ABSTRACT
The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
Subject(s)
Molecular Docking Simulation , Receptors, Opioid, kappa/chemistry , Analgesics/chemistry , Analgesics/pharmacology , Animals , Binding Sites , HEK293 Cells , Humans , Molecular Dynamics Simulation , Morphinans/chemistry , Morphinans/pharmacology , Protein Binding , Protein Stability , Receptors, Opioid, kappa/agonists , Receptors, Opioid, kappa/metabolism , Sf9 Cells , SpodopteraABSTRACT
Over the last several years we have synthesized and studied the in vitro and in vivo nAChR pharmacological properties of epibatidine (4) analogs. In this study we report the synthesis, nAChR in vitro and in vivo pharmacological properties of 3'-(substituted pyridinyl)-deschloroepibatidine analogs (5a-e and 6a-e). All of the analogs had high binding affinity for α4ß2(∗)-nAChRs. Several of the analogs were potent antagonists of α4ß2-nAChRs in in vitro efficacy tests and were potent antagonists of nicotine-induced antinociception in the mouse tail-flick test. Compound 6b had a Ki = 0.13 nM in the binding assay, 25- and 46-fold selectivity for the α4ß2(∗)-nAChR relative to the α3ß4- and α7-nAChR, respectively, in the in vitro efficacy test and an AD50 = 0.13 µg/kg in the tail-flick test. Combined with favorable calculated physiochemical properties compared to varenicline, our findings suggest that 6b should be considered for development as a potential pharmacotherapy for treating nicotine addiction and other CNS disorders.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Nicotiana/chemistry , Pyridines/chemical synthesis , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Humans , Mice , Molecular Structure , Pyridines/chemistry , Pyridines/pharmacology , Rats , Receptors, Nicotinic/metabolism , Structure-Activity RelationshipABSTRACT
Low-nanomolar binding constants were recorded for a series of six 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues with acetylcholine-binding protein (AChBP). The crystal structures of three complexes with AChBP reveal details of molecular recognition in the orthosteric binding site and imply how the other three ligands bind. Comparisons exploiting AChBP as a surrogate for α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the key interactions are conserved. The ligands interact with the same residues as the archetypal nAChR agonist nicotine yet display greater affinity, thereby rationalizing their in vivo activity as potent antagonists of nicotine-induced antinociception. An oxyanion-binding site is formed on the periphery of the AChBP orthosteric site by Lys42, Asp94, Glu170 and Glu210. These residues are highly conserved in the human α4, ß2 and α7 nAChR sequences. However, specific sequence differences are discussed that could contribute to nAChR subtype selectivity and in addition may represent a point of allosteric modulation. The ability to engage with this peripheral site may explain, in part, the function of a subset of ligands to act as agonists of α7 nAChR.
Subject(s)
Receptors, Nicotinic , Acetylcholine/chemistry , Acetylcholine/metabolism , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carrier Proteins/chemistry , Humans , Pyridines , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolismABSTRACT
The synthesis of novel fused-ring bicyclic (4-6) and tricyclic (7-10) nicotine derivatives from natural (S)-nicotine are described. Enantiopure bicyclic dioxino, dihydrofuro, and dihydropyranol nicotine derivatives as well as tricylic benzofuro and benzopyran derivatives were synthesized from simple alkoxy or halonicotine intermediates. Attempts to synthesize furonicotines (11, 12) resulted in formation of the furonicotine dimers 42 and 49.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Dioxins/chemical synthesis , Nicotine/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cyclization , Dioxins/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Nicotine/chemistry , StereoisomerismABSTRACT
Past studies have shown that it has been difficult to discover and develop potent and selective κ opioid receptor antagonists, particularly compounds having potential for clinical development. In this study, we present a structure-activity relationship (SAR) study of a recently discovered new class of tetrahydroisoquinoline κ opioid receptor antagonists which led to (3 R)-7-hydroxy- N-{(1 S)-2-methyl-1-[(-4-methylpiperidine-1-yl)methyl]propyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (12) (4-Me-PDTic). Compound 12 had a Ke = 0.37 nM in a [35S]GTPγS binding assay and was 645- and >8100-fold selective for the κ relative to the µ and δ opioid receptors, respectively. Calculated log BB and CNS (central nervous system) multiparameter optimization (MPO) and low molecular weight values all predict that 12 will penetrate the brain, and pharmacokinetic studies in rats show that 12 does indeed penetrate the brain.
Subject(s)
Brain/drug effects , Narcotic Antagonists/chemistry , Narcotic Antagonists/pharmacology , Receptors, Opioid, kappa/antagonists & inhibitors , Tetrahydroisoquinolines/chemistry , Animals , CHO Cells , Cricetulus , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Male , Narcotic Antagonists/metabolism , Radioligand Assay , Rats, Sprague-Dawley , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, delta/genetics , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/genetics , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/antagonists & inhibitors , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Structure-Activity RelationshipABSTRACT
Animal pharmacological studies suggest that potent and selective κ opioid receptor antagonists have potential as pharmacotherapies targeting depression, anxiety, and substance abuse (opiates, alcohol, nicotine, cocaine). We recently reported lead compound 1 as a new class of κ opioid receptor antagonists with only one basic amine group. Analogues were synthesized and evaluated for their in vitro opioid receptor antagonist properties using a [35S]GTPγS binding assay. All analogues were pure opioid receptor antagonists with no agonist activity. Compounds 1, 8, 9, 13, and 14 ( Ke values 0.058-0.64 nM) are highly potent and highly selective for the κ relative to the µ and δ opioid receptors. Favorable calculated physiochemical properties were confirmed in rat PK studies, demonstrating brain penetration for selected compounds 1, 9, and 13. High κ opioid receptor potency and selectivity and highly favorable calculated physiochemical and PK properties for brain penetration suggest these compounds should be considered for further development.
Subject(s)
Narcotic Antagonists/chemistry , Narcotic Antagonists/pharmacology , Receptors, Opioid, kappa/antagonists & inhibitors , Tetrahydroisoquinolines/chemistry , Animals , CHO Cells , Cricetulus , Dose-Response Relationship, Drug , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Male , Narcotic Antagonists/pharmacokinetics , Radioligand Assay , Rats, Sprague-Dawley , Receptors, Opioid, delta/genetics , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/genetics , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Structure-Activity RelationshipABSTRACT
The synthesis, nAChR in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted thiophenyl)deschloroepibatidine analogues (5a-f, 6a-d, and 7a-c) are presented herein. All had subnanomolar affinity at α4ß2*-nAChRs. Contrary to lead structure epibatidine, a potent nAChR agonist, all were potent α4ß2- and α3ß4-AChR antagonists in an in vitro functional assay. In vivo, the compounds were also nAChR antagonists with various degrees of agonist activity. Compounds 5e, 5f, 6a, 6c, 6d, and 7c had no agonist effects in the tail-flick, hot-plate, hypothermia, or spontaneous activity tests, whereas 5a-d, 7a and 7b did not have agonist activity in the tail-flick and hot-plate tests but, like varenicline, were agonists in the hypothermia and spontaneous activity tests. Compound 6b had agonist activity in all four in vivo tests. All the compounds were antagonists of nicotine-induced antinociception in the tail-flick test, and all except 5c, 5d, 5f, and 6b were antagonists of nicotine-induced antinociception in the hot-plate test. Compound 7c, which had a Ki = 0.86 nM in the binding assay similar potency at α4ß2/α3ß4 with selectivity relative to α7 nAChRs, had an AD50 value of 0.001 µg/kg in the tail-flick test with no agonist activity in the in vitro or in vivo test had one of the more interesting profiles.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Nicotinic Antagonists/chemical synthesis , Nicotinic Antagonists/pharmacology , Pyridines/chemical synthesis , Pyridines/therapeutic use , Receptors, Nicotinic/metabolism , Animals , Body Temperature/drug effects , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Disease Models, Animal , Locomotion/drug effects , Male , Mice , Mice, Inbred ICR , Models, Chemical , Nicotinic Antagonists/therapeutic use , Oocytes , Pain/drug therapy , Protein Binding/drug effects , Pyridines/chemistry , Pyridines/pharmacokinetics , Radioligand Assay , Rats , Structure-Activity Relationship , Tritium/pharmacokinetics , Xenopus laevisABSTRACT
Potent and selective κ opioid receptor antagonists have been derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid receptor antagonists. In order to determine if the 3-hydroxyphenyl and/or the piperidine amino groups are required for obtaining the pure opioid antagonists, (3R)-7-hydroxy-N-[(1S)-2-methyl-1-(piperidine-1-ylmethyl)propyl]-1,2,3,4-tetrahydroiosquinoline-3-carboxamide (1), which does not have a 4-(3-hydroxyphenyl) group, and (3R)-N-(1R)-1-(cyclohexylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (2), which does not have a 4-hydroxylphenyl or a piperidine amino group, were synthesized and evaluated for their [35S]GTPγS binding properties at the µ, δ, and κ opioid receptors. Surprisingly compound 1 remained a pure opioid antagonist with a Ke = 6.80 nM at the κ opioid receptor and is 21- and 441-fold selective for the κ receptor relative to the µ and δ opioid receptors, respectively. Even more unexpected and novel is the finding that 2 has a Ke = 0.14 nM at κ and is 1730- and 4570-fold selective for κ relative to the µ and δ opioid receptors, respectively.
ABSTRACT
In this study, we report the synthesis, nAChR in vitro and in vivo pharmacological properties of 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues (5, 6a,b, and 7a,b), which are analogues of our lead structure epibatidine. All of the analogues had subnanomolar binding affinity for α4ß2*-nAChRs, and all were potent antagonists of α4ß2-nAChRs in an in vitro functional assay. Analogues 6a,b were also highly selective for α4ß2- relative to α3ß4- and α7-nAChRs. Surprisingly, all of the analogues were exceptionally potent antagonists of nicotine-induced antinociception in the mouse tail-flick test, relative to standard nAChR antagonists such as DHßE. 2'-Fluoro-(4-carbamoyl-3-pyridinyl)deschloroepitabidine (6a) displayed an attractive combination of properties, including subnanomolar binding affinity (Ki = 0.07 nM), submicromolar inhibition of α4ß2-nAChRs in the functional assay (IC50 = 0.46 µM) with a high degree of selectivity for α4ß2- relative to the α3ß4/α7-nAChRs (54-/348-fold, respectively), potent inhibition of [(3)H]dopamine release mediated by α4ß2*- and α6ß2*-nAChRs in a synaptosomal preparation (IC50 = 21 and 32 nM, respectively), and an AD50 of 0.007 µg/kg as an antagonist of nicotine induced antinociception in the mouse tail-flick test which is 64â¯250 times more potent than DHßE. These data suggest that compound 6a will be highly useful as a pharmacological tool for studying nAChRs and merits further development.
Subject(s)
Analgesics , Bridged Bicyclo Compounds, Heterocyclic , Pyridines , Receptors, Nicotinic/metabolism , Analgesics/chemical synthesis , Analgesics/chemistry , Analgesics/pharmacology , Animals , Body Temperature/drug effects , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cholinergic Agents/pharmacology , Disease Models, Animal , Dopamine/metabolism , Dose-Response Relationship, Drug , Hyperalgesia/drug therapy , Locomotion/drug effects , Mice , Molecular Structure , Nicotine/pharmacology , Protein Binding/drug effects , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/pharmacology , Structure-Activity Relationship , Tritium/metabolism , Xenopus laevisABSTRACT
RATIONALE: Receptor mechanisms underlying the in vivo effects of nicotinic acetylcholine receptor (nAChR) drugs need to be determined to better understand possible differences in therapeutic potential. OBJECTIVE: This study compared the effects of agonists that are reported either to differ in intrinsic activity (i.e., efficacy) at α4ß2 nAChR in vitro or to have in vivo effects consistent with differences in efficacy. The drugs included nicotine, varenicline, cytisine, epibatidine, and three novel epibatidine derivatives: 2'-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102), 2'-fluorodeschloroepibatidine (RTI-7527-36), and 3'-(3â³-dimethylaminophenyl)-epibatidine (RTI-7527-76). METHODS: Mice discriminated nicotine base (1 mg/kg base) from saline; other mice were used to measure rectal temperature. RESULTS: In the nicotine discrimination assay, the maximum percentage of nicotine-appropriate responding varied: 92 % for nicotine, 84 % for epibatidine, 77 % for RTI-7527-36, and 71 % for varenicline and significantly less for RTI-7527-76 (58 %), RTI-7527-102 (46 %), and cytisine (33 %). Each drug markedly decreased rectal temperature by as much as 12 ºC. The rank-order potency in the discrimination and hypothermia assays was epibatidine > RTI-7527-36 > nicotine > RTI-7527-102 > varenicline = cytisine = RTI-7527-76. The nAChR antagonist mecamylamine (3.2 mg/kg) antagonized the discriminative stimulus effects of epibatidine and RTI-7527-102, as well as the hypothermic effects of every drug except cytisine. The ß2-subunit selective nAChR antagonist dihydro-ß-erythroidine (DHßE; up to 10 mg/kg) antagonized hypothermic effects but less effectively so than mecamylamine. CONCLUSIONS: The marked hypothermic effects of all drugs except cytisine are due in part to agonism at nAChR containing ß2-subunits. Differential substitution for the nicotine discriminative stimulus is consistent with differences in α4ß2 nAChR efficacy; however, collectively the current results suggest that multiple nAChR receptor subtypes mediate the effects of the agonists.
Subject(s)
Body Temperature/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Discrimination Learning/drug effects , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Pyridines/pharmacology , Receptors, Nicotinic/metabolism , Animals , Dihydro-beta-Erythroidine/pharmacology , Male , Mecamylamine/pharmacology , Mice , Nicotinic Antagonists/pharmacologyABSTRACT
2'-Fluoro-3-(substituted pyridine)epibatidine analogues 7a-e and 8a-e were synthesized, and their in vitro and in vivo nAChR properties were determined. 2'-Fluoro-3'-(4â³-pyridinyl)deschloroepibatidine (7a) and 2'-fluoro-3'-(3â³-pyridinyl)deschloroepibatidine (8a) were synthesized as bioisosteres of the 4'-nitrophenyl lead compounds 5a and 5g. Comparison of the in vitro nAChR properties of 7a and 8a to those of 5a and 5g showed that 7a and 8a had in vitro nAChR properties similar to those of 5a and 5g but both were more selective for the α4ß2-nAChR relative to the α3ß4- and α7-nAChRs than 5a and 5g. The in vivo nAChR properties in mice of 7a were similar to those of 5a. In contrast, 8a was an agonist in all four mouse acute tests, whereas 5g was active only in a spontaneous activity test. In addition, 5g was a nicotine antagonist in both the tail-flick and hot-plate tests, whereas 8a was an antagonist only in the tail-flick test.
Subject(s)
Analgesics/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Pyridines/chemical synthesis , Receptors, Nicotinic/metabolism , Analgesics/chemistry , Analgesics/pharmacology , Animals , Body Temperature/drug effects , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Contraindications , Female , Male , Mice , Nicotinic Agonists/chemical synthesis , Nicotinic Agonists/chemistry , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/chemical synthesis , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/pharmacology , Oocytes/drug effects , Oocytes/physiology , Pain/drug therapy , Pain/physiopathology , Patch-Clamp Techniques , Pyridines/chemistry , Pyridines/pharmacology , Radioligand Assay , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , XenopusABSTRACT
Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines 5b-g, analogues of 3'-(4-nitrophenyl) compound 5a. All compounds had high affinity for α4ß2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4ß2-, α3ß4-, and α7-nAChRs. The 4-carbamoylphenyl analogue 5g was highly selective for α4ß2-nAChR over α3ß4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using the agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and 5a-g. In particular, a unique binding mode for 5g was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of 5g for α4ß2-nAChR combined with its nAChR functional antagonist properties suggest that 5g will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other central nervous system disorders.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Nicotinic Agonists/chemical synthesis , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/chemical synthesis , Nicotinic Antagonists/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Receptors, Nicotinic/metabolism , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Chemistry Techniques, Synthetic , Electrophysiological Phenomena/drug effects , Humans , Male , Mice , Models, Molecular , Nicotinic Agonists/chemistry , Nicotinic Antagonists/chemistry , Protein Conformation , Pyridines/chemistry , Receptors, Nicotinic/chemistryABSTRACT
A concise synthesis of (S)-macrostomine has been accomplished in five steps from natural nicotine in 19% overall yield via a pyridyne Diels-Alder cycloaddition reaction as the key step. A Kumada cross-coupling reaction on a 1-chloroisoquinoline intermediate provided the natural product.