Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist–Neurokinin Antagonist Peptidomimetics

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.


Introduction
From the ancient use of the plant alkaloid morphine up until the discovery of modern medicine technologies, opioid receptor-targeting analgesics still occupy a prominent place in the management of acute or chronic pain. Through a competition with endogenous neuropeptides (e.g., endorphin, enkephalins, and dynorphins), these small molecule drugs work by primarily activating the µ-opioid receptor (MOR) [1], and to a lesser extent, the δ-opioid (DOR), κ-opioid (KOR), and nociceptin (NOP) receptors. Aside from their pivotal role in nociception, these receptors modulate other vital biological processes such as respiration, gastrointestinal transit, stress responses, neuroendocrine, and immune functions [2,3]. This variety in physiological processes, combined with the multitude of neurotransmitters and associated receptors involved in pain pathways, explain the challenges that have to be overcome when designing new analgesic drugs. Decades of research unveiled that despite their undeniable efficiency in pain control, prolonged use of agonist pharmacophores Dmt-DALDA and KGOP01 were covalently linked to a selection of peptidic, pseudo-peptidic, and non-peptidic NK2 and NK3 pharmacophores SBL-OPNK-1, SBL-OPNK-2, and SBL-OPNK-3 ( Figure 1B). As described below, the design of these neurokinin-targeting moieties was inspired by the structures of reported and approved NK2 and NK3 antagonists exhibiting good biological activity. The first-generation peptidic NK2 ligand MEN 10,376 ( Figure 1A) displayed good antagonistic activity at the NK2 receptor (pA 2 = 8.08 ± 0.1 in RPA-endothelium-deprived rabbit pulmonary artery against neurokinin A as an agonist), hence offering an adequate peptide-based candidate for this study [25]. Exhibiting subnanomolar and selective NK2 binding affinity, and investigated in clinical phases for irritable bowel syndrome treatment [26], Ibodutant, and one of its parents, MEN 14,268, were selected as starting scaffolds for peptidomimetic NK2 ligand design ( Figure 1A). Finally, as a major representative of the NK3 antagonist family, and as a compound examined in clinical trials for treatment of CNS disorders, Talnetant exhibited high affinity for human NK3 and long-lasting in vivo activity, justifying its selection as a starting scaffold for non-peptidic NK3 ligand design [27]. As the first challenge in multifunctional analgesic ligand design is to maintain affinity to both targets and agonist efficacy at the opioid receptors, this initial report aimed to investigate the binding affinity and opioid receptor activation of the newly designed opioid-NK2 and -NK3 hybrids. series of multifunctional opioid agonist-neurokinin NK2 or NK3 antagonist ligands were designed, synthesized, and evaluated in vitro in light of exploring new therapeutic pathways in pain and related disorders. Reported for their high MOR binding affinity [23,24], the two putative opioid agonist pharmacophores Dmt-DALDA and KGOP01 were covalently linked to a selection of peptidic, pseudo-peptidic, and non-peptidic NK2 and NK3 pharmacophores SBL-OPNK-1, SBL-OPNK-2, and SBL-OPNK-3 ( Figure 1B). As described below, the design of these neurokinin-targeting moieties was inspired by the structures of reported and approved NK2 and NK3 antagonists exhibiting good biological activity. The first-generation peptidic NK2 ligand MEN 10,376 ( Figure 1A) displayed good antagonistic activity at the NK2 receptor (pA2 = 8.08 ± 0.1 in RPA-endothelium-deprived rabbit pulmonary artery against neurokinin A as an agonist), hence offering an adequate peptide-based candidate for this study [25]. Exhibiting subnanomolar and selective NK2 binding affinity, and investigated in clinical phases for irritable bowel syndrome treatment [26], Ibodutant, and one of its parents, MEN 14,268, were selected as starting scaffolds for peptidomimetic NK2 ligand design ( Figure 1A). Finally, as a major representative of the NK3 antagonist family, and as a compound examined in clinical trials for treatment of CNS disorders, Talnetant exhibited high affinity for human NK3 and long-lasting in vivo activity, justifying its selection as a starting scaffold for non-peptidic NK3 ligand design [27]. As the first challenge in multifunctional analgesic ligand design is to maintain affinity to both targets and agonist efficacy at the opioid receptors, this initial report aimed to investigate the binding affinity and opioid receptor activation of the newly designed opioid-NK2 and -NK3 hybrids.

Chemistry
The structures of the targeted opioid agonist-neurokinin NK2 or NK3 antagonist ligands are presented in Figure 2.

Calcium Mobilization Assay
In the calcium mobilization assay, all compounds were assessed under the same experimental conditions and their effects were compared to the reference compounds: dermorphin, DPDPE, and dynorphin A for MOR, DOR, and KOR, respectively (Table 2 and Figures 3 and 4).

Calcium Mobilization Assay
In the calcium mobilization assay, all compounds were assessed under the same experimental conditions and their effects were compared to the reference compounds: dermorphin, DPDPE, and dynorphin A for MOR, DOR, and KOR, respectively (Table 2 and Figures 3 and 4).
In the NK3 radioligand binding assay, the parent NK3 pharmacophore SBL-OPNK-3 showed a slightly reduced affinity compared to the reference compound, while the corresponding hybrid ligand SBL-OPNK-9 presented again a 1.5-fold increase of its NK3 affinity and potency compared to the reference compound SB 222200. At MOR, compound Dmt-DALDA mimicked the stimulatory effect of dermorphin in the calcium release test and KGOP01 showed full efficacy, but potency about 8-folds higher than the standard compound (α = 1.04, EC 50 = 0.54). Hybrids SBL-OPNK-5 and SBL-OPNK-7 showed slightly reduced potencies compared to the reference compound dermorphin (2-and 3-fold, respectively) and to the opioid parent pharmacophore KGOP01 (18and 24-fold, respectively). Hybrid SBL-OPNK-9 moderately stimulated calcium release, while the remaining compounds showed strong (>200-fold) reduction in potencies compared to dermorphin (Table 2 and Figure 3A). With regard to DOR agonism, compounds KGOP01, SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 evoked calcium signaling responses with a stimulatory effect of the reference DPDPE, but with 2-3-fold decreased potencies (Table 2 and Figure 3B). As for potency at KOR, compounds Dmt-DALDA and SBL-OPNK-5 were able to elicit a weak stimulatory response at micromolar concentrations. Compound SBL-OPNK-7 was completely inactive, while the other tested compounds weakly stimulated calcium release only at the highest dose (10 µM), providing incomplete concentration-response curves ( Table 2 and Figure 3C).
In the NK3 radioligand binding assay, the parent NK3 pharmacophore SBL-OPNK-3 showed a slightly reduced affinity compared to the reference compound, while the corresponding hybrid ligand SBL-OPNK-9 presented again a 1.5-fold increase of its NK3 affinity and potency compared to the reference compound SB 222200.

Discussion
To address the side effects associated with the long-term use of opioid agonists, the design of multifunctional ligands has emerged as a valuable strategy in the treatment of chronic pain. More precisely, µ-opioid pharmacophores were covalently combined to a series of antagonists targeting G protein-coupled receptors (here, the NK2 and NK3 receptors) known to cooperatively act with the opioid system or more widely described as nociception modulators. Following this approach, we previously reported the design of opioid agonist-NK1 antagonist hybrids, composed out of the putative opioid pharmacophore KGOP01 H-Dmt-D-Arg-Aba-βAla-NH 2 . As a second benchmark MOR ligand, the Dmt-DALDA pharmacophore was included, since this reference compound has been described as a highly µ-selective agonist with a favorable amphipathic profile for BBB crossing and improved metabolic stability [31,32]. The design of the constrained analogue KGOP01 addressed the beneficial effect of a concomitant activation of DOR for application in chronic pain treatment [28,33,34]. Because the opioid pharmacophores are primarily recognized through the N-terminus, they were covalently linked to a selection of NK2 and NK3 ligands through their C-terminus. The NK2 and NK3 ligands were selected based on their structure, featuring either a peptidic or a non-peptidic scaffold, and depending on a good biological affinity, selectivity, and activity. Such a differentiated nature of the NK pharmacophores could eventually lead to distinct pharmacokinetic properties.
The Menarini Lab was a pioneer in the development of NK receptor ligands and the initial efforts were logically directed toward sequential modifications of the endogenous neurokinins SP, NKA, and NKB. In the first generation of patented NK2 receptor antagonists, the peptidic MEN 10,376 resulted from a D-tryptophan insertion in the truncated endogenous NKA(4-10) sequence and exhibited selectivity and nanomolar affinity toward NK2 [25]. Given this selectivity profile, it constituted an adequate peptidic neurokinin pharmacophore in the context of opioid-NK ligands. To answer metabolic issues, peptide scaffolds were then gradually modified into pseudo-peptidic drugs, and a new generation of NK antagonists was disclosed. Among those, MEN 15,596 or Ibodutant stood out as a NK2 antagonist with great potential for the treatment of abdominal pain [35,36], which ended up in clinical trials for IBS (Irritable Bowel Syndrome) therapy. Its parent compound, MEN 14,268, which mainly differs at the C-terminal basic appendage, displayed slightly reduced antagonism and NK2 affinity, but higher apparent permeability [13]. Considering synthetic feasibility, MEN 14,268 appeared more attractive. For the purpose of the current study, MEN 14,268 was eventually simplified by replacing the benzothiophene unit with a simple benzylamine group, leading to a pseudo-peptidic NK2 antagonist pharmacophore SBL-OPNK-2 ( Figure 1). Finally, as a representative of a non-peptidic scaffold, Talnetant matched, as this compound combines good affinity (nanomolar range), selectivity, and antagonist activity of the NK3 receptor [13]. This small molecule, bearing a central quinoline core, exhibited great potential in inhibiting nociception associated with intestinal distension [37]. To avoid any potent steric hindrance between the opioid and the NK3 pharmacophore SBL-OPNK-3, it was decided to insert a short ether linker in place of the hydroxyl group on the quinoline ring ( Figure 1 and Scheme 2).
Synthesis of all pharmacophores and hybrids proceeded smoothly following adapted reported procedures, allowing access to an adequate amount of material for biological evaluation.
In vitro binding studies demonstrated that the conjugation of the NK2 and NK3 pharmacophores generally allows for the selectivity toward MOR and DOR originally acquired by the individual parent opioid pharmacophores to be maintained, while still being much less active on KOR. In the same way as their opioid parent Dmt-DALDA, the three hybrids SBL-OPNK-4, SBL-OPNK-6, and SBL-OPNK-8 all exhibited selectivity toward MOR, while unfortunately, the affinity and potency were reduced. In our hands, fusion of the Dmt-DALDA sequence with additional pharmacophores at the opioid's C-terminal end generally led to such a decrease in affinity and potency (unpublished results). On the other hand, the KGOP01-based hybrids, SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9, all displayed both good affinity and activity toward MOR and DOR, despite slightly lowered K i and EC 50 values; results that can be correlated to the fusion of an additional pharmacophore. It could, therefore, be suggested that the impact of NK2 and NK3 pharmacophores on opioid activity will strongly depend on the nature of the opioid pharmacophore. The data suggest that anchorage of any C-terminal appendage to the constrained structure of KGOP01 is more tolerated, which is in line with previous studies [19,38,39]. In light of the promising opioid data, the KGOP01-based hybrids were therefore selected for further biological evaluation, and NK2 and NK3 receptor binding assays were performed. NK pharmacophores SBL-OPNK-1 and SBL-OPNK-3 both displayed similar affinity and potency as reported in the literature, with the important side note that compound SBL-OPNK-3 represents an altered form of the literature compound (see Figure 1). Compound SBL-OPNK-2, however, failed to reach sufficient effects at the highest test concentrations. This result could be correlated to the development history of the FDA-approved Ibodutant and MEN 14,268, for which structural examination of the 'N-terminal' fragment was subjected to extensive efforts, underlining its critical impact on neurokinin efficacy [26]. Unlike the original benzothiophene moiety, the N-acetylbenzylamine group thus appeared detrimental for the activity and potency of SBL-OPNK-2 on NK2. It was therefore highly satisfying to note that this negative effect disappeared for the corresponding NK2 hybrid SBL-OPNK-7, which displayed the best biological profile with subnanomolar affinity and activity. The latter compound even outperformed hybrid SBL-OPNK-5 in terms of binding. It might be hypothesized that the KGOP01 pharmacophore adds extra key and stabilizing contacts with the neurokinin receptor binding site, a finding which was also previously noted for the opioid-neurokinin 1 receptor hybrid, SBCHM01 [28]. Structural analysis might be implemented in future studies to confirm this, but the observation of enhanced and (sub)nanomolar IC 50 and K i values, demonstrated for NK2 hybrid SBL-OPNK-5 and NK3 hybrid SBL-OPNK-9, also supports this hypothesis. It can be noted that the increase in affinity was slightly less important for SBL-OPNK-9, suggesting that the binding ability of the more compact NK3 moiety is less influenced by the opioid unit.
In this study, we disclosed, for the first time, a series of novel opioid agonist-nonopioid NK2 and NK3 receptor antagonist hybrid compounds as an unprecedented, yet promising approach toward innovative pain therapeutics. The NK2 and NK3 receptors have been overlooked and might provide beneficial effects compared to opioid-NK1 receptor ligands. As for any new multifunctional drugs, the challenge of conserving affinity and activity to both or more targets had to first be resolved, and the herein disclosed hybrids have eventually fulfilled this criterion. The covalent combination of neurokinin pharmacophores to the opioid agonist moiety globally improves neurokinin binding affinity, while maintaining low nanomolar µ and δ-opioid affinity and functional activity. These highly promising results will lead to further in vivo evaluation, which will be reported in due time.

Chemistry Peptide Synthesis
Peptide compounds KGOP01, SBL-OPNK-4, and SBL-OPNK-5 were assembled on Rink Amide resin (ChemImpex, Wood Dale, IL, USA-Loading 0.47 mmol/g) following iterative couplings of the required Fmoc-protected residues. Canonical L-amino acids (four equivalents with respect to the resin) were coupled in 30 min using a mixture of four equivalents HBTU/DIPEA in DMF. D-amino acids (2 equivalents with respect to the resin) were coupled for 1 h using a mixture of two equivalents HBTU/DIPEA in DMF. Fmoc-Aba-βAla-OH (1 equivalent), previously prepared according to the reported procedure [28], was coupled overnight using HBTU/DIPEA (one equivalent). Ultimately, 1.5 equivalents of Boc-Dmt-OH (commercially available) was coupled at the C-terminal position using DIC/Oxyma (1.5/3 equivalents) and stirred overnight. Full cleavage from the resin was carried out in standard conditions using a cocktail cleavage of TFA/TIS/H 2 O (95:2.5:2.5, v/v/v). Fully protected opioid pharmacophores Boc-Dmt-D-Arg(Pbf)-Phe-Lys(Boc)-OH 1 and Boc-Dmt-D-Arg(Pbf)-Aba-βAla-OH 2 were assembled on 2-chlorotrityl resin following standard conditions as previously described for Rink Amide resin procedures. Cleavage from the resin was carried out using HFIP/DCM (1:4, v/v), in order to preserve protecting groups for further coupling (detailed data available in the Supplementary Materials).

Drugs
Cell culture media and fetal bovine serum were obtained from Euroclone (Pero, Italy) and supplements were purchased from Invitrogen (Paisley, UK). Standard ligands (dermorphin, dynorphin A, and DPDPE) were from Sigma-Aldrich Chemical Co.  Membrane preparations were incubated at 25 • C for 120 min with an appropriate concentration of a tested compound in the presence of 0.5 nM radioligand in a total volume of 0.5 mL of Tris/HCl (50 mM, pH 7.4), containing bovine serum albumin (BSA, 1 mg/mL), bacitracin (50 mg/L), bestatin (30 µM), and captopril (10 µM). Non-specific binding was determined in the presence of 10 µM of naloxone. Incubations were terminated by rapid filtration through Whatman GF/B (Brentford, UK) glass fiber strips, which were presoaked for 2 h in 0.5 % polyethylamine using Millipore Sampling Manifold (Billerica, MA, USA). The filters were washed three times with 4 mL of ice-cold Tris buffer solution. The bound radioactivity was measured in Packard Tri-Carb 2100 TR liquid scintillation counter (Ramsey, MN, USA) after overnight extraction of the filters in 4 mL of Perkin Elmer Ultima Gold scintillation fluid (Wellesley, MA, USA). Three independent experiments for each assay were carried out in duplicate.
The data were analyzed by a nonlinear least square regression analysis computer program Graph Pad PRISM 6.0 (GraphPad Software Inc., San Diego, CA, USA). The IC 50 values were determined from the logarithmic concentration-displacement curves, and the values of the inhibitory constants (K i ) were calculated according to the equation of Cheng et al. [40].

Calcium Mobilization Assay
CHO cells stably co-expressing human recombinant MOR or KOR opioid receptors and the C-terminally modified Gα qi5 , and CHO cells co-expressing the human recombinant DOR opioid receptor and the Gα qG66Di5 chimeric protein were a generous gift from Prof. Girolamo Calo', Ferrara University, Italy and have been successfully used in our laboratory [30].
The tested compounds were dissolved in 5% DMSO in bi-distilled water to the final concentration of 1 mM. The successive dilutions were made in the HBSS/HEPES (20 mM) buffer (containing 0.005% BSA fraction V).
For the experiment, cells were seeded at a density of 50,000 cells/well into 96-well black, clear-bottom plates. After 24 h incubation, the cells were treated with the loading solution of the culture medium supplemented with 2.5 mM probenecid, 3 µM of the calcium-sensitive fluorescent dye Fluo-4 AM, and 0.01% pluronic acid for 30 min at 37 • C. The loading solution was aspirated and a 100 µL/well of the assay buffer (Hank's Balanced Salt Solution (HBSS) supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 µM Brilliant Black) was added.
After placing both plates (cell culture and compound plate) into the FlexStation III plate reader, the on-line additions were carried out in a volume of 50 µL/well and fluorescence changes were measured at 37 • C.
Agonist potencies were given as EC 50 representing the molar concentration of an agonist that produces 50% of the maximal possible effect. Concentration-response curves were fitted with the four parameters logistic nonlinear regression model: where X is the agonist concentration and n is the Hill coefficient. Ligand efficacy was expressed as intrinsic activity (α) calculated as the E max of the ligand to E max of the standard agonist ratio.
At least five independent experiments for each assay were carried out in duplicate. Curve fittings were performed using Graph Pad PRISM 6.0 (GraphPad Software Inc., San Diego, CA, USA). Data were statistically analyzed with one way ANOVA followed by the Dunnett's test for multiple comparisons; p values less than 0.05 were considered significant.

NK2 and NK3 Binding Assays
Binding affinities for neurokinin receptors, NK2 and NK3, were carried out through Eurofins' service according to standard procedures, in which affinities were determined by displacing [ 125 I]-labelled NKA and [ 3 H]-labeled SR 142801 (Table 4) [41,42]. The IC 50 values (concentration causing a half-maximal inhibition of control specific binding) and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting: where Y = specific binding; A = left asymptote of the curve; D = right asymptote of the curve; C = compound concentration; C 50 = IC 50 ; and nH = slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot ® 4.0 for Windows ® (© 2021 by SPSS Inc., Chicago, IL, USA).
The inhibition constants (K i ) were calculated using the Cheng Prusoff equation where L = concentration of radioligand in the assay; and K D = affinity of the radioligand for the receptor. A Scatchard plot is used to determine the K D .
Supplementary Materials: The following are available online, Scheme S1: Solid-phase peptide and peptidomimetic synthesis; Scheme S2: Solution-phase peptides and peptidomimetics synthetic pathways; Scheme S3: NK3 pharmacophore and hybrids synthetic pathway; Figure S1: Correlation between radioligand binding and calcium mobilization assays for MOR and DOR.