech- nologies, West Warwick, RI, USA). A tested compound at increasing 3. Results − − concentrations (10 12 to 10 6 M) was added cumulatively into the fi organ bath. Tissue was incubated with each concentration for 8 min. As 3.1. In vitro pro le of Dmt-c[D-Lys-Phe-Asp]NH2 an internal control, the mean amplitude of initial 3 successive con- tractions was used. Changes in contractility (after a tested compound Our previously reported mu selective cyclopeptide Tyr-c[D-Lys-Phe- fi addition) were measured and reported as percentage of internal control. Asp]-NH2 (1) [35], was modi ed by introduction of Dmt into position 1. The obtained analog Dmt-c[D-Lys-Phe-Asp]NH2 (2), was examined at 2.7. Animal tests the mu, delta and kappa opioid receptors by the competitive binding 3 3 2 against [ H]DAMGO, [ H][D-Ala ]deltorphin-2, and U-69,593, respec- 2.7.1. Animals tively, and the results are summarized in Table 1. Introduction of Dmt Male Balb/C mice (Institute of Occupational Medicine, Lodz, resulted in transforming a mu selective analog 1 into a peptide 2 with ffi Poland), weighing 22–24 g, were used for the study. The animals were very high a nity for mu and delta receptors. fi housed in a room with controlled temperature (22 ± 1 °C), humidity The pharmacological pro le of the new analog was further eval- (70 ± 5%) and light/dark cycle conditions (12/12 h), with free access uated in vitro at all three opioid receptors in the calcium mobilization to laboratory chow and tap water. All procedures were approved by the Local Ethical Committee for Animal Research with the following Table 1 Opioid receptor binding affinities of cyclopeptides. numbers: 29/ŁB662/2013 and 20/ŁB708/2014. a No. Sequence Ki [nM] 2.7.2. Drugs and pharmacological treatment mu delta kappa The tested compounds were dissolved in DMSO, further diluted with

0.9% NaCl solution to the final concentration of 5% DMSO. Animals EM-2 Tyr-Pro-Phe-Phe-NH2 2.50 ± 0.05 > 1000 > 1000 without treatment (control group) received vehicle alone (5% DMSO in 1 Tyr- c[D-Lys-Phe-Asp]NH2 0.21 ± 0.02 461 ± 63 684 ± 59 0.9% NaCl solution). The vehicle given alone had no effects on the 2 Dmt-c[D-Lys-Phe-Asp]NH2 0.24 ± 0.01 1.33 ± 0.06 79 ± 2 observed parameters. All values are expressed as mean ± SEM, n ≥ 3. a Displacement of [3H]DAMGO (mu-selective), [3H]deltorphin-2 (delta-se- 2.7.3. Assessment of antinociception lective) and [3H]U-69593 (kappa-selective) from human opioid receptor The antinociceptive effects of peptides were assessed in the hot- membrane binding sites.

53 K. Gach-Janczak et al. Peptides 105 (2018) 51–57

Table 2 Effects of the reference agonists and cyclopeptides at human recombinant opioid receptors coupled with calcium signaling via chimeric G proteins.

mu delta kappa

a b pEC50 (CL95%) α ± SEM pEC50 (CL95%) α ± SEM pEC50 (CL95%) α ± SEM

EM-2 7.83 (7.69–7.96) 1.00 Inactive inactive DPDPE inactive 7.33 (7.20–7.47) 1.00 inactive 6.67 (6.17–7.17) 0.83 ± 0.10 7.73 (7.46–8.00) 0.99 ± 0.04 8.81 (8.73–8.90) 1.00 1 8.56 (8.32–8.79) 1.01 ± 0.07 6.62 (6.23–7.01) 1.00 ± 0.13 crc incomplete 2 8.53 (8.25–8.81) 1.03 ± 0.06 7.65 (6.74–8.55) 0.98 ± 0.05 6.11 (5.80–6.42) 0.5 ± 0.02

The crc incomplete means that the maximal effect could not be determined due to the low potency of a compound. EM-2, DPDPE, and dynorphin A were used as reference agonists for calculating intrinsic activity at the mu, delta and kappa opioid receptors, respectively. a Agonist potency values (pEC50). b Efficacy values (α). assay in which CHO cells expressing human recombinant opioid re- 3.2. In vivo profile of cyclopeptide analogs ceptors and chimeric G proteins were used to monitor calcium changes. The concentration-response curves of the tested compounds and the Assessment of antinociception of cyclopeptide analogs was studied standards were obtained and the calculated agonist potencies (pEC50) in the mouse hot-plate test (supraspinally mediated analgesia), after and efficacies (α) are shown in Table 2. At the mu receptor analog 2 i.c.v. administration. Both compounds showed an extremely strong displayed even higher potency than the reference mu agonist, en- analgesic effect, which was dose-dependent (Fig. 3). Analog 2 with the domorphin-2 (EM-2) and the same maximal effect. At the delta receptor mu/delta profile exerted stronger antinociceptive effect than the parent

2 showed the same potency and maximal effect as the selective delta compound 1, which was mu selective. The ED50 were 0.6 ng and 2.9 ng agonist DPDPE. Compared to dynorphin A, the kappa receptor re- for analog 2 and analog 1, respectively. ference compound, cyclopeptide 2 displayed very low potency and re- The antinoceptive activity of analog 2 was effectively reversed by duced efficacy. In agreement with the binding assay results, introduc- preemptive i.c.v. injection of β-FNA (mu selective antagonist) or NLT tion of Dmt transformed analog 1 with high mu potency into a non- (delta selective antagonist) (Fig. 4), indicating that the action of this selective peptide 2 which showed high potency at the mu and delta peptide was mediated by the mu and delta opioid receptors in the brain. receptors. To compare the inhibitory effect of analogs 1 and 2 on gastro- Then, the interaction of the mu opioid receptor with G-protein and intestinal (GI) transit, the mouse WGT test was performed. Tested β-arrestin 2 were evaluated in the BRET assay, measuring the energy compounds were administered at three doses (0.1; 0.3; and 1 mg/kg, transfer between Renilla Luciferase (RLuc) linked to the mu opioid re- i.p.) For comparison, loperamide, the well-known antidiarrheal agent, ceptor and Renilla Green Fluorescent Protein (RGFP) linked to the was used. Analog 2 with biased profile toward β-arrestin 2/mu exerted signal transducer proteins (G-protein or β-arrestin 2). In membrane the strongest effect. This compound significantly decreased the WGT at extracts taken from SH-SY5Y cells co-expressing the mu/RLuc and the all three concentrations. At a dose of 0.1 mg/kg, it delayed excretion of

Gβ1-RGFP fusoproteins, the mu receptor agonist EM-2 promoted re- the colored boluses up to 193.7 ± 24.7 min, in comparison to control ceptor/G protein interaction in a concentration dependent manner with (90.9 ± 5.0 min) and loperamide (130.0 ± 3.0 min). Analog 1 and potency value of 6.97 and maximal effect (Emax) of 0.80 ± 0.11 sti- loperamide were active only at the highest used dose, which was 1 mg/ mulated BRET ratio (Fig. 1A). Analog 1 mimicked the maximal effects kg (Fig. 5). of EM-2, being 4 fold more potent (Fig. 1B). In a separate series of experiments, EM-2 promoted mu/G-protein interaction in a con- 4. Discussion centration-dependent manner with high potency (pEC50 7.32) and maximal effect of 1.48 ± 0.16 stimulated BRET ratio (Fig. 1C). Under the same experimental conditions cyclopeptide 2 behaved as a full Most of the currently available pain-relieving opioids exert their ff agonist and was 8 fold more potent than EM-2 (Fig. 1D). analgesic but also adverse e ects primarily through the activation of In SH-SY5Y cells stably co-expressing the mu/RLuc and the β-ar- the mu receptor. However, a large number of biochemical and phar- restin 2/RGFP fusoproteins, EM-2 promoted receptor/arrestin interac- macological studies provide evidence that there are strong modulatory tion in a concentration dependent manner with a potency value of 6.91, interactions between mu and delta opioid receptors. Several studies and E of 0.35 ± 0.09 stimulated BRET ratio (Fig. 1A). Analog 1 indicate that delta receptor agonists as well as antagonists can sig- max fi ff showed higher maximal effects and was 4 fold more potent than EM-2 ni cantly improve the pharmacological e ects exerted by mu agonists. (Fig. 1B). In the second series of experiments, EM-2 promoted mu/β- In particular, delta agonists can enhance the analgesic potency and ffi arrestin interaction in a concentration dependent manner with a po- e cacy of mu agonists, and delta antagonists can prevent or diminish the development of tolerance and physical dependence induced by mu tency value of 7.20, and Emax of 0.38 ± 0.04 stimulated BRET ratio (Fig. 1C). Compound 2 was 16 fold more potent and exhibited higher agonists. maximal effect than EM-2 (Fig. 1D). These results, which are shown in Therefore, the use of agents that simultaneously activate more than ffi Fig. 1, were used for calculating bias factors of the ligands: analog 1 had one opioid receptor in order to enhance e cacy and/or reduce side ff a bias factor not statistically different from 0, while analog 2 displayed e ects is a promising approach in the search for innovative analgesics a modest (14-fold) but significant bias toward β-arrestin 2 (Table 3). [3,48]. In further in vitro experiments, the effect of analog 2 was evaluated As is well known, opioid agonists interact with GPCRs which upon on EFS-induced smooth muscle contractility in the mouse distal colon. activation, are phosphorylated by GPCR kinases and subsequently bind β Analog 2 significantly inhibited EFS-induced colonic contractions in a -arrestins, which prevent further coupling of the receptor to G protein − − concentration-dependent manner (10 9 to 10 6 M) (Fig. 2). [49]. GPCRs can then be internalized and are either recycled to the plasma membrane or degraded. However, β-arrestins are not only ne- gative regulators of the G protein signaling but can promote distinct intracellular signals of their own. Experiments with the β-arrestin 2 knockout (KO) mice showed that signaling mechanism of morphine

54 K. Gach-Janczak et al. Peptides 105 (2018) 51–57

Fig. 1. Comparison of the effect of EM-2 (panels A and C), Tyr- c[D-Lys-Phe-Asp]NH2 (1; panel B), and Dmt-c[D-Lys-Phe-Asp]NH2 (2; panel D) at the mu/G protein and mu/β-arrestin 2 interaction. Data are mean ± SEM of at least 5 experiments. engage both G protein coupling and β-arrestin recruitment and that The discovery of β-arrestin-mediated signaling pathways opened blocking the β-arrestin pathway can reduce unwanted side effects of new possibilities in opioid drug development [54–56]. Agonists biased opioids, such as respiratory depression, inhibition of gastrointestinal to either G protein or β-arrestin may be used to segregate physiological motility, and tolerance liability. In the β-arrestin 2 KO mice morphine responses downstream of the receptor [57–59]. induced, via the mu opioid receptor, enhanced and longer-lasting an- Molinari et al. [42] measured the possible differential ability of tinociceptive effects than in a wild type mice and almost no tolerance various opioid ligands of peptide and alkaloid structure (including EM- [50,51]. On the other hand, the antidiarrheal agent loperamide, which 2 and morphine) to induce G-protein and β-arrestin signaling at the mu is a peripherally restricted mu receptor agonist, significantly reduced and delta receptors. None of the tested ligands showed greater efficacy colonic propulsion in wild type mice, and this effect was completely for β-arrestin than for the G-protein. The authors suggested that the abolished in β-arrestin 2 KO mice [52]. This suggests that peripherally structure requirements for an agonist to trigger the interaction of the restricted β-arrestin-biased agonists might be useful in the treatment of receptor with β-arrestin are more stringent than those sufficient to in- diarrhea and other hypermotility disorders [53]. itiate G-protein coupling.

Table 3 Potencies, maximal effects, and bias factors of EM-2, cyclopeptides 1 and 2 in the BRET assay.

Compound mu/G-protein mu/β-arrestin 2 bias factor (CL95%)

pEC50 (CL95%) α ± SEM pEC50 (CL95%) α ± SEM

EM-2 6.97 (6.73–7.22) 1.00 6.91 (6.76–7.05) 1.00 0.00 1 7.56 (7.15–7.97) 1.08 ± 0.07 7.52 (6.85–8.20) 1.46 ± 0.03 −0.49 (−1.36 to 0.38) EM-2 7.32 (7.14–7.51) 1.00 7.20 (7.08–7.33) 1.00 0.00 2 8.23 (8.09–8.36) 1.05 ± 0.03 8.39 (8.33–8.45) 1.43 ± 0.06 −1.16 (−1.59 to −0.74)

55 K. Gach-Janczak et al. Peptides 105 (2018) 51–57

Fig. 5. Dose-response inhibitory effect of loperamide and peptides 1 and 2 on the mouse WGT. A peptide or vehicle were injected i.p. 15 min before the in- tragastric (i.g.) administration of a colored marker. Data represent

− − mean ± SEM of n =6–8 mice for each experimental group. Statistical sig- Fig. 2. The inhibitory effect of analog 2 (10 12 to 10 6 M) on EFS induced nificance was assessed using one-way ANOVA and a post hoc multiple com- longitudinal smooth muscle contractions in mouse colon. Data represent parison Student-Newman-Keuls test. ***p < 0.001, *p < 0.05, as compared mean ± SEM (n = 7-8). Statistical significance was assessed using one-way with control (vehicle treated mice). ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. *p < 0.05, **p < 0.01, ***p < 0.001, as compared with control (vehicle alone). interactions. The cyclic pentapeptide Dmt-c[D-Lys-Phe-Phe-Asp]NH2 activated both, G protein and β-arrestin pathways [40]. Reported here 1 tetrapeptide Tyr-c[D-Lys-Phe-Asp]NH2 (1) with Tyr and a shorter se- quence by one Phe residue was also unbiased. However, the 1 Dmt –containing analog Dmt-c[D-Lys-Phe-Asp]NH2 (2), activated G protein pathway similarly to EM-2 but promoted β-arrestin recruitment with a much higher maximal effect than EM-2 (1.46 and 1.0, respec- tively) and therefore showed 14-fold bias toward β-arrestin. To the best of our knowledge, analog 2 is the first reported β-arrestin biased opioid peptide. This ligand activated with high efficacy the mu and delta receptors. Compounds with such profile are known to exert stronger pharmaco- logical effects than the mu selective ligands. Indeed, analog 2 showed a 5-fold more pronounced antinociceptive effect in mice than its mu se- Fig. 3. The effect of different doses of the tested analogs in the mouse hot-plate lective parent 1. On the other hand, 2 was also found to strongly inhibit β test. Results are expressed as percentage (mean ± SEM) of the maximal pos- the WGT in mice which is consistent with activation of the -arrestin sible effect (%MPE) for the inhibition of hind paw licking induced by i.c.v. pathway. injection of a peptide. n =6–8 mice for each experimental group. These results are in accordance with earlier reports indicating that various in vivo activities of opioid agonists arise not only from the ac- tivation of one or more opioid receptors, but also from promoting G- protein or β-arrestin pathways. Therefore prediction of molecular in- teractions linked to the drug responses in vivo is very complex.

Conflict of interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Author contribution

K. G-J. designed the research study and performed radioligand binding assays, J. P-C. and F.F. performed functional assays, J.P-C. and J P-Z. performed in vivo studies, F.F. and G. C. analyzed calcium and BRET data, A. A-B. and K.W. performed peptide synthesis, A. S. per- Fig. 4. Antagonist effect of β-FNA and NLT (both at 1 μg/animal, i.c.v.) on the formed the ex vivo test, A. J. discussed the results and prepared the inhibition of hind paw licking induced by administration of analog 2 (1 ng/ manuscript. animal, i.c.v.) in the mouse hot-plate test. Results are expressed as percentage (mean ± SEM) of the maximal possible effect (%MPE). n =6–8 mice for each experimental group. Statistical significance was assessed using one-way ANOVA Acknowledgments and a post hoc multiple comparison Student–Newman–Keuls test. ***p < 0.001, as compared with analog 2. This work was supported by the Polpharma Scientific Foundation grant (11/XIII/2014 to KG) and grants from the Medical University of Recently, we have tested several of our opioid peptide analogs to- Lodz (No 503/1-156-02/503-11-002 and 502-03/1-156-02/502-14- wards their possible differential mu/G protein and mu/β-arrestin 301) and from the University of Ferrara (FAR grant to G.C.). KG is re- cipients of the Polish L’Oréal UNESCO Awards for Women in Science.

56 K. Gach-Janczak et al. Peptides 105 (2018) 51–57

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