Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1ß2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3ß2 â‰… α3ß4, indicating that ß2/ß4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9', 13', and 16', whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (-) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Potentiation of the GABAAR reveals variable energetic contributions by etiocholanolone and propofol.

The properties of a potentiator are typically evaluated by measuring its ability to enhance the magnitude of the control response. Analysis of the ability of drugs to potentiate responses from receptor channels takes place in the context of particular models to extract parameters for functional effects. In the often-used coagonist model, the agonist generating control activity and the potentiator enhancing the control activity make additive energetic contributions to stabilize the active state of the receptor. The energetic contributions are fixed and, once known, enable calculation of predicted receptor behavior at any concentration combination of agonist and potentiator. Here, we have examined the applicability of the coagonist model by measuring the relationship between the magnitude of receptor potentiation and the level of background activity. Ternary αßγ GABAA receptors were activated by GABA or the allosteric agonist propofol, or by a gain-of-function mutation, and etiocholanolone- or propofol-mediated potentiation of peak responses was measured. We show that the free energy change contributed by the modulators etiocholanolone or propofol is reduced at higher levels of control activity, thereby being in disagreement with basic principles of the coagonist model. Possible mechanisms underlying this discrepancy are discussed.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABAA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG ∼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.

Mutational Analysis of Anesthetic Binding Sites and Their Effects on GABAA Receptor Activation and Modulation by Positive Allosteric Modulators of the α7 Nicotinic Receptor.

The positive allosteric modulators (PAMs) of the α7 nicotinic receptor N-(5-Cl-2-hydroxyphenyl)-N'-[2-Cl-5-(trifluoromethyl)phenyl]-urea (NS-1738) and (E)-3-(furan-2-yl)-N-(p-tolyl)-acrylamide (PAM-2) potentiate the α1ß2γ2L GABAA receptor through interactions with the classic anesthetic binding sites located at intersubunit interfaces in the transmembrane domain of the receptor. In the present study, we employed mutational analysis to investigate in detail the involvement and contributions made by the individual intersubunit interfaces to receptor modulation by NS-1738 and PAM-2. We show that mutations to each of the anesthetic-binding intersubunit interfaces (ß+/α-, α+/ß-, and γ+/ß-), as well as the orphan α+/γ- interface, modify receptor potentiation by NS-1738 and PAM-2. Furthermore, mutations to any single interface can fully abolish potentiation by the α7-PAMs. The findings are discussed in the context of energetic additivity and interactions between the individual binding sites.