Noribogaine is a G-protein biased κ-opioid receptor agonist.

Noribogaine is the long-lived human metabolite of the anti-addictive substance ibogaine. Noribogaine efficaciously reaches the brain with concentrations up to 20 µM after acute therapeutic dose of 40 mg/kg ibogaine in animals. Noribogaine displays atypical opioid-like components in vivo, anti-addictive effects and potent modulatory properties of the tolerance to opiates for which the mode of action remained uncharacterized thus far. Our binding experiments and computational simulations indicate that noribogaine may bind to the orthosteric morphinan binding site of the opioid receptors. Functional activities of noribogaine at G-protein and non G-protein pathways of the mu and kappa opioid receptors were characterized. Noribogaine was a weak mu antagonist with a functional inhibition constants (Ke) of 20 µM at the G-protein and ß-arrestin signaling pathways. Conversely, noribogaine was a G-protein biased kappa agonist 75% as efficacious as dynorphin A at stimulating GDP-GTP exchange (EC50=9 µM) but only 12% as efficacious at recruiting ß-arrestin, which could contribute to the lack of dysphoric effects of noribogaine. In turn, noribogaine functionally inhibited dynorphin-induced kappa ß-arrestin recruitment and was more potent than its G-protein agonistic activity with an IC50 of 1 µM. This biased agonist/antagonist pharmacology is unique to noribogaine in comparison to various other ligands including ibogaine, 18-MC, nalmefene, and 6'-GNTI. We predict noribogaine to promote certain analgesic effects as well as anti-addictive effects at effective concentrations>1 µM in the brain. Because elevated levels of dynorphins are commonly observed and correlated with anxiety, dysphoric effects, and decreased dopaminergic tone, a therapeutically relevant functional inhibition bias to endogenously released dynorphins by noribogaine might be worthy of consideration for treating anxiety and substance related disorders.

Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs). Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles. Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster" from Augustin et al, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.