**1. Introduction**

Persistent pain affects more than 30% of North America's population throughout their life and it attributes to substantial expense in the US with annual costs ranging between \$560 and \$635 billion, which is larger than the cost of the nation's priority health conditions [1]. This main socio-economic issue is expected to have a two-fold increase within the next 10 years especially in the elderly, as reported by the 2010 Medical Expenditure Panel Survey (MEPS). Despite the life- threatening effect caused by respiratory depression in overdose and the potential of high abuse, opioid analgesics stand as the conventional choice of treatment for moderate to severe pain [2–6]. As a result of misuse and extensive diversion, the use of opioids has become a leading crisis in the US, which was declared by the United States Department of Health and Human Service (HHS) in 2017 [7,8]. According to the Centers for Disease Control and Prevention (CDC), a significant increase in overdoserelated deaths occurred in 2020 in which the involvement of synthetic opioids was over 60% [9]. For this reason, several research institutes have made it a priority to develop safe, effective, and non-addictive therapeutics for chronic pain management and address opioid-use disorders with innovative medications, to save lives and encourage recovery.

Opioids exert their effect via opioid receptors, a member of a large superfamily of seven-transmembrane-spanning (7TM) G-protein-coupled receptors (GPCRs), mu (MOP receptor), kappa (KOP receptor), delta (DOP receptor), and nociceptin (NOP receptor) [10]. Since NOP receptors are distributed in various regions (dorsal root ganglia (DRG), spinal dorsal horn (SDH), and brain) that are involved in pain transmission, NOP receptor ligands are under investigation primarily as an alternative for MOP receptor opioid analgesic in addition to their anxiolysis and antidepressant-like effect [11–13]. However, the NOP

receptor was considered a controversial drug target for analgesics because of its unique pharmacological effects in pain modulation (antinociceptive vs. nociceptive effects) in the earlier phases of discovering nociceptin [14–19]. Currently, the NOP receptor has become a main focus as a promising target for analgesics as NOP receptor ligands have reported to show antinociceptive effects in non-human primates regardless of their administered doses and administration routes (spinal or supraspinal).

Moreover, the bifunctional and multifunctional NOP/opioid receptor agonists have recently displayed potent antinociceptive activity with favorable side effect profiles. Among these agonists, cebranopadol represents a promising therapeutic candidate for pain, according to the results of its clinical trials. In this article, the current literature for NOP receptor's crystal structure, distribution, signaling pathway, and the rational design of NOP receptor ligands with various pharmacological profiles as a promising alternative for conventional opioid analgesic is reviewed to assess its therapeutic potential as analgesics.

#### **2. Structure of NOP Receptor**

In the mid 1990s, the human cDNA clone that encodes the NOP receptor was first isolated from the human and mouse brainstem and was then identified in several murine genomes including rat, pig, and guinea pig [20–25]. It was previously known as "orphanin FQ", "nociceptin,", or "ORL-1" for opioid receptor-like 1 receptor, due to the lack of its endogenous peptide ligand in the binding assays; however, nociceptin or orphanin FQ (N/OFQ) that closely resemble dynorphin A, a selective KOP receptor endogenous peptide, was characterized a year later by applying reverse pharmacology as the endogenous neuropeptide for NOP receptor [14,15]. This endogenous neuropeptide has 17 amino acids, Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln, which have quite unique features. The Phe-Gly-Gly-Phe amino terminal is noticeably comparable to the Tyr-Gly-Gly-Phe that is conserved in other classical opioid peptides [26,27]. Moreover, the number of Lys and Arg residues that are found in N/OFQ are similar to dynorphin A. Along with this resemblance, the gene structure of opioid peptide genes (preprodynorphin and preproenkephalin) and nociceptin precursor gene are also similar [27,28]. Multiple conserved amino acid residues and motifs specifically in the transmembrane helices and the intracellular loops have been identified by comparing the cDNA-derived amino acid sequence of the NOP receptor protein with that of other opioid receptors, indicating that NOP receptor belongs to GPCR Class A (rhodopsin-like) receptors, as the fourth and last characterized opioid receptor [29]. Consequently, the IUPHAR nomenclature defined this receptor and its peptide which are currently named NOP receptor and N/OFQ [30].

To date, three crystal structures of human NOP receptor have been solved with three piperidine-based antagonists (Banyn Compound-24 (C24), SB-612111, and Compound-35 (C-35)) at a resolution of 3 Å [31,32]. These crystal structures provide a perspective into the atomic details of the molecular structure of the NOP receptor and support previous homology models developed to further understand the functional mechanism of NOP receptor. In all three structures, the protonated nitrogen of the piperidine interacts with the D1303.32 (superscripts indicate the Ballesteros Weinstein TM helix residue numbering) residue in NOP receptor which leads to the formation of a salt bridge, implying the high affinity for these ligands. Consistent with NOP receptor crystal structure in its inactive state, the previous homology models of NOP receptor in complex with N/OFQ further support that the N-terminal amino groups of an endogenous neuropeptide agonist, N/OFQ, interact with D1303.32 [33–35]. This indicates the important role of this residue which is conserved in other canonical opioid receptors on the binding of NOP receptor ligands. Moreover, the replacement of D1303.32 into alanine or asparagine in the mutagenesis studies abolished N/OFQ binding, emphasizing the negative charge essentiality at this location [32].

Computer aided molecular docking studies of the selective NOP receptor agonist Ro 64-6198 into the first active state NOP receptor homology model, have also indicated signs for the mentioned NOP receptor selectivity enhancing of interactivity [34]. In this model, the amide hydrogen in Ro 64-6198 directly interacts with T3057.39 to form a hydrogen

bond that takes place at the extracellular end of the orthosteric binding site, while the phenalenyl ring of Ro 64-6198 and the hydrophobic V2796.51 residue interact together inside the binding site.

Despite these studies that have highlighted the key residues and structures that are involved with ligand binding, receptor activation, and signaling, the determination of NOP receptor in its active state is required to illuminate the conformational changes in receptor's architecture.

#### **3. The Distribution and Signaling Pathway of NOP Receptor**

Several techniques and animal model including in situ hybridization, immunohistochemistry, autoradiography, RT-PCR, knock-in mice with a fluorescent-tagged NOP receptor (NOP receptor-eGFP) in place of the native NOP receptor, and [35S]-GTPγS assay were employed to reveal the tissue distribution of NOP receptor. It is widely expressed in the human and other animal species both in the central and peripheral nervous systems [12,36]. Peripherally, the human immune cells (lymphocytic B and T-cell lines, monocytic cell lines, and circulating lymphocytes and monocytes) express NOP receptor mRNA [37]. Centrally, the NOP receptor mRNA is detected in the cortical areas, hypothalamus, mammillary bodies, the substantia nigra, thalamus nuclei, limbic structures (the hippocampus and amygdala), brainstem (colliculi, the ventral tegmental area, the locus coeruleus), and the pituitary gland [37,38].

Because NOP receptor activation modulates several physiological functions and pharmacological roles including, but not limited to, pain sensation, mood, learning, memory, cardiovascular control, and immune response [39], it is important to understand its signaling pathways and subsequent trafficking events. NOP receptor has shown a high sequence identity and homology in the TM helices and intracellular loops with other classical opioid receptors (DOP receptor, MOP receptor, and KOP receptor) which couple to inhibitory G proteins, suggesting a similar activation mechanism upon ligand binding. This binding triggers the heterotrimeric dissociation of Gαβγ subunits following the replacement of guanosine diphosphate (GDP) by guanosine triphosphate (GTP) at Gα subunit and subsequently induces multiple intracellular signaling pathways [40,41]. The dissociated Gα subunit suppresses adenyl cyclase and cAMP production, while Gβγ subunits directly couple with different ion channels such as Ca2+ and Kir3 [42–44]. NOP receptor also regulates the voltage-dependent Ca2+ channels by modulating Rho-associated coiled-coil-containing protein kinase (ROCK) and LIM domain kinase (LIMK) [45]. Like canonical opioid receptor, the suppression of pre and postsynaptic Ca2+ influx, the activation of G protein gated inward rectifying potassium (GIRKs) conductance, as well as the inhibition of various ions channels such as Na<sup>+</sup> channel resulted in cellular hyperpolarization and attenuation of neuronal excitability and nociceptive stimuli transmission, thus producing antinociceptive effects [46]. In addition to ion channels, the activation of NOP receptors also modulates all three mitogen-activated protein kinase (MAPK). MAPK activity thereby regulates cell proliferation, progression, and differentiation (ERK1/ERK2), as well as the response to cellular stressors (p38 and JNK1/JNK2/JNK3) [47,48]. Moreover, the neurotransmitter release of serotonin, noradrenaline and glutamate, as well as the phospholipase (PLC) A2 and C signaling, are induced by NOP receptor activation [49–52].

Within minutes of NOP receptor activation, the uncoupling of NOP receptor to G protein is facilitated by a desensitization process, a feedback mechanism to control the receptor overstimulation during acute and chronic exposure to the ligand [53]. This process is regulated by various kinases such as GPCR kinases (GRKs) that mediate the phosphorylation, and the arrestin ligation to the C-terminus of the opioid receptor. Besides GRKs, second messenger-dependent protein kinases including protein kinase A (PKA), protein kinase C (PKC), and calcium/calmodulin-dependent protein kinase II have also been shown to phosphorylate and desensitize the NOP receptor [54,55]. The receptor desensitization is suppressed through the inhibition of mitogen activated protein dependent kinase that could interfere with the arrestin recruitment. After the GRK/arrestin recruitment, the

NOP receptor is translocated into the intracellular compartment through clathrin-mediated endocytosis into which the receptor is recycled and re-sensitized to restore the receptor function back again.

#### **4. Ligands of NOP Receptor**

Analgesia is one of the potential clinical indications of NOP receptor due to its wide distribution in the nervous system (central and peripheral) which are involved in the pain processing pathways. In this review, NOP receptor ligands including N/OFQ-related peptides, nonpeptidic, and bifunctional compounds with different pharmacological profiles (full agonist, partial agonist, and antagonist) that represent viable drug target for pain are spotlighted. Initially, intrathecally (i.t) administered N/OFQ produces dose-dependent analgesia in the tail flick assay and flinching behavior in the formalin test without causing sedation as well as promotes antinociceptive effect of morphine in both rats and monkey [16–19]. Whereas opposite effects like hyperalgesia and a decrease in locomotor activity are produced as a result of the intracerebroventricular (i.c.v) N/OFQ administration in the hot plate test and the tail flick test in mice [14,15]. The unexpected action of i.c.v. N/OFQ administration resulted from both the anti-opioid activity (antagonizing MOP receptor, DOP receptor, and KOP receptor antinociception activity) via NOP receptor stimulation in the periaqueductal gray (PAG) neurons and the reversal of opioid induced analgesia of N/OFQ opposed to the nociceptive activity as proposed previously [56–58]. These findings indicate the dual actions of N/OFQ that mainly depend on the administered dose, pain models (chronic or acute), examined species, and the route of administration as illustrated in Figure 1. The reason behind this discrepancy across species is not known yet; however, some studies (reviewed in [29,59]) suggest that the difference in neuronal circuitry of pain between different species could be the reason for NOP receptor system having opposite effects in pain processing. Furthermore, the effectiveness of NOP receptor agonists in addressing chronic pain (over acute pain), can be explained by the varying levels of NOP receptor mRNA and NFQ peptide induced by chronic inflammation.

**Figure 1.** N/OFQ effect in rodent and non-human primates on pain response. (**A**) Supraspinal administration of N/OFQ produces hyperalgesia and blocks morphine-induced analgesia in rodent, whereas the opposite effect of analgesia and the promotion of an antinociceptive effect are produced in non-human primates. (**B**) Spinal administration of N/OFQ produces dose-dependent analgesia in both rodent (nanomoles and higher doses) and non-human primates (nanomoles and ultra-low doses) as well as promotes an antinociceptive effect of morphine, while ultra-low doses of N/OFQ induce hyperalgesia in rodent.

In this section, the relevant pharmacological features of NOP receptor ligands including peptide, nonpeptide, and bifunctional and mixed NOP receptor compounds are explored with a focus on their role in modulating pain to further comprehend the nature of the N/OFQ–NOP receptor system within these processes.

### *4.1. Peptide Ligands Related to N/OFQ Targeting Pain*

Earlier systematic SAR studies revealed that both truncation and amidation of N/OFQ are required to sustain the biological activity of N/OFQ and avoid the N-terminus degradation by proteases, respectively [60]. As a result, N/OFQ(1-13)-NH2, which is the shortest peptide sequence that maintains the potency, efficacy, and affinity as N/OFQ, has been used as a template to design a new series of N/OFQ analogues. In the frame of SAR studies, several peptides with distinct pharmacological activity have been identified such as [Phe1Ψ(CH2-NH)Gly2]N/OFQ(1-13)NH2, UFP-101, and [Nphe1]N/OFQ(1-13)NH2 (NOP receptor antagonists), UFP-112 (highly potent NOP receptor agonist), and UFP-113 (partial NOP receptor agonist) [61–65]. The peptides that have antinociceptive activity are summarized in Table 1 and described below.

**Table 1.** The peptides that have antinociceptive activity are summarized.



### **Table 1.** *Cont.*

#### 4.1.1. [Nphe<sup>1</sup> ]N/OFQ(1-13)NH2

A preliminary hypothesis regarding the behavior of NOP receptor-active compounds stated that if N/OFQ induces pain, antagonists are likely to exhibit antinociceptive activity. To test this, [Nphe<sup>1</sup> ]N/OFQ(1-13)-NH2, the first reported peptide with antagonist activity, was generated by shifting the side chain of Phe<sup>1</sup> from C to N atom in the amidated N/OFQ. A binding assay using Chinese hamster ovary (CHO) cells that express recombinant human NOP receptor and cyclic AMP accumulation in CHO cells identified the antagonistic properties of [Nphe<sup>1</sup> ]N/OFQ(1-13)-NH2. The mouse tail withdrawal assay revealed that a single i.c.v. administration of [Nphe<sup>1</sup> ]N/OFQ(1-13)NH2 increased the tail withdrawal latency time, while a combinational administration of [Nphe<sup>1</sup> ]N/OFQ(1-13)NH2 with N/OFQ and morphine inverted the reduction of tail withdrawal latency time latency and promoted the antinociceptive effect of morphine, respectively, implying the analgesic action of this ligand [61,72].

#### 4.1.2. [Nphe<sup>1</sup> , Arg14, Lys15]N/OFQ-NH<sup>2</sup> (UFP-101)

Previous studies have shown that the binding of C-terminus-amidated N/OFQ to the acidic restudies at the ECL2 of NOP receptor was enhanced by inserting Arg and Lys [35,73]. Combination of [Nphe<sup>1</sup> ]N/OFQ(1-13)-NH<sup>2</sup> and [Arg14, Lys15]N/OFQ-NH<sup>2</sup> led to the generation of a new peptide [Nphe<sup>1</sup> , Arg14, Lys15]N/OFQ-NH2, also called UFP-101 [62]. In vitro assays including functional binding assays using (CHO cells expressing human NOP receptor and [35S]-GTPγS), cyclic AMP accumulation experiment, and Schild regression analysis of electrically stimulated isolated peripheral (rats, mice, and guinea pigs) and central tissues (rat) showed that UFP-101 competitively antagonized the effects of N/OFQ. Similar to [Nphe<sup>1</sup> ]N/OFQ(1-13)-NH2, i.c.v. administration of 10 nmol UFP-101 produced antinociceptive effect in the mouse tail withdrawal assay, but with higher potency and longer duration of action, indicating that the presence of Arg<sup>14</sup> and Lys<sup>15</sup> may promote either the binding of UFP-101 to NOP receptor and/or the UFP-101 metabolic stability. Since UFP-101 is a selective NOP receptor antagonist, it has been also used as a research tool to confirm that NOP receptor mediates both the inhibition of spinal excitatory transmission in vitro as well as the spinal antinociception in vivo [66].
