Lisa Benson

Discovery of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (TC-5619), a selective α7 nicotinic acetylcholine receptor agonist, for the treatment of cognitive disorders.

(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7a, TC-5619), a novel selective agonist of the α7 neuronal nicotinic acetylcholine receptor, has been identified as a promising drug candidate for the treatment of cognitive impairment associated with neurological disorders. 7a demonstrated more than a thousand-fold separation between the affinities for the α7 and α4β2 receptor subtypes and had no detectable effects on muscle or ganglionic nicotinic receptor subtypes, indicating a marked selectivity for the central nervous system over the peripheral nervous system. Results obtained from homology modeling and docking explain the observed selectivity. 7a had positive effects across cognitive, positive, and negative symptoms of schizophrenia in animal models and was additive or synergistic with the antipsychotic clozapine. Compound 7a, as an augmentation therapy to the standard treatment with antipsychotics, demonstrated encouraging results on measures of negative symptoms and cognitive dysfunction in schizophrenia and was well tolerated in a phase II clinical proof of concept trial in patients with schizophrenia.

Differential Pharmacologies of Mecamylamine Enantiomers: Positive Allosteric Modulation and Noncompetitive Inhibition

(±)-Mecamylamine is a racemic mixture of a widely used brain-permeant noncompetitive inhibitor of muscle-type and neuronal nicotinic receptors (NNRs). The present studies evaluated whether the stereoisomers of this drug show different profiles for inhibition of the high-sensitivity (HS) and low-sensitivity (LS) isoforms of the human α4β2 NNR subtype expressed in subclonal human epithelial 1 cells. We found that at low concentrations (micromolar range), TC-5214 [S-(+)-mecamylamine] was more effective than TC-5213 [R-(-)-mecamylamine] in inhibiting the LS α4β2 NNRs. In addition, we demonstrated that TC-5214 potentiated and TC-5213 inhibited agonist-induced activation of HS α4β2 NNRs. The stereoselectivity of mecamylamine enantiomers at HS and LS α4β2 receptors demonstrates that TC-5214 is the preferred stereoisomer for selective activation of HS, whereas it is more effective in suppressing LS receptor function. This feature could be relevant to therapeutic applications where such a selective mechanism of action is required.

Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3β4 and hα4β2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC50 values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHβE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

Functional and Structural Interaction of (-)-Reboxetine with the Human α4β2 Nicotinic Acetylcholine Receptor

The interaction of the selective norepinephrine reuptake inhibitor (−)-reboxetine with the human α4β2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca2+-influx results indicate that (−)-reboxetine does not activate hα4β2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4β2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (−)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4β2 nAChR function. We tested whether (−)-reboxetine binds to the luminal [3H]imipramine site. The results indicate that, although (−)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4β2 nAChR ion channels. Patch-clamp results also indicate that (−)-reboxetine progressively inhibits the hα4β2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (−)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6′ and 14′. In addition, we found a (−)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (−)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (−)-reboxetine may be produced (at least partially) by its inhibitory action on hα4β2 nAChRs.