Pain remains a major global health concern affecting the worldwide population. Moderate to severe pain is nowadays treated by opioid analgesics such as morphine, oxycodone, and fentanyl that activate the μ-opioid receptor (MOR). Nonetheless, opioid drugs are causing several adverse effects including constipation, respiratory depression, sedation, and nausea, and their chronic administration is associated with dependence liability and analgesic tolerance. (1) Consequently, the treatment of chronic pain by opioids remains troublesome. In addition, the growing number of overdoses and deaths caused by misuse of and addiction to opioids is a public health concern. (2) To overcome the aforementioned opioid-related limitations and to combat the current opioid crisis, several strategies have emerged during the past decades. These include the development of drugs with opioid-independent actions (e.g., neurotensin, neuropeptide FF, cannabinoids, melanocortin, and substance P analogues), (3) the design of chimeric chemical entities showing two pharmacophores involved in pain regulation/signaling, most commonly an opioid coupled to a nonopioid part, (4) and generation of G protein-biased opioid agonists. (5)
As one of the nonopioid pharmacophores, neurotensin (NT) receptor ligands can be considered. NT is a natural neuropeptide composed of 13 amino acids, isolated by Carraway and Leeman in 1973 from bovine hypothalamus and later on from the bovine small intestine. (6) This neuropeptide is the endogenous ligand of three receptors: NTS1 and NTS2 belonging to the GPCR superfamily, whereas NTS3 also called sortilin is a single transmembrane domain receptor. (7) Over the past decades, NT and its receptors were shown to be responsible for or involved in various biological effects, such as food intake regulation, modulation of pituitary hormone release, and opioid-independent antinociception. (7) The discovery that the C-terminus hexapeptide, namely, NT(8-13) (H-Arg-Arg-Pro-Tyr-Ile-Leu-OH), could serve as NT’s minimal active sequence for receptor binding and activation led to increasing interest for NT analogues with improved pharmacokinetic features. (8)
Figure 1. Chemical structure of neurotensin and NT(8-13).
Although generally characterized by a rapid proteolytic degradation and poor blood–brain barrier (BBB) permeation, NT(8-13) analogues with potent antinociceptive activity have been described, particularly in acute, tonic, and neuropathic pain models. (9) Among the NT receptors, NTS1 and NTS2 were extensively associated with NT-induced analgesia, exerting naloxone and naltrexone-independent analgesic responses. (10) While NTS1 was initially identified as the main target for antinociception and several potent ligands were discovered, the focus gradually shifted to NTS2 to prevent NTS1-related physiological effects, such as hypotension and hypothermia. (9f) Hence, NTS2-selective, but also more generally NTS1/2, agonists represent a promising alternative to opioid analgesics for the treatment of chronic pain. As recently reported by our groups, modifications within the NT native sequence such as Tle 12, Dmt 11, or (6-OH)Tic 11 have a significant effect on NTS2/NTS1 selectivity and plasma stability. (11) Substitution of the two native basic residues Arg 8 and Arg 9 by β 3 hLys 8 and Lys 9 improves even more both selectivity and stability, as exemplified by peptide 2 (Figure 2A; NTS2/NTS1 selectivity > 1300; t 1/2> 24 h). (11)
Figure 2. (A) Previously described NT(8-13) analogues. (B) Chemical structure of the OP-NT chimeric peptide, PK20.
Ever since Morphy and Rankovich introduced the concept of designed multiple ligands (DMLs) or multitarget drugs, the field of medicinal chemistry has seen an extensive effort for developing more efficient and safer treatments for human diseases using such an approach. (12) Because pain is a highly complex physiological and psychological phenomenon involving different molecular targets, the use of multitarget compounds for effective analgesia has been shown to be a successful strategy. This is illustrated by several examples, particularly applying the fusion of opioid (OP) with nonopioid pharmacophores, such as substance P, NT, cholecystokinin, cannabinoids, melanocortin ligands, and their respective analogues. (4) More recently, our groups developed a new OP-neuropeptide FF ligand showing agonism at the MOR and antagonism at the NPFF receptors, exhibiting effective and potent analgesia in mouse models of acute and inflammatory pain as well as reduced opioid-induced adverse effects, including respiratory depression, hyperalgesia, tolerance, and withdrawal syndrome. (13) Several studies have also highlighted the potency of combining opioid and NT pharmacophores in order to obtain superior analgesia and reduced unwanted side effects. (14) Accordingly, Eiselt et al. recently described that the coadministration of morphine and a brain-penetrant Angiopep-2-conjugated NT(8-13) improved the analgesic/adverse effect ratio. (15)
Since peptidic opioid ligands such as endomorphin-2 or dermorphin derivatives exert their opioid activity through their N-terminal residues and NT(1-13) via its six amino acids at the C-terminus, those two pharmacophores could potentially be fused in a straightforward fashion. To date, only one chimeric OP-NT peptide between a modified endomorphin-2 pharmacophore and an NT(8-13) analogue was described, namely, PK20 (Figure 2B). (16) To yield PK20 starting from the native NT(8-13) sequence, Lys 8 and Lys 9 replaced the native Arg residues, Tyr 11 was substituted by a Phe, and Ile was changed for a Tle in order to improve enzymatic stability. Additionally, the N-terminal endomorphin-2 pharmacophore was modified to improve both enzymatic stability and affinity to the opioid receptors via incorporation of a 2′,6′-dimethyl-tyrosine residue (Dmt) and substitution of Pro for d-Lys. (16) This decapeptide was tested in vivo in the rat tail-flick test and showed a long-lasting, time-dependent antinociceptive activity. Antinociception resulted from an additive effect of the two antinociceptive systems, opioid and NT, since naltrexone administration only partially reduced the antinociceptive activity of PK20. (16) More recently, it was reported that the analogue [Ile 9]PK20 (Figure 2B), which shows a lowered potency in vivo when compared to the combination of both pharmacophores, causes less side effects, such as motor incoordination. (17)
Herein, we describe the design, synthesis, pharmacological evaluation, and structure–activity relationship (SAR) studies of a series of new OP-NT analogues (Figure 3). The OP-NT hybrid peptides were designed by fusing an MOR agonist derived from dermorphin and featuring an aminobenzazepinone (Aba) as a constrained Phe mimetic, KGOP01 (H-Dmt-d-Arg-Aba-β-Ala-NH 2), and NT(8-13) derivatives (Figure 3). This specific opioid segment was previously described as a balanced MOR/DOR agonist with better affinities and activities than the reference peptide [Dmt 1]DALDA. (18) The important role of the Dmt 1 residue in KGOP01 on ligand binding and activation of the MOR was also highlighted in a very recent molecular modeling study. (18c) In addition to a high metabolic stability and BBB permeation, the opioid tetrapeptide KGOP01 has been efficiently fused to other pharmacophores, including neuropeptide FF, neurokinin, and nociceptin antagonists, leading to bifunctional ligands with interesting pharmacological profiles. (18b,19) In the present study, in the search for potent MOR-NTS ligands, several NT sequence modifications, particularly on Tyr 11 and the two basic residues Arg 8 and Arg 9, were targeted in order to retain binding at the MOR and NT receptors. To this aim, pharmacological investigations were undertaken to evaluate the consequences of merging NT(8-13) analogues with the opioid agonist KGOP01 pharmacophore on binding to the opioid and NT receptors as well as activities at the opioid and NTS1 receptor, and the emerged SARs are reported.
Figure 3. Design strategy of new OP-NT hybrid peptides (with the 4-amino-1,2,4,5-tetrahydro-3 H-benzo[c]azepin-3-one “Aba” residue in the third position of the sequence).
In this study, we have designed OP-NT chimeric ligands based on a combination of the MOR/DOR agonist KGOP01 and NT(8-13) analogues (Figure 3 and Table 1). In order to preserve binding to both receptor types, the two pharmacophores were fused via a peptide bond between the C-terminal β-Ala residue of KGOP01 and the first basic N-terminal residue of the NT(8-13) analogues.
All sequence modifications are highlighted in bold.
Based on previous studies, published by our group and others, several modifications were introduced within the NT(8-13) native sequence. (20) For example, the benefit of a Tle residue in lieu of the native Ile 12 to improve proteolytic stability had been previously demonstrated. (21) Moreover, several studies highlighted the crucial importance of the aromatic residue in position 11 for NTS2 selectivity. (22) Since our recent results indicated that Dmt, (6-OH)Tic, and m-Tyr substitutions were well tolerated and led to an increased NTS2 selectivity, (11) those modifications were also introduced in the OP-NT chimeric analogues. For the same reason, modification was attempted within the dibasic N-terminal motif of NT(8-13) through insertion of a β 3-homo amino acid in position 8. (23) Knowing that the substitution of Arg 8-Arg 9 for Lys residues has been shown to be beneficial for NTS2 selectivity and activity in nonconjugated NT analogues lacking a second pharmacophore, (11) this modification was also introduced in the design and synthesis of OP-NT hybrid peptides (Table 1).
Here, all chimeric structures were synthesized by solution phase peptide synthesis (SPPS) following the Fmoc/t-Bu methodology using HBTU/DIPEA or DIC/Oxyma Pure as coupling mixtures. (24) The first attempts were performed with a preloaded Fmoc-Leu-Wang resin (0.83 mmol/g loading), resulting in inefficient coupling from the Pro residue onward. This low reactivity became even more critical during the opioid tetrapeptide assembly on resin with coupling conversions remaining under 60%. This resulted in very complex mixtures of deletion peptides and cumbersome purification by RP-HPLC, therefore tremendously reducing the synthetic yield.
In order to facilitate the purification, a fragment strategy was attempted. The opioid tetrapeptide was thus assembled on a 2-chlorotrityl resin and cleaved with an HFIP/DCM mixture. The resulting fully protected peptide was obtained in good yield without need for further purification and coupled on the resin-bound NT sequence. Although it resulted in a facilitated purification, the conversion was limited to 40% and the fragment approach was not retained as a decent alternative. In an attempt to improve the peptide assembly, the polystyrene-based Wang resin was replaced with a 0.25 mmol/g Fmoc-Leu-Wang TentaGel resin. Gratefully, due to the reduced loading and PEG enting, this resin gave easier coupling steps (2 to 4 h with 1.5 equiv of amino acid for challenging peptide bond formations) with both HBTU/DIPEA and DIC/Oxyma Pure as coupling cocktails. All the analogues prepared in this work were cleaved from the resin with a TFA/TIS/H 2 O 95:2.5:2.5 cleavage cocktail. After preparative HPLC purification, all NT analogues were obtained in yields ranging from 2.5 to 31% with an excellent purity (>95%).
Binding affinities at the human opioid receptors (μ-(MOR), δ-(DOR), and κ-(KOR)) and NT receptors (NTS1 and NTS2) of the new OP-NT hybrid peptides (Table 1) were first determined in competitive radioligand binding assays using membranes from Chinese hamster ovary (CHO) or 1321N1 astrocytoma cells stably expressing one of the recombinant human receptors (CHO cells for opioid and NTS1 receptors and 1321N1 cells for NTS2 receptors), according to the described procedures. (11,25) For comparison purposes, bindi
