Novel N-Substituted benzomorphan-based compounds: from MOR-agonist/DOR-antagonist to biased/unbiased MOR agonists

Modifications at the basic nitrogen of the benzomorphan scaffold allowed the development of compounds able to segregate physiological responses downstream of the receptor signaling, opening new possibilities in opioid drug development. Alkylation of the phenyl ring in the N-substituent of the MOR-agonist/DOR-antagonist LP1, resulted in retention of MOR affinity. Moreover, derivatives 7a, 7c and 7d were biased MOR agonists towards ERK1,2 activity stimulation, whereas derivative 7e was a low potency MOR agonist on adenylate cyclase inhibition. They were further screened in the mouse tail flick test and PGE2- induced hyperalgesia and drug-induced gastrointestinal transit

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the “one-molecule-multiple targets” strategy nowadays is highly recognized. Indeed, multitarget ligands displaying a better antinociceptive activity with fewer side effects, combined with favorable pharmacokinetic and pharmacodynamic characteristics, have already been shown. Multitarget ligands possessing non-opioid/opioid and opioid/opioid mechanisms of action are considered as potential drug candidates for the management of various pain conditions. In particular, dual-target MOPr (mu opioid peptide receptor)/DOPr (delta opioid peptide receptor) ligands exhibit an improved antinociceptive profile associated with a reduced tolerance-inducing capability. The benzomorphan-based compounds LP1 and LP2 belong to this class of dual-target MOPr/DOPr ligands. In the present manuscript, the structure–activity relationships and the pharmacological fingerprint of LP1 and LP2 compounds as suitable drug candidates for persistent pain relief is described

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists

The opioid pharmacological profile of cis-(−)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a–d and 6a–d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(−)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50 of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(−)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment

Synthesis and Structure-Activity Relationships of (-)-cis-N-Normetazocine-Based LP1 Derivatives

(−)-cis-N-Normetazocine represents a rigid scaffold able to mimic the tyramine moiety of endogenous opioid peptides, and the introduction of different N-substituents influences affinity and efficacy of respective ligands at MOR (mu opioid receptor), DOR (delta opioid receptor), and KOR (kappa opioid receptor). We have previously identified LP1, a MOR/DOR multitarget opioid ligand, with an N-phenylpropanamido substituent linked to (−)-cis-N-Normetazocine scaffold. Herein, we report the synthesis, competition binding and calcium mobilization assays of new compounds 10⁻16 that differ from LP1 by the nature of the N-substituent. In radioligand binding experiments, the compounds 10⁻13, featured by an electron-withdrawing or electron-donating group in the para position of phenyl ring, displayed improved affinity for KOR (Ki = 0.85⁻4.80 μM) in comparison to LP1 (7.5 μM). On the contrary, their MOR and DOR affinities were worse (Ki = 0.18⁻0.28 μM and Ki = 0.38⁻1.10 μM, respectively) with respect to LP1 values (Ki = 0.049 and 0.033 μM). Analogous trends was recorded for the compounds 14⁻16, featured by indoline, tetrahydroquinoline, and diphenylamine functionalities in the N-substituent. In calcium mobilization assays, the compound 10 with a p-fluorophenyl in the N-substituent shared the functional profile of LP1 (pEC50MOR = 7.01), although it was less active. Moreover, the p-methyl- (11) and p-cyano- (12) substituted compounds resulted in MOR partial agonists and DOR/KOR antagonists. By contrast, the derivatives 13⁻15 resulted as MOR antagonists, and the derivative 16 as a MOR/KOR antagonist (pKBMOR = 6.12 and pKBKOR = 6.11). Collectively, these data corroborated the critical role of the N-substituent in (−)-cis-N-Normetazocine scaffold. Thus, the new synthesized compounds could represent a template to achieve a specific agonist, antagonist, or mixed agonist/antagonist functional profile

Expression of <i>A. gambiae</i> OR1, OR2 and OR7 in insect cells.

<p>(<b>A</b>) Schematic representation of the basic backbone vector (pEIA) used for the heterologous expression of various forms (tagged and untagged) of ORs in lepidopteran cells. <i>hr3</i> enhancer, baculoviral (BmNPV) homologous region 3 enhancer sequence; pActin, <i>Bombyx mori</i> A3 cytoplasmic actin promoter; MCS, multiple cloning site; actin pA, 3′untranslated region of <i>B. mori</i> actin gene containing polyadenylation signals; IE1 cassette, baculoviral (BmNPV) DNA fragment containing the <i>ie-1</i> transactivator gene under the control of its native viral promoter; OR; <i>A. gambiae</i> odorant receptor ORF; Myc, Flag and MycHis, epitope tags. (<b>B</b>) Detection of heterologous expression of C-terminally MycHis-tagged ORs in transfected Hi5 cells using Myc monoclonal antibody. (<b>C</b>) Detailed western blot analysis of OR2. Hi5 cells were transfected with plasmids expressing different versions of OR2, and lysates were analyzed using a specific polyclonal antibody against OR2 (left panel, lanes 1–5) or monoclonal antibodies against the Myc (middle panel, lanes 6–9) or the Flag epitope (right panel, lanes 10–11). Arrowheads and arrows indicate major bands corresponding to monomers and putative dimers, respectively. (<b>D</b>) Detailed western blot analysis of OR1. Hi5 cells were transfected with plasmids expressing different versions of OR1, and lysates were analyzed using monoclonal antibodies against the Myc (middle panel, lanes 1–4) or the Flag epitope (right panel, lanes 5–6). In the left panel immunoreactivity of the specific polyclonal antibody against OR1 is shown, with lysates from cells expressing mycOR1 after treatment with the proteasome inhibitor MG132. Molecular weight markers are shown on the left. (<b>E</b>) and (<b>F</b>) Effect of coexpression of OR7 on the expression levels of OR1 and OR2. Hi5 cells were transfected with constant amounts (45% of total DNA) of Myc-tagged OR1 or OR2, along with equal amounts of Flag-tagged OR7 or empty vector (pEIA), and pEIA-GFP (10% of total DNA, for evaluation of the efficiency of transfection). Whole cell lysates (<b>E</b>) and membrane fractions (<b>F</b>) were analysed by SDS-PAGE and western blot. Detection of OR1, OR2 and OR7 was done using the anti-Myc and anti-Flag antibodies either consecutively (in <b>E</b>) or simultaneously (in <b>F</b>). To control for loading, the whole lysate fractions were also probed with an anti-tubulin antibody.</p

Topology assays for the mosquito OR1 and OR2.

<p>Chimeric receptor proteins fused at either their N- or the C- terminus to the TEV cleavage sequence (THE) and the HR3 transcription factor are co-expressed in Hi5 cells with a GFP reporter construct together with or without TEV protease. For both OR1 (<b>A</b>) and OR2 (<b>B</b>) N-terminal fusions (HR3-THE-OR1 and HR3-THE-OR2), expression of the TEV protease resulted in increase of fluorescence of the cells. No significant increase of fluorescence was detected when the C-terminal fusions of ORs were used (OR1-THE-HR3 and OR2-THE-HR3). Similar constructs of the human opioid receptor δ (δOR) that was used as control (<b>C</b>) give opposite results with an increased fluorescence for the C-terminal fusion (δOR-THE-HR3). For each chimeric receptor protein, both representative images (left) and quantitative results (right, with values representing the mean ± S.E.M. of four experiments) from the fluorometric analysis are shown.</p