Progress in the development of more effective and safer analgesics for pain management

European Journal of Medicinal Chemistry 183 (2019) 111701

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Progress in the development of more effective and safer analgesics for pain management Rita Turnaturi a, *, Santina Chiechio b, c, Loredana Salerno a, Antonio Rescifina d,
a, Giuseppina Cantarella e, Emilia Tomarchio f, Carmela Parenti b, 1, Valeria Pittala Lorella Pasquinucci a, 1 a

Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria 6, 95125, Catania, Italy Department of Drug Sciences, Pharmacology and Toxicology Section, University of Catania, Viale A. Doria 6, 95125, Catania, Italy Oasi Research Institute-IRCCS, Troina, Italy d Department of Drug Sciences, Chemistry Section, University of Catania, Viale A. Doria, 95125, Catania, Italy e Department of Biomedical and Biotechnological Sciences, Pharmacology Section, University of Catania, Catania, Italy f
1 Clinica Ortopedica, ASST Gaetano Pini-CTO, Milan, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 June 2019 Received in revised form 26 August 2019 Accepted 12 September 2019 Available online 16 September 2019

Opioid analgesics have been used for thousands of years in the treatment of pain and related disorders, and have become among the most widely prescribed medications. Among opioid analgesics, mu opioid receptor (MOR) agonists are the most commonly used and are indicated for acute and chronic pain management. However, their use results in a plethora of well-described side-effects. From selective delta opioid receptor (DOR) and kappa opioid receptor (KOR) agonists to multitarget MOR/DOR and MOR/KOR ligands, medicinal chemistry provided different approaches aimed at the development of opioid analgesics with an improved pharmacological and tolerability fingerprint. The emergent medicinal chemistry strategy to develop ameliorated opioid analgesics is based upon the concept that functional selectivity for G-protein signalling is necessary for the therapeutic effect, whether b-arrestin recruitment is mainly responsible for the manifestation of side effects, including the development of tolerance after repeated administrations. This review summarises most relevant biased MOR, DOR, KOR and multitarget MOR/ DOR ligands synthesised in the last decade and their pharmacological profile in “in vitro” and “in vivo” studies. Such biased ligands could have a significant impact on modern drug discovery and represent a new strategy for the development of better-tolerated drug candidates. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Mu opioid receptor Delta opioid receptor Kappa opioid receptor SAR Analgesia

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1. MOR biased ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2. DOR biased ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.3. KOR biased ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4. Multitarget MOR/DOR biased ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.5. Molecular modelling and computational studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

* Corresponding author. E-mail address: [email protected] (R. Turnaturi). 1 C.P. and L.P. contributed equally to this work. https://doi.org/10.1016/j.ejmech.2019.111701 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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Abbrevation KOR DOR MOR GPCR KO SAR s.c. FDA MAP ERK1/2 EM1 EM2

kappa opioid receptor delta opioid receptor mu opioid receptor G-protein coupled receptor knockout structure activity relationship subcutaneous food and drug administration mitogen-activated protein extracellular signal-regulated protein kinases 1 and 2 endomorphin 1 endomorphin 2

1. Introduction Clinically used opioid drugs, including morphine, provide strong analgesic effect, but their use is also associated with tolerance, physical dependence, constipation, respiratory depression, and nausea [1]. Opioid rotation or opioid switching, the process of changing one opioid to another, is a therapeutic strategy aiming a favourable balance between benefits and risks over time [2]. However, the efficacy of this approach remains unclear since there are no opioid compounds devoid of unfavourable side-effects observed after chronic administration. Thus, it remains an unmet need the study and development of opioid analgesics. To address this issue, the medicinal chemistry provided different approaches. Pioneering tactic consisted in the synthesis of selective kappa and delta opioid receptors (KOR and DOR) agonists to overcome mu opioid receptor (MOR) agonists adverse effects. However, this strategy resulted unsuccessful [3], and their development was discouraged because, KOR agonists elicit, together with a consistent analgesic effect, the simultaneous occurrence of dysphoria. On the other hand, the weak effect of DOR agonists to counteract acute pain made their development useless. Next strategy adopted by medicinal chemistry to develop better analgesics is the well-known multitarget opioid approach [4,5] to overcome typical side effects associated with selective opioid agonists. This approach, based on the paradigm “one molecule, multiple targets”, in the opioid research field came out by the numerous experimental data showing the co-localization and the existence of physical and functional modulatory interactions between MOR/DOR and MOR/KOR [6]. For instance, an improved tolerability profile was reported for multitarget MOR/KOR compounds [7,8]. Potent analgesic effect with lower propensity to produce tolerance and physical dependence was reported for both multitarget MOR/DOR agonist [9,10], and MOR agonist/DOR antagonist ligands [11e13]. Recently, the concept of biased agonists [14e16], able to differentially activate G-protein coupled receptor (GPCR) downstream pathways, became a new approach in the design of novel drug candidates. Classical opioid drugs mediate their effects through the activation of GPCRs. According to traditional receptor theory, activation of a GPCR produces signalling through G-proteins to regulate second messengers and its prolonged activation, by repeated agonist exposure, causes b-arrestin-mediated desensitisation and internalisation. Recently, the receptor theory was enlarged, including the concept of functional selectivity, also known as biased agonism. This theory is based on the feature of different ligands of the same receptor to stabilise various receptor active states, which determines the activation of diverse signalling pathways. Thus, a

Dmt dimethyltyrosine Map 2-methylene-3-amino propanoic acid i.c.v. intracerebroventricular i.v. intravenous GPI guinea pig ileum MVD mouse vas deferens TM transmembrane EC extracellular CCI chronic constriction injury Cx43 connexion 43 PRESTO-TANGO parallel receptor-ome expression and screening via transcriptional output-transcriptional activation following arrestin translocation PAM positive allosteric modulator

so-called “biased agonist” preferentially activates one signalling pathway over another [17,18]. Referring to opioid pharmacology, a lot of different studies have highlighted the benefits of biased signalling that prefers G-protein signalling to b-arrestin engagement. Using b-arrestin-2 knockout (KO) mice, it was established that specific opioid side effects could be attendant with b-arrestin-2 recruitment [19]. MOR b-arrestin recruitment has been associated with tolerance, constipation and respiratory depression development. For DOR, G-protein activation reduces alcohol intake in mice, whereas b-arrestin increased alcohol use. Moreover, for this receptor, b-arrestin recruitment is involved in its internalisation that could result either in lysosomal trafficking and subsequent degradation [20] or in receptor recycling. For KOR, it has been demonstrated that the dysphoric effects of KOR agonists are mediated by the arrestin-dependent activation of p38 mitogen-activated protein kinase, while the analgesic effects are mediated only through G-protein signalling [21]. Although several drug targets have been emerging as new therapeutic options to treat different types of pain [22e26], the development of opioid ligands which exclusively or preferentially activate G-protein signalling without eliciting b-arrestin recruitment, represent a new goal in opioid medicinal chemistry. The purpose of this review is to highlight recent advances in the development of opioid biased ligands with a potential safer analgesic profile for the management of different pain states, according to discoveries achieved during the last ten years. 1.1. MOR biased ligands Through the screening of the RIKEN Natural Products Depository (NPDepo) chemical library, constituted by 5848 compounds, Nikaido et al. [27] identified the compound 2-[({2-[2ethyl-4-(2-methoxyphenyl)-2-methyl-2H-3,4,5,6tetrahydropyran-4-yl] ethyl} amino) methyl] phenol, named GUM1 (1, Fig. 1), as a structurally unique opioid ligand with an unusual and new structural scaffold. Indeed, it does not possess the morphine structural requirements for MOR interaction. Using Sf9 cells expressing MORGi1a, KOR-Gi1a, or DOR-Gi1a, the [35S]GTPgS binding of GUM1 was measured and compared to reference compounds. GUM1 showed a functional profile similar to morphine, being able to stimulate MOR (EC50 ¼ 1.1 mM) and KOR (EC50 ¼ 3.4 mM) in the micromolar range. In rat plantar test this compound, subcutaneously (s.c.) administered, elicited dose-dependent naloxone-reversed antinociception in a dose range lower than morphine (0.3e3 mg/kg vs. 1e10 mg/kg). Interestingly, GUM1 and morphine produced a similar EC50 but a different ED50, being GUM1 more potent than morphine. In

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Fig. 1. GUM1 structure.

regimen of daily injection GUM1, similarly to morphine, showed tolerance-inducing capability. However, this compound had significant analgesic properties in morphine-tolerant rats. This evidence prompted authors to hypothesise for GUM1 a way to bind opioid receptor such to induce biased signal transduction. By screening a private compound collection, Chen et al. [28] identified the hit compound 2 (Fig. 2), a 4-phenyl-4-(2benzylaminoethyl)-tetrahydropyran analogue, as a MOR agonist with the capability to promote the G-protein recruitment over barrestin in the submicromolar range. Structure-activity relationship (SAR) studies demonstrated the importance of alkyl substituents at position 2 of the tetrahydropyran ring. An increased G-protein and mostly b-arrestin MOR recruitment were rescued for the derivative 3, with fluorine at the position 4 of the phenyl ring (Fig. 2). Based on the increased MOR potency, compound 4, an analogue of 3 with a diethyl group at position 2 of the tetrahydropyran ring (Fig. 2), was synthesised. The good MOR G-protein and b-arrestin profile of compound 4 led to the development of analogues bearing spirocyclic groups at position 2 of the tetrahydropyran ring. Among them, compound 5 (Fig. 2) retained the functional profile, and its R enantiomer was 16-fold more potent in the cAMP assay with a 5fold lower b-arrestin efficacy than its antipode. Replacement of the 4-fluorophenyl group with 2-pyridine ring conducted to compound R-6 (Fig. 2) with an improved in vitro profile. In compound R6 the replacement of the phenyl group with 2-chlorophenyl, 2-, 3or 4-pyridyl, and 2-pyrazyl, gave MOR partial agonists. Potent MOR G-protein agonist activity with low b-arrestin recruitment capability was found for derivatives with 4-pyrimidyl, 2-thiophenyl and 3-thiophenyl. Among them, the 2-thiophenyl derivative R-7 (Fig. 2), after s. c. administration, showed a more potent antinociceptive effect than morphine in the mouse hot plate test with an ED50 5fold lower than morphine. Through the mouse glass bead retention model of colonic motility and the mouse fecal boli accumulation model, R-7, at equianalgesic morphine dose, induced less constipation. In light of the promising in vitro and in vivo profile of R-7, further SARs were performed replacing the 2-thiophene with 2-furan and introducing methyl or methoxyl groups in different positions. In general, the resulting derivatives showed an ameliorated MOR Gprotein profile and reduced b-arrestin recruitment. Similarly to R-7, compound R-8 (Fig. 2), named TRV130, resulted more potent than morphine in rodent acute nociceptive models. Moreover, TRV130 possess a better therapeutic index, inducing less constipation, respiratory depression and cardiovascular liability [29]. TRV130, known as oliceridine, finished phase 3 clinical trials for the treatment of moderate to severe acute pain [30e32] and it is awaiting

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the approval from U.S. Food and Drug Administration (FDA) [33]. By this investigation, the tetrahydropyran scaffold was optimised as new MOR chemotype, and its versatility was outlined. Indeed, the chance to structurally modify the tetrahydropyran scaffold to selectively address the efficacy profile versus G-protein over barrestin was demonstrated, consistently with the idea that distinct receptor conformations are stabilised by biased and unbiased ligands. Contrarily to previous SAR stating that the methyl substitution at the benzylic position of R-6 resulted in 640-fold potency loss, Xin et al. [34] fused the benzylic position to the aromatic ring obtaining compound 9, named SHR9352 (Fig. 3). This structural modification afforded a remarkably high MOR potency (EC50 ¼ 0.77 nM), with minimal b-arrestin recruitment (Emax ¼ 18.8%). The resulting MOR biased agonist profile was tested in vivo. SHR9352 was effective in the rat incision assay at a dose lower than TRV130 and, in the mouse charcoal meal model, at the same dose, it did not produce constipation. This study demonstrated that biased ligands could be discovered through empirical SAR-based lead optimisation. The structural modification of the TRV130 thiophene ring deepens better the interaction with amino acids relevant to the biased G-protein signalling. An interesting MOR biased agonist emerged by SAR conducted on Salvinorin A (10, Fig. 4), a natural diterpene KOR agonist isolated from Salvia divinorum [35]. In Salvinorin A, the replacement of acetoxy group in C-2 with the phenoxy one led to Herkinorin (11, Fig. 4) that evidenced 47-fold affinity loss at KOR (Ki ¼ 90 nM) and 25-fold affinity increase at MOR (Ki ¼ 12 nM) in comparison to Salvinorin A [36]. Functional studies showed Herkinorin, a full MOR (Emax ¼ 130%) and KOR (Emax ¼ 140%) agonist, as the first example of a nonnitrogenous MOR agonist. Herkinorin does not promote the recruitment of b-arrestin-2 or MOR internalisation [37]. Moreover, compound 11 induces MOR signalling through MAP kinase activation as revealed by the increased ERK1/2 phosphorylation Herkinorin-induced. The Herkinorin structure, as well as Salvinorin A, is unique among opioid receptor ligands since they lack a basic nitrogen. Its chemical backbone could provide the framework for the generation of both new opioid agonists and pharmacological tools. Indeed, its novel chemical structure could help to identify the molecular determinants promoting the G-protein opioid receptor signalling to obtain opioid analgesics with limited adverse effects. A naltrexamine derivative with functional selectivity for Gprotein activation was recently reported by Zhang et al. [38]. The naltrexamine derivative NAP (12, Fig. 5) displayed high MOR affinity and high degree of selectivity being its MOR Ki (0.37 nM) 700and 150-fold lower than DOR and KOR Ki, respectively [39]. NAP acted as a MOR partial agonist since it stimulated [35S]GTPgS binding with low efficacy, and in vivo it did not elicit antinociceptive effect but was able to reverse the analgesic effect, morphine-induced, with an AD50 of 4.51 mg/kg after s. c. injection. The discrepancy between in vitro and in vivo evidences prompted investigators to deeply study NAP functional profile evaluating the compound ability to recruit G-protein and/or b-arrestin signalling. By using a more downstream measure of G-protein-mediated effector activity, the Ca2þ mobilisation assay, NAP did not increase intracellular Ca2þ level but, on the contrary, it determined a reduction of Ca2þ flux DAMGO-induced, showing a MOR antagonist profile [38]. Interestingly, NAP did not promote b-arrestin signalling, but it blocked DAMGO-induced b-arrestin-2 recruitment. Altogether, these evidences suggested for NAP a potential profile of biased antagonist at MOR against b-arrestin recruitment consistent with the compound capability to reverse the reduced intestinal motility caused by morphine in mice [40].

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Fig. 2. SAR on tetrahydropyran-based compounds.

To optimise drug-like properties in order to separate antinociception from adverse effects, Zadina et al. [41] synthesised cyclized endomorphin 1 and 2 (EM1 and EM2) analogues, containing a D-amino acid (13e16, Fig. 6). In particular, 13, 15 and 16 were EM1 analogues, while 14 was an EM2 analogue. In comparison to the leads EM1 and EM2, all four

analogues maintained MOR affinity and selectivity, although their Ki values were about 2e3-folds lower. By [35S]GTPgS binding assay, 13e16 resulted in MOR agonists more potent than EM1 and EM2. Their pharmacodynamic profile was also established in vivo, where their antinociceptive effect was reversed by naloxone but not by the selective DOR and KOR antagonists, naltrindole and nor-BNI,

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Fig. 5. NAP structure.

Fig. 3. Structure of SHR9352.

respectively. Moreover, in mice tail-flick test, after intravenous (i.v.), s.c., and oral administration, all EM1 and EM2 analogues elicited potent antinociception with reduction or absence of abuse liability, respiratory depression, impairment of motor coordination, tolerance, hyperalgesia and glial activation. In this investigation, it has been demonstrated that the side-chain cyclized structures of compounds 13e16, allowing amidation or extension of the C-terminus, provided MOR selectivity, improved solubility, high stability, and favourable bioavailability, thus producing compounds with a better pharmacological profile. Another strategy to obtain EMs analogues with improved druglike properties was adopted by Liu et al. [42]. They performed a multisite combinatorial modification with the introduction of nonnatural amino acid, such as 2,6-dimethyltyrosine (Dmt), D-NMeAla, 2-methylene-3-amino propanoic acid (Ph)Map, and (2-furyl)Map, into the sequence of peptides EM1 and EM2 (17e24, Fig. 7). In comparison to the lead EM1, in MEL-N1601 (17) the introduction of NMeAla2 and (Ph)Map4 determined a 5-fold increased MOR affinity. A similar trend was reported for compounds MELN1602e1604 (18e20). The structural modifications maintained the MOR agonist profile of the leads, although the relative EC50 (1.45, 4, and 39 nM) were lower, as detected by cAMP accumulation measurements assay. In the same investigation, MEL-N1605e1608

(21e24), in which the Tyr1 was replaced with Dmt1, were also synthesised and characterised. This structural modification conducted to an upgraded opioid profile. Indeed, MEL-N1605-MELN1608 (21e24) exhibited MOR affinity (Ki in the range 0.0109e0.0511 nM) and efficacy (EC50 in the range 0.00177e0.00515 nM) in the low picomolar range. The Dmt1 introduction led to an ameliorated DOR profile. However, MELN1605eN1608 (21e24) maintained the functional profile of MOR selective agonists. The most potent compounds, MEL-N1606 and MEL-N1608 (22 and 24), stimulated b-arrestin-2 signalling in a concentration-dependent manner and produced a bias factor, indicating that the analogues preferentially activate the downstream G-protein pathways. In vivo studies performed by mouse tail-flick test showed that MEL-N1606 and MEL-N1608 (22 and 24) elicited a naloxone-reversed antinociceptive effect after intracerebroventricular (i.c.v.) administration, with an ED50 of 15.2 and 22.6 nmol/kg, and after i.v. administration, with an ED50 of 0.382 and 0.489 mg/kg, respectively. Moreover, comparable ED50 values were detected for both compounds when they were tested in mice formalin test, an animal model of inflammatory pain. Novel EMs analogues were also able to reduce the number of writhes at a dose of 3 mg/kg in the mouse writhing test. Moreover, in a regimen of repeated administration at a dose of 6 mg/kg, both compounds maintained a strong antinociceptive effect resulting in low tolerance-inducing capability. Also, MEL-N1606 showed a reduced tendency to induce constipation. Thus, EM analogues containing unnatural amino acid resulted in potent MOR biased agonists with improved therapeutic index. In this study, the enhancement in

Fig. 4. Structures of the naturally diterpene KOR agonist Salvinorin A and its semi-synthetic MOR agonist derivative Herkinorin.

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Fig. 6. Endomorphin analogues structures.

Fig. 7. The aminoacidic sequence of MEL-N16 peptides.

opioid receptor affinity by Tyr1 replacement with Dmt1 was corroborated. The non-natural amino acid introduction, a structural modification reducing the stacking effect between peptide sidechain groups, not only improved pharmacokinetic properties but also the pharmacodynamic profile because of its contribution to stabilise the dominant conformation of the polypeptide. Through the computational docking of 3 million molecules versus MOR, new scaffolds, unrelated to known opioids, were identified by Manglik et al. [16]. One of them (25, Fig. 8) showed MOR micromolar affinity and represented the hit by which other analogues, that retained the key recognition groups but added packing substituents or extended towards the extracellular side of

the receptor, were docked. Among them, compound 26 (Fig. 8) showed an improved MOR affinity with an about 60-fold increased Ki. Compound 26 strongly activated G-protein with low levels of barrestin-2 recruitment. With the aim to improve its pharmacological fingerprint, the (S,S)-isomer 27 of compound 26 was synthesised (Fig. 9). Compound 27 was featured by a 40-fold increased MOR affinity and a more pronounced G-protein activation capability (EC50 ¼ 4.6 nM; Emax ¼ 76%). A further efficacy improvement was rescued by the introduction of a phenolic hydroxyl group in the structure of 27, leading to the success compound known as PZM21 (28, Fig. 8). PZM21 resulted in a potent, selective, G-protein biased MOR agonist that showed a dose-dependent analgesic effect in the mouse hot plate assay but not in the tail-flick test, indicating that the compound confers analgesia to the affective component of pain. Moreover, PZM21 resulted effective also in the inflammatory pain model induced by formalin injection. PZM21-induced analgesia was MOR-mediated as demonstrated in MOR KO mice. Its biased MOR agonist profile reflected in low constipation, respiratory depression, lack of locomotor potentiation, and conditioned place preference inducing capabilities in comparison to morphine and TRV130. In this study, besides the importance of structure-based approach for GPCR, new scaffolds and chemotypes discovery was

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Fig. 8. Structure of 25 and its optimised derivatives 26e28.

highlighted. Indeed, the structure-based approach allowed identifying a hit subsequently optimised to drug candidate and pharmacological tool. To detail SAR of PZM21, an attractive new and unique chemical scaffold for the presence of the tertiary amine and urea groups not present in conventional opioid analgesics, Ma et al. [43] synthesised analogues by replacing the benzene ring with an aromatic naphthalene scaffold, changing the thiophene ring position and unchanging the specific dimethyl amino and urea pharmacophore groups. Additionally, the influence of methyl group chirality was also investigated. Novel synthesised PZM21 derivatives were tested in vitro in order to establish their functional profile by measuring intracellular cAMP levels and b-arrestin-2 recruitment. Compounds 29 (Fig. 9, EC50 ¼ 203.4 nM), 30 (EC50 ¼ 242.8 nM), and 31 (EC50 ¼ 455.6 nM) were weaker MOR agonists than PZM21 (EC50 ¼ 52.41 nM). Other PZM21 analogues, 32e35, showed the same potency of the lead with EC50 ranging from 52.41 to 91.14. Except compound 31, all PZM21 analogues strongly activated barrestin-2 signalling. Compounds 29, 30, and 32 were stronger analgesics than PZM21 in both mouse formalin and writhing test. Compound 29 was the most potent analogue, requiring a dose that was 1/16th to 1/4th than PZM21 and could represent a template for the synthesis of effective biased MOR agonists. In this SAR study, it was demonstrated that the phenol group replacement with a naphthalene group determines loss of functional selectivity. Moreover, its position, as well as thiophene position, affects potency and efficacy. Biased MOR agonists with N-benzyl cycloamino benzimidazolone scaffold were recently developed by Kennedy et al. [44]. Considering the high potential of the benzimidazolone nucleus as a scaffold [45], the parent compound 36 (Fig. 10) was a weaker MOR

agonist biased toward b-arrestin-2. Introduction of o-Cl, p-Cl and m-Cl led to an 11-, 2-, and 4-fold increased MOR potency, and just the o-Cl derivative 37 (Fig. 10) possessed a low b-arrestin-2 recruitment capability. The para introduction of Br, Me, OMe, NHC(O)Me, CN, C(O)NH2, conducted to analogues with similar or lower G-protein efficacy and unbiased profile. The o-Me and p-Cl substituted derivative 38 (Fig. 10) resulted in a more potent biased MOR agonist, as well as the o-F and p-Br substituted derivative 39 (Fig. 10). Further improvement of biased MOR profile was achieved by introducing an N-benzylic methyl group (40, Fig. 11) in compound 39. Compound 40 was chosen as the new lead compound. The o-Cl or p-Cl introduction in compound 40 led to a 3- and 6fold increased G-protein signalling activation although a significative bias factor was possessed only by compound 41 (Fig. 11). In compound 42 (Fig. 11), the replacement of p-Cl with p-Br significantly improved the biased MOR agonist profile. Contrarily, the p-Cl replacement with p-OMe, p-OEt or p-OiPr retained or lowered the G-protein activation of 39 and worse the biased MOR profile with the exception of compound 43, with the p-OiPr substituent (Fig. 11). Compound 44 (Fig. 11), with a p-OCF3 substituent, was featured of highest biased MOR agonist profile with a bias factor of 8.3. The effect of changing the substituents on benzimidazolone moiety was also investigated. In compounds 45e47 (Fig. 12), the Cl introduction conferred a full MOR agonist profile with bias factors ranging from 5.8 to 7.1. A similar trend was also reported for compound 48, although it resulted less biased. Finally, the effect of changing the central ring size as well as the benzimidazolone position was also probed. In comparison to the corresponding 4-substituted piperidine derivatives, the 7membered 51 (Fig. 14) and 5-membered 52 and 53 ring

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Fig. 9. Structures of PZM21 analogues.

analogues, (Fig. 14), were less potent in the [35S]GTPgS binding assay and were less biased. By these extensive SAR studies, it has been demonstrated that slight structural changes notably affect biased signalling. For instance, it emerged that halogen atoms in the distal benzimidazolone moiety and the N-benzyl ring, are essential for G-protein coupling, as well as, a benzylic methyl group that improves the potency and has a small but typically beneficial impact upon biased profile. Moreover, the presence of a central piperidine nucleus is also preferred. All this evidence allowed authors to establish a pharmacophore model to design MOR biased agonists. Piperidine compounds such as SR-17018 (54), SR-15099 (55) and SR-15098 (56) resulted MOR G-protein biased agonists (Fig. 15) [46]. SR-17018 (54), SR-15099 (55), and SR-15098 (56) systemically administered, elicited, in the tail-flick test, a dose-dependent antinociceptive effect with an ED50 of 7.7, 7.4 and 13 mg/kg respectively. Even when administered at doses higher than their ED50 (48 mg/kg), SR-17018 (54), SR-15099 (55) and SR-15098 (56) determined negligible respiratory depression. In this study, it was demonstrated that the piperidine compounds, featured by biased

G-protein signalling over b-arrestin-2 recruitment, have much broader therapeutic windows.

1.2. DOR biased ligands Naturally occurring peptides that selectively activate G-protein signalling pathways at DOR, endowed with minimal b-arrestin recruitment, were discovered by Cassell et al. [47]. Rubiscolin peptides YPLDL (rubiscolin-5, 57 Fig. 16) and YPLDLF (rubiscolin-6, 58 Fig. 16), identified from spinach rubisco, have micromolar afDOR finity (ICDOR 50 x2 mM and IC50 x0.9 mM, respectively, in radioligand DOR binding assay) and potency (ICDOR 50 ¼ 51.0 mM and IC50 x24.4 mM, respectively, in MVD assay) at DOR and x500 fold selectivity in DOR binding over MOR (ICDOR 50 ¼ 1085 mM and IC50 >2000 mM, respectively) [48]. Moreover, rubiscolin-5 and -6 have an antinociceptive effect in mice after i.c.v. at the doses of 3 nmol/mouse and 1 nmol/ mouse, respectively, or oral administration at higher doses of 300 and 100 mg/kg, respectively. Based upon the experimental observation that DOR internalisation is linked to b-arrestin recruitment and that rubiscolin-6 did not promote DOR internalisation, in a model of skin inflammation

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Fig. 10. Structure of N-benzyl cycloamino benzimidazolone-based compounds.

[49], both peptides were investigated for their potential DOR agonist G-protein biased profile. Rubiscolin-5 and rubiscolin-6 inhibited forskolin-stimulated intracellular cAMP accumulation resulting full DOR agonist (pIC50 ¼ 5.2 and 5.8 with a ¼ 85 and 77, respectively). Importantly, b-arrestin-1 (pEC50 not detectable) and 2 (pEC50 ¼ 4.1 and 5.1 with a ¼ 15 and 20, respectively) recruitment at DOR ranged from undetectable to low levels even at very high peptides concentrations [47]. By the structure analysis was established no conformational differences between rubiscolin peptides and known DOR peptides such as Leu-enkephalin or deltorphin II. However, the presence of a proline residue, due to its cyclic structure, could confer a minor degree of flexibility to rubiscolin peptides, with respect to Leu-enkephalin or deltorphin II, inducing a DOR conformation that makes rubiscolin-5 and -6 the first naturally occurring peptides that selectively activate G-protein signalling pathways at DOR with minimal b-arrestin recruitment. Recently, Stanczyk et al. [50] reported the biased DOR G-protein profile of BMS-986187 (59, Fig. 17). BMS-986187 emerged by a high throughput screening (HTS) conducted to identify a chemotype for DOR positive allosteric modulators (PAM) [51] being able to inhibit adenylyl cyclase in the absence of an orthosteric agonist. The compound was also able to stimulate [35S]GTPgS binding, however with a low potency. The [35S]GTPgS binding stimulation BMS986187-induced, was reduced by the orthosteric DOR selective antagonist naltrindole and by the non-selective antagonist naloxone. By the parallel receptor-ome expression and screening via transcriptional output-transcriptional activation following

arrestin translocation (PRESTO-TANGO) assay, BMS-986187 (59) recruited b-arrestin-2 very weakly with an EC50 of 578.5 mM. Thus, BMS-986187 (59) activated DOR G-protein signalling through an allosteric DOR site. This evidence was confirmed by the low BMS986187 (59) capability to cause DOR internalisation, phosphorylation and desensitisation. 1.3. KOR biased ligands Using in silico and parallel screening approaches, White et al. [52] identified several KOR-selective scaffolds with a range of biased signalling in vitro which allowed to obtain structural information useful to optimise KOR biased ligands. In this study different KOR chemotypes such as dynorphins, benzomorphans, benzodiazepines, diterpenes, and arylacetamides were screened and, among them, the RB family of salvinorin derivatives were found to be the first identified KOR G-protein biased ligands centrally acting. In particular, RB-64 (60) and RB-48 (61, Fig. 18), deriving by C-2 Salvinorin A modification, were extraordinarily potent and selective KOR agonists. RB-64 (60) and RB-48 (61) KOR affinity were 0.59 nM and 2.1 nM, respectively, and their EC50 values were 0.077 nM with Emax of 95% for the former and 0.19 nM with Emax of 85% for the latter [53]. RB 64 (60) and RB 48 (61), the first identified most potent compounds in activating G-protein signalling with a high degree of bias factor of 35 and 25, respectively, can be employed for in vivo probing of KOR-mediated Gprotein signalling.

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Fig. 11. Structures of compound 40 analogues.

The triazole hit was undertaken to a systematic SAR study by which the crucial role of the linker length, aromatic ring substitution and nitrogen emerged. The aromatic ring substitution conducted to the triazole derivative (63, Fig. 19) able to induce biased receptor signalling toward G-protein over b-arrestin-2 recruitment [55]. Indeed, in the mouse tail-flick test, after i.p. injection (30 mg/ kg), the compound produced significant antinociception. The 4-N introduction of 2-furanyl, 2-thiophenyl and 2-pyridyl substituents to the triazole scaffold led to a series of compounds that maintained the bias profile between G-protein signalling and b-arrestin-2 recruitment, but with variations between G-protein signalling and

ERK1,2 activation. In particular, an improved functional selectivity for G-protein signalling over both b-arrestin and HERK1,2 was recorded when the furan ring was replaced with a pyridine ring (64, Fig. 19) [56]. New triazole-based compounds could be important pharmacological tools to investigate the pharmacological and physiological impact of biased signalling at KOR. Recently, noribogaine (65, Fig. 20) e the metabolite of ibogaine, an alkaloid derived from the African shrub, iboga (Tabernanthe iboga) e was found to be a KOR biased agonist [57]. Noribogaine exhibited a Ki value for KOR of 720 nM and a 2-fold higher Ki value for MOR. As assessed by [35S]GTPgS binding on CHO

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Fig. 12. Structures of compound 37 analogues with different benzimidazolone substituents. In compounds 49 and 50 (Fig. 13) the Cl substituents led to most pronounced biased profiles.

Fig. 13. Structures of compound 37 analogues with different benzimidazolone substituents.

cells stably expressing KOR, noribogaine was a partial agonist producing an Emax of 72% and an EC50 of 8.75 mM. Noribogaine marginally stimulated [35S]GTPgS binding via MOR with an EC50 of 16 mM and, in the presence of MOR agonists such as DAMGO and Met-enkephalin, right-shifted their curve response profile resulting in a MOR antagonist. Noribogaine exhibited a robust functional biased profile at KOR and was marginally efficacious in recruiting b-

arrestin with an Emax of 13% and an EC50 of 110 nM. Moreover, it was able to inhibit b-arrestin recruitment induced by agonists of this pathway, such as dynorphin A. It was hypothesised that noribogaine, promoting a set of KOR conformations, induces functional selectivity to dynorphin A. This peculiar KOR functional profile could contribute to the positive effects against stress, anxiety and atypical depression.

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Fig. 14. Structures of 51e53 analogues with different size of the central ring.

Fig. 15. SR-17018 (54), SR-15099 (55) and SR-15098 (56) structures.

Fig. 16. Structures of rubiscolin-5 (57) and rubiscolin-6 (58) peptides.

The functional selectivity of Nalfurafine [58,59] (66, Fig. 21), a known anti-pruritus agent [60], was recently investigated [61]. Nalfurafine (66) resulted in a G-protein biased ligand at KOR. Indeed, it was able to activate ERK1,2 as a measure for G-protein signalling, whereas it showed a lower potency for p38 activation, as a measure for b-arrestin mediated signalling. This in vitro profile was coherent with the in vivo collected data. Indeed, the compound (66), administered at the dose of 50e150 mg/kg, elicited an antinociceptive effect reversed by pre-treatment with the KOR antagonist nor-BNI, with low tolerance-inducing capability in a regimen

of repeated administration and locomotor impairment. Recently, a new scaffold of the class of diphenethylamines was reported for KOR ligands [62]. To investigate the role of the Nsubstitution on KOR interaction, a series of N-substituted diphenethylamine compounds have been synthesised. In this SAR study, Spetea et al. [62] demonstrated how an N-cyclopropylmethyl or Ncyclobutylmethyl substitution is more favourable for KOR interaction than N-alkyl groups. Indeed, the N-cyclobutylmethyl derivative HS665 (67, Fig. 22) and the N-cyclopropylmethyl derivative HS666 (68, Fig. 22) resulted in biased KOR agonist toward G-protein as

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Fig. 20. Noribogaine (65) structure.

1.4. Multitarget MOR/DOR biased ligands

Fig. 17. Structure of BMS 986187 (59).

assesses in vitro by [35S]GTPgS and the PathHunter b-arrestin-2 recruitment assays [63]. In wild type mice, both compounds, i.c.v. administered, produced a KOR mediated antinociceptive effect (ED50 of 3.74 nmol for HS665 and 6.02 nmol for HS666) as demonstrated by the lack of pharmacological effects in KOR KO mice. Moreover, at higher doses, in the rotarod test, both HS665 (67) and HS666 (68) did not impact on motor performance and in the place conditioning paradigm they did not result aversive.

Through high-throughput screening of a small-molecule library, compounds targeting MOR/DOR heteromers were identified [64] and, among them, CYM51010 (69, Fig. 23) was found to be a MOR/ DOR biased ligand. CYM51010 (69) causes a robust increase in [35S]GTPgS binding in MOR/DOR UO5S cell membranes (EC50 ¼ 54 nM and Emax ¼ 168%). This [35S]GTPgS binding stimulation was completely blocked by MOR/DOR mouse antibody. In the same cell-line expressing MOR/ DOR was also established the CYM51010 (69) low capability to recruit b-arrestin (EC50 ¼ 8300 nM and Emax ¼ 1197%). Thus, CYM51010 (69) resulted, in vitro, a MOR/DOR heteromer biased ligand. Investigators examined if CYM51010 (69) exhibited MOR/ DOR heteromer-mediated activity also in vivo. By tail-flick assay, the compound elicited an antinociceptive effect similar to morphine. Unlike morphine, whose effect was completely

Fig. 18. Structures of RB-64 (60) and RB-48 (61) By HTS, Frankowski et al. [54] identified a new KOR triazole-based chemotype (62, Fig. 19) possessing high KOR selectivity and moderate functional potency in the DiscoveRx barrestin PathHunter assay.

Fig. 19. Structures of compounds 62e64.

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Fig. 21. Nalfurafine (66) structure.

Fig. 23. CYM51010 (69) structure.

naltrexone reversed, CYM51010 (69) antinociception was partially reversed by the MOR antagonist and entirely blocked by the MOR/ DOR mouse antibody, supporting the idea that its effect is prevalently mediated by the MOR/DOR heteromer. In contrast to morphine, CYM51010 (69), in a regimen of repeated administration, at the dose of 10 mg/kg, maintained a significant antinociceptive effect until the 8th day of observation. Moreover, in naloxone precipitated withdrawal assay, repeated CYM51010 (69) administration (6 mg/kg) shows less severe signs of withdrawal for diarrhoea and body weight loss if compared with morphine. The MOR/DOR biased agonist CYM51010 (69) could provide a chemical scaffold for the development of therapeutics with reduced side effects commonly associated with chronic morphine use. Two monoterpene indole alkaloids, structurally not closely related to morphine [65], were isolated from the plant Mitragyna speciosa: mitragynine (70) and its naturally occurring oxidation product, 7-hydroxy-mitragynine (7-OH-mitragynine, 71, Fig. 24). In homogenates of guinea pig brain, the affinity of mitragynine was 7.2 nM at MOR, 60 nM at DOR, and >1000 nM at KOR. The affinity of 7-OH-mitragynine (71) was 13 nM at MOR, 155 nM at DOR, and 123 nM at KOR. Both mitragynine (70) and 7-OH-mitragynine (71) have MOR affinity comparable to that of morphine (Ki ¼ 3.5 nM). Mitragynine displayed about 10-fold MOR selectivity over DOR and more 1000-fold selectivity over KOR. 7-OH-mitragynine (71) displayed more 10-fold MOR selectivity over DOR and KOR. In electrically stimulated guinea pig ileum (GPI), mitragynine (70) resulted in a full agonist with 1/4 potency than morphine. 7-OHmitragynine (71) exhibited 10-fold higher potency than morphine and the same intrinsic activity. Mitragynine (70) and 7-OH-

mitragynine (71) displayed centrally mediated (supraspinal and spinal) antinociceptive activity in various pain models [65]. Selected modifications in the mitragynine (70) and 7-OHmitragynine structure (71) identified the most critical molecular groups affecting compounds’ profile: (1) C-9 position, (2) C-7 position, (3) Nb lone pair and b-methoxyacrylate moiety, (4) epimerization of ethyl group in position 19 (Fig. 24). The rearrangement product of 7-OH-mitragynine (71) with a spiro-pseudoindoxyl core is mitragynine pseudoindoxyl (72, Fig. 24). Mitragynine pseudoindoxyl (72) produced a 10-fold higher analgesic effect than mitragynine. In the binding assay, mitragynine pseudoindoxyl showed an affinity profile versus MOR and DOR similar to DAMGO (pIC50 ¼ 8.18 vs. 8.77) and DPDPE (pIC50 ¼ 9.55 vs. 8.90), respectively, but a negligible affinity versus KOR (pIC50 ¼ 6.88) [66]. Mitragynine pseudoindoxyl inhibited the electrically stimulated GPI and mouse vas deferens (MVD) contraction in a concentration-dependent manner with a pD2 value of 8.96 and 7.40, significantly reversed by naloxone and naltrindole, respectively. These data suggested that conversion of the indole to indolenine ring and further to spiro-pseudoindoxyl core increase affinity for opioid receptors. In [35S]GTPgS functional assay mitragynine pseudoindoxyl (72) was a potent full agonist at MOR and antagonist at both DOR and KOR. In contrast to classical MOR agonists, mitragynine pseudoindoxyl (72) failed to recruit b-arrestin2 and reduced DAMGO-induced b-arrestin-2 stimulation Moreover, mitragynine pseudoindoxyl developed analgesic tolerance more slowly than morphine and showed a limited capability to induce physical dependence, respiratory depression, constipation and reward or aversive behaviour in the conditioned place preference

Fig. 22. HS665 (67) and HS666 (68) structures.

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Fig. 24. Structure of monoterpene indole alkaloids isolated from Mitragyna speciosa and their analogues.

paradigm. By these investigation mitragynine pseudoindoxyl (72) emerged for its advantageous pharmacological profile to whom contribute both the multitarget MOR agonist/DOR antagonist profile and the failed b-arrestin-2 recruitment. V aradi et al. [67] investigated the SAR of mitragynine (70) designing and synthesising pseudoindoxyl and semisynthetic analogues. All C-9-modified mitragynine pseudoindoxyl compounds maintained high affinity at both MOR and DOR. On the contrary, the

alkyl substitution at N-1 eliminated opioid affinity. None of the derivatives stimulated b-arrestin recruitment. Unlike C-9 mitragynine modifications that altered MOR efficacy, C-9 mitragynine pseudoindoxyl substituents maintained a full MOR agonist profile. C-9 substituted derivatives resulted in DOR antagonists except 9phenyl analogue 73 (Fig. 24) that was a DOR agonist. Compound 73 was featured by high MOR (Ki ¼ 0.91 nM), DOR (Ki ¼ 0.8 nM) and low KOR (Ki ¼ 51 nM) affinity and is a potent and full MOR

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Fig. 25. LP2 (74) and (2S)-LP2 (75) structures.

(EC50 ¼ 1.4 nM, Emax ¼ 123%) and DOR (EC50 ¼ 0.83 nM, Emax ¼ 89%) agonist. Compound 73 also determined the same antinociceptive effect of parent mitragynine pseudoindoxyl (ED50 ¼ 1 mg/kg s.c. vs. 0.76 mg/kg s.c., respectively). These results suggested that compounds with pseudoindoxyl scaffold showed different SAR features if compared to the natural Mitragyna alkaloid. Mitragynine pseudoindoxyl C-9 substituted derivative 73 was able to produce robust antinociception with a low incidence of side effects reflecting not only the lack of contribution in the b-arrestin-recruitment but also its capability to simultaneously targeting MOR and DOR that make it a suitable template. Extensive and systematic SAR studies [68e71] exploring the critical role of the N-substituent nature in the benzomorphan scaffold [72,73] conducted to a MOR/DOR biased agonist. Recently, a series of 6,7-benzomorphan-based compounds bearing short and flexible substituents at the basic nitrogen were synthesised [74]. Among them, the compound with the (R/S)-2-methoxy-2phenylethyl group as N-substituent, named LP2 (74, Fig. 25), was characterized by nanomolar affinity for MOR (Ki ¼ 1.08 nM), DOR (Ki ¼ 6.61 nM) and KOR (Ki ¼ 15.22 nM). Moreover, in GPI and MVD assays, LP2 (74) showed MOR/DOR agonist profile (IC50 ¼ 21.5 nM and 4.4 nM, respectively). In tail-flick test, after intraperitoneal (i.p.) administration, LP2 (74) produced long-lasting antinociception, naloxone-reversed, with an ED50 of 0.9 mg/kg. A later investigation also highlighted the LP2 (74) capability to preferentially activate MOR (pEC50 ¼ 7.90) and DOR (pEC50 ¼ 7.02) G-protein signalling over the b-arrestin signalling recruitment (pEC50 ¼ 6.56 and 5.65 at MOR and DOR, respectively) [75]. The MOR/DOR biased agonist profile of LP2 (74) was tested in an animal model of inflammatory pain induced by intraplantar (i.pl.) injection of formalin [76]. LP2 (74) (0.75e1.00 mg/kg, i.p.), dose-dependently, counteracted both phases of formalin test and its effects were completely blocked by a pre-treatment with the opioid antagonist naloxone (3 mg/kg, i.p.). The pre-treatment with naloxone methiodide (5 mg/kg, i.p.), a peripherally restricted opioid antagonist, only blocked the lower analgesic dose of LP2 (74) (0.75 mg/kg) revealing a peripheral analgesic component of it. In the same animal pain model, the effect of LP2 (74) on the rotarod test, to exclude motor impairments, was investigated. LP2 (74) analgesic dose did not produce typical motor deficits usually observed with opioid compounds. In an experimental model of neuropathic pain induced by the unilateral sciatic nerve chronic constriction injury (CCI) on male Sprague-Dawley rats, LP2 (74) significantly ameliorated mechanical allodynia from the early

phase of treatment up to 21 days post-ligatures [77,78]. Moreover, LP2 (74) prevented CCI-induced Cx43 alterations and pro-apoptotic signalling in the central nervous system. Based upon the importance of stereocenter for the ligand/opioid receptor interaction, through an asymmetric approach, both LP2 diastereoisomers were synthesised and evaluated both in vitro and in vivo [75]. It was found an improved pharmacological fingerprint for the (2S)-LP2 (75) isomer, featured by an increased affinity profile at MOR (Ki ¼ 0.5 nM) and DOR (Ki ¼ 2.59 nM). The compound, evaluated for its ability to promote receptor/G-protein interaction through measurements of cAMP accumulation and receptor/b-arrestin interaction through the BRET assay, resulted a potent MOR/DOR biased agonist (pEC50 ¼ 8.33 and 7.49 for MOR- and DOR G-protein and pEC50 ¼ 6.82 and 5.73 for MOR- and DOR-b-arrestin, respectively) producing a significant bias factor at MOR and mainly at DOR (0.82 vs 2.31). The improved pharmacological profile was confirmed in vivo. Indeed, in comparison to LP2 (74), (2S)-LP2 (75) in mice tail-flick test elicited a 1.5-fold more potent antinociceptive effect without determining neither sedation nor alteration in locomotor activity. 1.5. Molecular modelling and computational studies Considering that chemical scaffold of TRV130 differs significantly from the canonical morphinan structure, Schneider et al. [79], using long-scale unbiased molecular dynamics simulations, explored how TRV130 binds and stabilises an activated MOR conformational state. This study gave important details about the

Fig. 26. 3D-model of MOR (PDB ID: 5C1M) with the co-crystallized pose of BU72 (green) and the docked pose of TRV130 (yellow) in the orthosteric site. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 27. Interactions of the TRV130 (yellow, ball and stick) with selected amino acid residues (magenta, stick) into the orthosteric binding pocket of MOR (PDB ID: 5C1M). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 28. Best docked poses of compounds TRV130 (yellow) and SHR9352 (red) in the orthosteric pocket of the active MOR (PDB ID: 5C1M). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

binding pathway of MOR biased agonists. In the so-called vestibule receptor region, the ligand assumed several metastable bound states implied in modulating ligand binding kinetics. One of these intermediate states is stabilised by the interaction with the residue N127 (asparagine) that seem to be important in determining TRV130 MOR selectivity. In MOR orthosteric site, TRV130 adopts an energetically favourable pose that partially overlaps with the binding pose of the co-crystallized ligand BU72 (Fig. 26).

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Contrary to morphine, for which a strong coupling between the binding pocket and the intracellular ends of transmembrane (TM) 3 and 6 was observed, TRV130-bound binding pocket stabilises a communication with the intracellular end of TM3. Moreover, unlike morphine, TRV130 engages residues in the extracellular (EC) 2 and EC3 loops forming polar interactions that extend to the extracellular ends of TM3 and TM5. Finally, it has been observed that in morphine and TRV130 intracellular region stability some residues are common (such as Y1062.42, W133 (EC1), Y3267.43, F343 (H8), W2936.48, Y3367.53, F135 (EC1), D1643.49, and I1443.29) (Fig. 27). Other residues are involved only when TRV130 is bound to the receptor (such as W3187.35, R1653.50, Y1493.34, F347(H8), and Y911.55) (Fig. 27) or only when morphine is bound to receptor (such as F1082.44, I1072.43, N1884.46, and R2776.32). Altogether, all this information about the different interactions formed at the orthosteric binding pocket provide a new rationale for the design of drugs with tailored pharmacologic profiles. Recently, through a series of molecular dynamics simulations, Cheng et al. [80] identified the interactions that down-regulated barrestin signalling or up-regulated the G-protein signalling of TRV130, useful for the design of new MOR biased ligands. Differently from unbiased ligands analysed in this study, in TRV130 binding, the triplet residues Q2.60, D3.32 and Y7.43 interactions were abrogated. The TRV130 interaction with Y7.43 stabilised W6.48, as well as its ring structure also stabilised W7.35. On MOR agonist cocrystal structure, the TRV130 derivative SHR9352 [34] was modelled into the ligand-binding site. SHR9352 maintained all key interactions of the lead TRV130, showing the structural parts in common almost completely overlapping (Fig. 28). Through the structure-based docking against the orthosteric pocket of inactive MOR of 3 million commercially available leadlike compounds, PZM21 [16] was identified. By this investigation, it has been possible to identify the most relevant interactions of the potent MOR biased agonist PZM21 with MOR binding pocket. PZM21 hydroxyl phenolic group forms the classic water-mediated hydrogen bonds with two consensus-water molecules, which involve, in addition, another consensus-water molecule and the amino acids Y1483.33 and H2976.52 (Fig. 29). The ionic interaction between PZM21 tertiary amine and D1473.32 is relevant for the ligand potency, as well as, the hydrophobic interaction between the N-methyl group and M1513.36 and W2936.48, and the hydrogen bonding interaction between the urea and D1473.32, Y3267.43 and Q1242.60. The PZM21 thiophene comes within 6 A of N1272.63 in the active MOR. Analogous modelling studies indicated that the epoxymorphinan skeleton of NAP resided in an identical binding pocket of b-FNA, while their side chains assumed insignificantly different

Fig. 29. 3D (left) and 2D (right) sketch of the principal interactions of compound PMZ21 within the orthosteric pocket of the active MOR (PDB ID: 5C1M).

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References

Fig. 30. 3D-model of MOR (PDB ID: 4DKL) with the co-crystallized pose of b-FNA (green) and the docked pose of NPA (yellow) in the receptor binding pocket. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

orientations in the binding site (Fig. 30) [38]. The NAP side chain interacts with non-conserved amino acid residues W318, K303, and V300, not involved in opioid receptor activation and located in TM3, 5 and 6 and not in ECL2 and TM7, as expected for the classic opioid receptor activation mechanisms [81]. All this information seems to be in agreement with the biased modulation properties of NAP on MOR.

2. Conclusion After opioid receptors isolation, much information has been gained about their expression, signalling, and modulation. Substantial efforts were extensively addressed to the development of potent and safer opioid analgesics and, in this perspective, the idea to dissociate the beneficial from the undesirable opioid effects by biased agonists stimulated medicinal chemistry research during the last decade. In fact, biased opioid ligands, with tailored signalling properties, could physiologically segregate analgesia from constipation, respiratory depression, and other side effects, by preferentially engaging G-protein over the b-arrestin pathway. The continually increasing number of novel biased opioid agonists and the unmasking of an unidentified biased profile of many marketed drugs will help us to better understand the structural requirements for the biased signalling properties. Meanwhile, therapeutically useful and innovative drugs with new scaffolds are required to design safer drugs for the management of pain. In this regard, the available opioid receptors crystal structure [82e84] and the modern computational methodologies can be applied to aid in the development of new opioid ligands via structure-based drug design. Indeed, computational modelling and X-ray crystallography could elucidate the drug conformation inducing specific receptor states that are more or less susceptible to G-protein pathway activation. Future explorations will benefit from the availability of biased ligands as tool compounds to dissociate physiological outcomes downstream of activated opioid receptor, to study the barrestin-mediated role in the adverse effects associated with opioid receptors, and thereby to provide better therapeutic possibilities.

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