Reanalysis of a µ opioid receptor crystal structure reveals a covalent adduct with BU72.

BACKGROUND: The first crystal structure of the active µ opioid receptor (µOR) exhibited several unexplained features. The ligand BU72 exhibited many extreme deviations from ideal geometry, along with unexplained electron density. I previously showed that inverting the benzylic configuration resolved these problems, establishing revised stereochemistry of BU72 and its analog BU74. However, another problem remains unresolved: additional unexplained electron density contacts both BU72 and a histidine residue in the N-terminus, revealing the presence of an as-yet unidentified atom. RESULTS: These short contacts and uninterrupted density are inconsistent with non-covalent interactions. Therefore, BU72 and µOR form a covalent adduct, rather than representing two separate entities as in the original model. A subsequently proposed magnesium complex is inconsistent with multiple lines of evidence. However, oxygen fits the unexplained density well. While the structure I propose is tentative, similar adducts have been reported previously in the presence of reactive oxygen species. Moreover, known sources of reactive oxygen species were present: HEPES buffer, nickel ions, and a sequence motif that forms redox-active nickel complexes. This motif contacts the unexplained density. The adduct exhibits severe strain, and the tethered N-terminus forms contacts with adjacent residues. These forces, along with the nanobody used as a G protein substitute, would be expected to influence the receptor conformation. Consistent with this, the intracellular end of the structure differs markedly from subsequent structures of active µOR bound to Gi protein. CONCLUSIONS: Later Gi-bound structures are likely to be more accurate templates for ligand docking and modelling of active G protein-bound µOR. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions, and in the interpretation of excess or unexplained density.

Selective κ opioid antagonists nor-BNI, GNTI and JDTic have low affinities for non-opioid receptors and transporters.

BACKGROUND: Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. RESULTS: In binding assays, the three antagonists showed no detectable affinity (K(i)≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over µ and δ (3 to 44-fold). Nor-BNI bound weakly to the α(2C)-adrenoceptor (K(i) = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α(1A)-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (K(B) = 3.7 µM). JDTic bound to the noradrenaline transporter (K(i) = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K(i) = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. CONCLUSIONS: Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α(1A)-adrenoceptors). This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

Functional selectivity of 6'-guanidinonaltrindole (6'-GNTI) at κ-opioid receptors in striatal neurons.

There is considerable evidence to suggest that drug actions at the κ-opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gαi/o protein coupling and the recruitment of ß-arrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and ß-arrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as "functional selectivity" or "signaling bias." Recently, a KOR agonist, 6'-guanidinonaltrindole (6'-GNTI), was shown to display bias toward the activation of G protein-mediated signaling over ß-arrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. Although the reference KOR agonist U69,593 induces the phosphorylation of ERK1/2 and Akt, 6'-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and ß-arrestin2 knock-out mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires ß-arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.

Studies toward bivalent κ opioids derived from salvinorin A: heteromethylation of the furan ring reduces affinity.

The recent crystal structure of the κ-opioid receptor (κ-OR) revealed, unexpectedly, that the antagonist JDTic is a bivalent ligand: in addition to the orthosteric pocket occupied by morphinans, JDTic also occupies a distinct (allotopic) pocket. Mutagenesis data suggest that salvinorin A (1) also binds to this allotopic pocket, adjacent to the aspartate residue that anchors the basic nitrogen atom of classical opiates (Asp138). It has been suggested that an H-bond donor appended to 1 might interact with Asp138, increasing affinity. Such a bivalent ligand might also possess altered functional selectivity. Based on modeling and known N-furanylmethyl opioid antagonists, we appended H-bond donors to the furan ring of 1. (Dimethylamino)methyl groups at C-15 or C-16 abolished affinity for κ-OR. Hydroxymethylation at C-16 was tolerated, but 15,16-bis-hydroxymethylation was not. Since allosteric modulators may go undetected in binding assays, we also tested these and other low-affinity derivatives of 1 for allosteric modulation of dynorphin A in the [(35)S]GTPγS assay. No modulation was detected. As an alternative attachment point for bivalent derivatives, we prepared the 2-(hydroxyethoxy)methyl ether, which retained high affinity for κ-OR. We discuss alternative design strategies for linked, fused or merged bivalent derivatives of 1.

Long-acting κ opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity.

BACKGROUND: Nor-BNI, GNTI and JDTic induce κ opioid antagonism that is delayed by hours and can persist for months. Other effects are transient. It has been proposed that these drugs may be slowly absorbed or distributed, and may dissolve in cell membranes, thus slowing elimination and prolonging their effects. Recent evidence suggests, instead, that they induce prolonged desensitization of the κ opioid receptor. METHODS: To evaluate these hypotheses, we measured relevant physicochemical properties of nor-BNI, GNTI and JDTic, and the timecourse of brain and plasma concentrations in mice after intraperitoneal administration (using LC-MS-MS). RESULTS: In each case, plasma levels were maximal within 30 min and declined by >80% within four hours, correlating well with previously reported transient effects. A strong negative correlation was observed between plasma levels and the delayed, prolonged timecourse of κ antagonism. Brain levels of nor-BNI and JDTic peaked within 30 min, but while nor-BNI was largely eliminated within hours, JDTic declined gradually over a week. Brain uptake of GNTI was too low to measure accurately, and higher doses proved lethal. None of the drugs were highly lipophilic, showing high water solubility (> 45 mM) and low distribution into octanol (log D7.4 < 2). Brain homogenate binding was within the range of many shorter-acting drugs (>7% unbound). JDTic showed P-gp-mediated efflux; nor- BNI and GNTI did not, but their low unbound brain uptake suggests efflux by another mechanism. CONCLUSIONS: The negative plasma concentration-effect relationship we observed is difficult to reconcile with simple competitive antagonism, but is consistent with desensitization. The very slow elimination of JDTic from brain is surprising given that it undergoes active efflux, has modest affinity for homogenate, and has a shorter duration of action than nor-BNI under these conditions. We propose that this persistence may result from entrapment in cellular compartments such as lysosomes.

Salvinorin B meth-oxy-methyl ether.

The title compound [MOM-SalB; systematic name: methyl (2S,4aR,6aR,7R,9S,10aS,10bR)-2-(3-fur-yl)-9-meth-oxy-meth-oxy-6a,10b-dimethyl-4,10-dioxo-2,4a,5,6,7,8,9,10a-octa-hydro-1H-benzo[f]isochromene-7-carboxyl-ate], C(23)H(30)O(8), is a deriv-ative of the κ-opioid salvinorin A with enhanced potency, selectivity, and duration of action. Superimposition of their crystal structures reveals, surprisingly, that the terminal C and O atoms of the MOM group overlap with the corresponding atoms in salvinorin A, which are separated by an additional bond. This counter-intuitive isosterism is possible because the MOM ether adopts the 'classic anomeric' conformation (gauche-gauche), tracing a helix around the planar acetate of salvinorin A. This overlap is not seen in the recently reported structure of the tetra-hydro-pyranyl ether, which is less potent. The classic anomeric conformation is strongly favoured in alk-oxy-methyl ethers, but not in substituted acetals, which may contribute to their reduced potency. This structure may prove useful in evaluating models of the activated κ-opioid receptor.

Lack of effect of sublingual salvinorin A, a naturally occurring kappa opioid, in humans: a placebo-controlled trial.

RATIONALE: Salvinorin A (SA) is a highly selective kappa opioid receptor agonist and the putative psychoactive compound in Salvia divinorum (SD), an increasingly abused hallucinogenic plant. OBJECTIVES: The objectives of this study were to characterize the physiological and subjective effects of SA versus placebo and measure drug and metabolite levels. METHODS: Sublingual SA doses up to 4 mg were administered in dimethyl sulfoxide/polyethylene glycol 400 solution to eight SD-experienced subjects using a placebo-controlled ascending-dose design. RESULTS: No dose of SA produced significantly greater physiological or subjective effects than placebo. Furthermore, effects did not resemble reported "typical" effects of smoked SD. SA was detectable in plasma and urine, but was, in most cases, below the reliable limit of quantification (0.5 ng/mL). CONCLUSIONS: Our results suggest that the sublingual bioavailability of SA is low. Higher doses, alternate formulations, or alternate routes of administration will be necessary to study the effects of SA in humans.

Salvinorin A and derivatives: protection from metabolism does not prolong short-term, whole-brain residence.

Salvinorin A (SA) is a potent kappa opioid agonist with a brief duration of action. Consistent with this, our previous positron emission tomography (PET) studies of carbon-11 labeled SA showed that brain levels decrease rapidly after intravenous administration. SA is rapidly metabolized, giving the much less potent salvinorin B (SB), which is presumed to be responsible in part for SA's brief duration of action. To test this, we labeled the metabolically stable methyl ester of SA and SB with carbon-11 and compared their pharmacokinetics by PET imaging after intravenous administration to baboons. Labeling of salvinorin B ethoxymethyl ether (EOM-SB), a derivative with greater potency and resistance to metabolism, provided an additional test of the role of metabolism in brain efflux. Plasma analysis confirmed that SB and EOM-SB exhibited greater metabolic stability than SA. However, the three compounds exhibited very similar pharmacokinetics in brain, entering and exiting rapidly. This suggests that metabolism is not solely responsible for the brief brain residence time of SA. We determined that whole-brain concentrations of EOM-SB declined more slowly than SA after intraperitoneal administration in rodents. This is likely due to a combination in EOM-SB's increased metabolic stability and its decreased plasma protein affinity. Our results suggest that protecting salvinorin A derivatives from metabolism will prolong duration of action, but only when administered by routes giving slow absorption.

Modification of the furan ring of salvinorin A: identification of a selective partial agonist at the kappa opioid receptor.

In an effort to find novel agents which selectively target the kappa opioid receptor (KOPR), we modified the furan ring of the highly potent and selective KOPR agonist salvinorin A. Introduction of small substituents at C-16 was well tolerated. 12-epi-Salvinorin A, synthesized in four steps from salvinorin A, was a selective partial agonist at the KOPR. No clear SAR patterns were observed for C-13 aryl ketones. Introducing a hydroxymethylene group between C-12 and the furan ring was tolerated. Small C-13 esters and ethers gave weak KOPR agonists, while all C-13 amides were inactive. Finally, substitution of oxadiazoles for the furan ring abolished affinity for the KOPR. None of the compounds displayed any KOPR antagonism or any affinity for mu or delta opioid receptors.

Standard protecting groups create potent and selective kappa opioids: salvinorin B alkoxymethyl ethers.

Protection of salvinorin B as standard alkoxyalkyl ethers yielded highly potent kappa opioid receptor agonists. Ethoxymethyl ether 6 is among the most potent and selective kappa agonists reported to date. Fluoroethoxymethyl ether 11 is the first potent, selective fluorinated kappa ligand, with potential use in MRI and PET studies. Further enlargement of the alkoxy group, alkylation of the acetal carbon, or heteroatom substitution all reduced activity. These protecting groups may prove useful in related work not only by enabling the use of harsher synthetic conditions, but potentially by optimizing the potency of the products.

N-methylacetamide analog of salvinorin A: a highly potent and selective kappa-opioid receptor agonist with oral efficacy.

Several preclinical studies indicate that selective kappa-opioid receptor (KOR) antagonists have antidepressant-like effects, whereas KOR agonists have opposite effects, suggesting that each might be useful in the treatment of mood abnormalities. Salvinorin A (salvA) is a valuable KOR agonist for further study due to its high potency and receptor selectivity. However, it has short lasting effects in vivo and limited oral bioavailability, probably due to acetate metabolism. We compared the in vitro receptor binding selectivity of salvA and four analogs containing an ethyl ether (EE), isopropylamine (IPA), N-methylacetamide (NMA), or N-methylpropionamide (NMP) at C-2. All compounds showed high binding affinity for the KOR (K(i) = 0.11-6.3 nM), although only salvA, EE, and NMA exhibited KOR selectivity. In a liver microsomal assay, salvA was least stable, whereas NMA and IPA displayed slower metabolic transformations. Intraperitoneal (i.p.) administration of salvA, NMA, and NMP dose-dependently elevated brain reward thresholds in the intracranial self-administration (ICSS) test, consistent with prodepressive-like KOR agonist effects. NMA and NMP were equipotent to salvA but displayed longer lasting effects (6- and 10-fold, respectively). A dose of salvA with prominent effects in the ICSS test after i.p. administration (2.0 mg/kg) was inactive after oral administration, whereas the same oral dose of NMA elevated ICSS thresholds. These studies suggest that, although salvA and NMA are similar in potency and selectivity as KOR agonists in vitro, NMA has improved stability and longer lasting actions that might make it more useful for studies of KOR agonist effects in animals and humans.

8-epi-Salvinorin B: crystal structure and affinity at the kappa opioid receptor.

There have been many reports of epimerization of salvinorins at C-8 under basic conditions, but little evidence has been presented to establish the structure of these compounds. We report here the first crystal structure of an 8-epi-salvinorin or derivative: the title compound, 2b. The lactone adopts a boat conformation with the furan equatorial. Several lines of evidence suggest that epimerization proceeds via enolization of the lactone rather than a previously proposed indirect mechanism. Consistent with the general trend in related compounds, the title compound showed lower affinity at the kappa opioid receptor than the natural epimer salvinorin B (2a). The related 8-epi-acid 4b showed no affinity.

Autoxidation of salvinorin A under basic conditions.

[reaction: see text] Treatment of salvinorin A (1a) with KOH in MeOH gave the enedione 3, for which the dienone structure 7 was recently proposed. Also isolated, after methylation, were the secotriesters 4a-c. A mechanism for this unusual series of autoxidations is proposed. Surprisingly, 4a showed weak affinity at the kappa-opioid receptor. Divinatorins A-C (2a-c) showed no affinity at opioid receptors. Attempted reduction of 3 to a novel salvinorin diol (9d) was unsuccessful, but careful deacetylation of salvinorin C (9a) provided a viable route to this compound. A general method for identifying salvinorin 8-epimers by TLC is also presented.

Studies toward the pharmacophore of salvinorin A, a potent kappa opioid receptor agonist.

Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.

Divinatorins A-C, new neoclerodane diterpenoids from the controlled sage Salvia divinorum.

Three new neoclerodane diterpenoids, divinatorins A-C (7-9), have been isolated from the leaves of Salvia divinorum. The compounds were identified by spectroscopic methods as derivatives of the antibiotic (-)-hardwickiic acid (10), which was also isolated, along with four other known terpenoids. Neither the crude extract nor 7-9 displayed antimicrobial activity.

Salvinorins D-F, new neoclerodane diterpenoids from Salvia divinorum, and an improved method for the isolation of salvinorin A.

Three new neoclerodane diterpenoids, salvinorins D-F (4-6), have been isolated from the leaves of Salvia divinorum. The structures were elucidated by chemical and spectroscopic methods, particularly 1D and 2D NMR. A simplified isolation method using chromatography on activated carbon also gave improved yields of the controlled substance salvinorin A (1) and of salvinorin C (3).