Feature Paper
Article
27 May 2021
Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund’s adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.
Effective pain treatment, particularly chronic pain, remains an unmet medical need at the beginning of the 21st century. While opioid-based pharmacotherapy is still the most powerful strategy for the treatment of moderate to severe pain, the risk–benefit ratio is suboptimal because of frequent and serious side effects [1]. The dramatic increase in the medical use and misuse of opioids with the concurrent rising number of overdose deaths and opioid-use disorders has led to the current opioid crisis [2,3]. Hence, research efforts are needed to overcome the limitations of present therapies with the aim to improve treatment efficacy and reduce complications.
The µ-opioid receptor (MOR) is a member of the opioid system, together with the δ- (DOR), κ- (KOR), and nociceptin (NOP) receptors and their endogenous peptides [4], modulating both nociception and reward systems [5]. The majority of clinically used opioids are agonists to the MOR, and these are vastly misused and abused [1]. Alternative chemical and pharmacological strategies are therefore evaluated to mitigate the deleterious effects of opioids, amongst which are multifunctional ligands, G protein-biased agonists, peripherally restricted opioids, and abuse-deterrent formulations of existing opioids [6,7,8,9,10]. Furthermore, the available structures of the MOR provide a valuable opportunity for computational drug design and discovery [11,12,13].
The concept of ‘one molecule, multiple targets’ received increased attention in the opioid research over the past years as a promising approach for the discovery of effective and safer opioids [6,8,14,15,16,17,18]. Bifunctional ligands targeting the MOR simultaneously with other (opioid/non-opioid) neurotransmitter systems implicated in pain processing and/or opioid-induced side effects are of particular interest [8,19,20,21].
The recently discovered bifunctional peptide-based hybrid KGNOP1, H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 (Dmt: 2′,6′-dimethyl-L-Tyr; Aba: 4-amino-tetrahydro-2-benzazepinone), combines a MOR pharmacophore, H-Dmt-D-Arg-Aba-β-Ala-NH 2, and a NOP receptor pharmacophore, H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 [22]. KGNOP1 was reported to produce effective antinociception in rodent models of acute nociception and neuropathic pain with a lower propensity for respiratory depression than conventional opioids [22,23,24]. In this study, we further investigated the in vivo effects of KGNOP1 in mouse models of acute and chronic inflammatory pain after subcutaneous (s.c.) administration and assessed potential opioid liabilities for locomotor dysfunction and rewarding and withdrawal effects after chronic treatment, in comparison to the clinically relevant morphine. Furthermore, we provide new understanding on KGNOP1’s mechanism of action and report the first structure-based investigation on KGNOP1 binding to the structures of the MOR [12] and NOP receptors [25].
We re-evaluated the binding properties of KGNOP1 to the human MOR, DOR, KOR, and NOP receptors by radioligand binding to membrane preparations from Chinese hamster ovary (CHO) cells overexpressing recombinant receptors, as described previously [26,27] (Figure 1A). KGNOP1 demonstrated subnanomolar affinity (K i = 0.42 nM) for the MOR, while having lower binding affinities for the DOR and KOR (Table 1). KGNOP1 also bound to the NOP receptor (K i = 141 nM), although with reduced affinity in comparison with the other opioid receptors, as determined in competitive radioligand binding assays, and also aligned with previously reported data [22].
Initial data on the in vitro functional activity of KGNOP1 were reported in guinea pig ileum and mouse vas deferens bioassays [22], with KGNOP1 depicted as a potent MOR agonist. In the current study, we assessed the in vitro activity of KGNOP1 to the human opioid receptors using the guanosine 5′-O-(3-[35 S]thio)triphosphate ([35 S]GTPγS) functional assay with membranes of CHO cells expressing the MOR, DOR, KOR, or NOP receptors [26], with agonist potency (ED 50) and efficacy (E max) values shown in Table 1. Stimulation of the [35 S]GTPγS binding induced by KGNOP1 was compared to the effect of reference full agonists [D-Ala 2,N-Me-Phe 4,Gly-ol 5]enkephalin (DAMGO, MOR), [D-Pen 2,D-Pen 5]enkephalin (DPDPE, DOR), U69,593 (KOR), and nociceptin (NOP receptor). As shown in Figure 1B, KGNOP1 produced a concentration-dependent increase in the [35 S]GTPγS binding with the highest potency and full efficacy to the MOR. At the DOR and KOR, KGNOP1 showed reduced potencies, while displaying full and partial agonism, respectively (Table 1). Furthermore, KGNOP1 showed full efficacy for the G protein activation using the [35 S]GTPγS binding assay in NOP receptor-expressing CHO cells (E max = 99%), albeit with a very low potency (Figure 1B, Table 1).
Intraplantar administration of the formalin solution to the mouse hindpaw induces a pain response in a biphasic manner [28]. The first phase is characterized by the acute activation of nociceptors, while the second phase involves an inflammatory reaction in the peripheral injured tissue. We used this preclinical pain model for acute inflammatory pain to assess the antinociceptive effects of KGNOP1 after s.c. administration to mice, and to compare its effect to that of morphine. Systemic administration of KGNOP1 and morphine produced a dose-dependent reduction in pain behavior of formalin-injected mice, determined as the amount of time (in seconds, sec) each animal spent licking, biting, lifting, and flinching the formalin-injected paw (Figure 2A,B and Table S1), with an almost complete inhibition of the pain response counted between 15 and 60 min in mice treated with KGNOP1 (1.22 µmol/kg) and morphine (15.5 µmol/kg). Both KGNOP1 and morphine attenuated the nociceptive response during Phase I of the formalin assay with a significant effect at all tested doses of KGNOP1, and at 7.77 and 15.5 µmol/kg of morphine (Figure 2C). During Phase II, KGNOP1 and morphine also produced a dose-dependent decrease in pain response with a significant effect at 0.49 and 1.22 µmol/kg of KGNOP1, and at 7.77 and 15.5 µmol/kg of morphine (Figure 2D). The calculated antinociceptive ED 50 values for KGNOP1 in the acute nociceptive Phase I and inflammatory Phase II of the formalin test were 1.17 µmol/kg (95% confidence limits, CL, 0.41–3.35) and 0.55 µmol/kg (95% CL, 0.22–1.33), respectively, and for morphine in Phase II was 6.44 µmol/kg (95% CL 3.20–12.7) (Table S1).
To evaluate the involvement of the MOR and/or NOP receptors in KGNOP1-induced antinociception in the formalin test, the effect of the MOR antagonist naltrexone and NOP receptor antagonist SB-612111 was tested [29,30] (Figure 2E,F). Pre-treatment of mice with naltrexone (2.6 µmol/kg, s.c.) resulted in a significant antagonism of the antinociceptive effect of KGNOP1 in both Phase I and II. The NOP receptor selective antagonist SB-612111 (6.6 µmol/kg, s.c.) also significantly reversed the antinociceptive response during the nociceptive and inflammatory phase of the formalin test (Figure 2E,F).
Chronic inflammatory pain was induced by injection of Complete Freund’s Adjuvant (CFA) [31] to the dorsal side of the right hindpaw, evidenced by a significant reduction at 72 h post-inoculation in paw withdrawal thresholds to thermal and mechanical stimulation (Figure 3). In this study, mice were treated s.c. with saline, KGNOP1 (0.49 and 1.22 µmol/kg), or morphine (15.5 µmol/kg), and tested for thermal and mechanical sensitivity using the Hargreaves (Figure 3A) and von Frey tests (Figure 3B), respectively.
