Wayne W. Harding

An opioid agonist that does not induce μ-Opioid receptor-arrestin interactions or receptor internalization

G protein-coupled receptor desensitization and trafficking are important regulators of opioid receptor signaling that can dictate overall drug responsiveness in vivo. Furthermore, different μ-opioid receptor (μOR) ligands can lead to varying degrees of receptor regulation, presumably because of distinct structural conformations conferred by agonist binding. For example, morphine binding produces a μOR with low affinity for β-arrestin proteins and limited receptor internalization, whereas enkephalin analogs promote robust trafficking of both β-arrestins and the receptors. Here, we evaluate μOR trafficking in response to activation by a novel μ-selective agonist derived from the naturally occurring plant product, salvinorin A. It is interesting that this compound, termed herkinorin, does not promote the recruitment of β-arrestin-2 to the μOR and does not lead to receptor internalization. Moreover, whereas G protein-coupled receptor kinase overexpression can promote morphine-induced β-arrestin interactions and μOR internalization, such manipulations do not promote herkinorin-induced trafficking. Studies in mice have shown that β-arrestin-2 plays an important role in the development of morphine-induced tolerance, constipation, and respiratory depression. Therefore, drugs that can activate the receptor without recruiting the arrestins may be a promising step in the development of opiate analgesics that distinguish between agonist activity and receptor regulation and may ultimately lead to therapeutics designed to provide pain relief without the adverse side effects normally associated with the opiate narcotics.

k Opioids as potential treatments for stimulant dependence

Stimulant abuse is a major problem in the United States and the development of pharmacological treatments for stimulant abuse remains an important therapeutic goal. Classically, the “dopamine hypothesis” has been used to explain the development of addiction and dependence of stimulants. This hypothesis involves the direct increase of dopamine as the major factor in mediating the abuse effects. Therefore, most treatments have focused on directly influencing the dopamine system. Another approach, which has been explored for potential treatments of stimulant abuse, is the use of κ opioid agonists. The κ receptor is known to be involved, via indirect effects, in synaptic dopamine levels. This review covers several classes of κ opioid ligands that have been explored for this purpose.

Herkinorin Analogues with Differential Beta-Arrestin-2 Interactions

Salvinorin A is a psychoactive natural product that has been found to be a potent and selective kappa opioid receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such as morphine in that it mediates potent kappa opioid receptor signaling yet leads to less receptor downregulation than observed with other kappa agonists. Our initial chemical modifications of salvinorin A have yielded one analogue, herkinorin ( 1c), with high affinity at the microOR. We recently reported that 1c does not promote the recruitment of beta-arrestin-2 to the microOR or receptor internalization. Here we describe three new derivatives of 1c ( 3c, 3f, and 3i) with similar properties and one, benzamide 7b, that promotes recruitment of beta-arrestin-2 to the microOR and receptor internalization. When the important role micro opioid receptor regulation plays in determining physiological responsiveness to opioid narcotics is considered, micro opioids derived from salvinorin A may offer a unique template for the development of functionally selective mu opioid receptor-ligands with the ability to produce analgesia while limiting adverse side effects.

Affinity of Aporphines for the Human 5-HT2A Receptor: Insights from Homology Modeling and Molecular Docking Studies

Analogs of nantenine were docked into a modeled structure of the human 5-HT(2A) receptor using ICM Pro, GLIDE, and GOLD docking methods. The resultant docking scores were used to correlate with observed in vitro apparent affinity (K(e)) data. The GOLD docking algorithm when used with a homology model of 5-HT(2A), based on a bovine rhodopsin template and built by the program MODELLER, gives results which are most in agreement with the in vitro results. Further analysis of the docking poses among members of a C1 alkyl series of nantenine analogs, indicate that they bind to the receptor in a similar orientation, but differently than nantenine. Besides an important interaction between the protonated nitrogen of the C1 alkyl analogs and residue Asp155, we identified Ser242, Phe234, and Gly238 as key residues responsible for the affinity of these compounds for the 5-HT(2A) receptor. Specifically, the ability of some of these analogs to establish a H-bond with Ser242 and hydrophobic interactions with Phe234 and Gly238 appears to explain their enhanced affinity as compared to nantenine.

(±)-Nantenine analogs as antagonists at human 5-HT2A receptors: C1 and flexible congeners

C1 and flexible analogs of (+/-)-nantenine were synthesized and evaluated for antagonist activity at human 5-HT(2A) receptors in a calcium mobilization assay. This work has resulted in the identification of the most potent 5-HT(2A) antagonist known based on an aporphine. Our results also suggest that the C1 position may be a key site for increasing 5-HT(2A) antagonist activity in this compound series. In addition, the structural rigidity of the aporphine core appears to be required for nantenine to function as a 5-HT(2A) antagonist.

New aporphinoid 5-HT2A and α1A antagonists via structural manipulations of nantenine.

A series of C1, C2, C3 and N6 analogs of nantenine (2) was synthesized and evaluated in 5-HT(2A) and α(1A) receptor functional assays. Alkyl substitution of the C1 and N6 methyl groups of nantenine provided selective 5-HT(2A) and α(1A) antagonists, respectively. The C2 alkyloxy analogs studied were generally selective for α(1A) versus 5-HT(2A). The C3 bromo analog 15 is one of the most potent aporphinoid 5-HT(2A) antagonists known presently.

Nantenine as an acetylcholinesterase inhibitor: SAR, enzyme kinetics and molecular modeling investigations.

Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman’s method. It was found that the rigid aporphine core of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ, a major causative factor in the progression of Alzheimer’s disease (AD).

Cytotoxicity of aporphines in human colon cancer cell lines HCT-116 and Caco-2: an SAR study.

A series of synthetic aporphine derivatives structurally related to domesticine and nantenine (ring A, N6 and ring C truncated analogs), was evaluated in MTS cytotoxicity assays against the human colon cancer cell lines, HCT-116 and Caco-2. In general, the C1 position of ring A is tolerant of alkoxy substituents as well as a benzoyl ester functionality. Other modifications evaluated resulted in a decrease in cytotoxic activity. The most potent compounds identified had IC(50) values in the range 23-38 μM, comparable to the known cytotoxic agent, etoposide.

Synthetic studies and pharmacological evaluations on the MDMA ('Ecstasy') antagonist nantenine.

The naturally occurring aporphine alkaloid nantenine, has been shown to antagonize behavioral and physiological effects of MDMA in mice. We have synthesized (+/-)-nantenine via an oxidative cyclization reaction with PIFA and evaluated its binding profile against a panel of CNS targets. To begin to understand the importance of the chiral center of nantenine with regards to its capacity to antagonize the effects of MDMA in vivo, (R)- and (S)-nantenine were prepared and evaluated in a food-reinforced operant task in rats. Pretreatment with either nantenine enantiomer (0.3mg/kg ip) completely blocked the behavioral suppression induced upon administration of 3.0mg/kg MDMA. (+/-)-Nantenine displayed high affinity and selectivity for the alpha(1A) adrenergic receptor among several other receptors suggesting that this alpha(1) subtype may be significantly involved in the anti-MDMA effects of the enantiomers.

Evaluation of structural effects on 5-HT2A receptor antagonism by aporphines: Identification of a new aporphine with 5-HT2A antagonist activity

A set of aporphine analogs related to nantenine was evaluated for antagonist activity at 5-HT2A and α1A adrenergic receptors. With regards to 5-HT2A receptor antagonism, a C2 allyl group is detrimental to activity. The chiral center of nantenine is not important for 5-HT2A antagonist activity, however the N6 nitrogen atom is a critical feature for 5-HT2A antagonism. Compound 12b was the most potent 5-HT2A aporphine antagonist identified in this study and has similar potency to previously identified aporphine antagonists 2 and 3. The ring A and N6 modifications examined were detrimental to α1A antagonism. A slight eutomeric preference for the R enantiomer of nantenine was observed in relation to α1A antagonism.