Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the α7 nicotinic acetylcholine receptor.

Several previous studies have demonstrated that the activity of neurotransmitters acting on ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR) can be altered by compounds binding to allosteric modulatory sites. In the case of α7 nAChRs, both positive and negative allosteric modulators (PAMs and NAMs) have been identified and have attracted considerable interest. A recent study, employing revised structural models of the transmembrane domain of the α7 nAChR in closed and open conformations, has provided support for an inter-subunit transmembrane allosteric binding site (Newcombe et al 2017). In the present study, we have performed virtual screening of the DrugBank database using pharmacophore queries that were based on the predicted binding mode of PAMs to α7 nAChR structural models. A total of 81 compounds were identified in the DrugBank database, of which the 25 highest-ranked hits corresponded to one of four previously-identified therapeutic compound groups (carbonic anhydrase inhibitors, cyclin-dependent kinase inhibitors, diuretics targeting the Na+-K+-Cl- cotransporter, and fluoroquinolone antibiotics targeting DNA gyrase). The top-ranked compound from each of these four groups (DB04763, DB08122, furosemide and pefloxacin, respectively) was tested for its effects on human α7 nAChR expressed in Xenopus oocytes using two-electrode voltage-clamp electrophysiology. These studies, conducted with wild-type, mutant and chimeric receptors, resulted in all four compounds exerting allosteric modulatory effects. While DB04763, DB08122 and pefloxacin were antagonists, furosemide potentiated ACh responses. Our findings, supported by docking studies, are consistent with these compounds acting as PAMs and NAMs of the α7 nAChR via interaction with a transmembrane site.

Depletion of Ca2+ from intracellular stores of the rat portal vein stimulates a tonic contraction.

The possibility that Ca2+ store depletion can stimulate contraction of the rat portal vein was investigated in functional experiments. Ca2+ stores were depleted with phenylephrine or cyclopiazonic acid in the absence of extracellular Ca2+ and then washed out for 30 min. Upon re-addition of extracellular Ca2+, a tonic contraction was produced, showing the stimulus for contraction was Ca2+ store depletion. The contractions were abolished by niflumic acid and nifedipine however, indicating they were dependent on depolarization resulting from opening of Ca2+-activated Cl- channels and Ca2+ influx through voltage-gated channels. Cumulative additions of phenylephrine below 3x10(-6) M did not produce tonic contractions but did in high K+ Krebs solution, where levcromakalim had no effect. This showed the tonic contractions were initially prevented by K+ channel opening. Increased Ca2+ entry through voltage-gated channels may therefore stimulate Ca2+-activated Cl- channels. Ca2+ store depletion could stimulate this by opening store-operated non-selective cation channels, resulting in depolarization.

Depletion of Ca2+ from intracellular stores potentiates spontaneous contractions of the rat portal vein.

Spontaneous contractions of the rat portal vein were potentiated in magnitude by phenylephrine, cyclopiazonic acid, ryanodine or caffeine. All these drugs can deplete Ca2+ from intracellular stores, which stimulates store-operated cation entry in some tissues. The possibility that depletion of Ca2+ from intracellular stores potentiates the spontaneous contractions was therefore investigated using functional experiments. Phenylephrine or cyclopiazonic acid was added to tissues in Ca2+-free Krebs solution, followed by a 30-min washout. After addition of extracellular Ca2+, the spontaneous contractions were potentiated. This showed the stimulus for potentiating the contractions remained so long as intracellular Ca2+ stores were depleted. Following phenylephrine washout in normal Krebs solution, potentiation of the spontaneous contractions was attenuated with time. This attenuation was abolished by the protein kinase C inhibitor calphostin C. These results show depletion of Ca2+ from intracellular stores potentiates spontaneous contractions of the portal vein. Protein kinase C may inhibit this mechanism.

Phasic contractions of the rat portal vein depend on intracellular Ca2+ release stimulated by depolarization.

The phasic contraction to phenylephrine of the rat isolated portal vein was investigated using functional studies. Phasic contractions to phenylephrine and caffeine could be produced after several minutes in Ca(2+)-free Krebs solution, which were inhibited by cyclopiazonic acid or ryanodine. The phenylephrine and caffeine contractions were abolished, however, within 10 min in Ca(2+)-free Krebs solution and by nifedipine. This indicated the Ca(2+) stores were depleted in the absence of Ca(2+) influx through voltage-gated channels. The phasic contraction to phenylephrine was also abolished by niflumic acid even in Ca(2+)-free Krebs solution. This showed that the response depended on intracellular Ca(2+) release stimulated directly by depolarization, resulting from opening of Ca(2+)-activated Cl(-) channels, but did not require Ca(2+) influx. In support of this, K(+)-induced phasic contractions were also produced in Ca(2+)-free Krebs solution. The phenylephrine but not K(+)-induced phasic contractions in Ca(2+)-free Krebs solution were inhibited by ryanodine or cyclopiazonic acid. This would be consistent with Ca(2+) release from more superficial intracellular stores (affected most by these agents), probably by inositol 1,4,5-trisphospate, being required to stimulate the phenylephrine depolarization.