In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

Electrophysiological and Pharmacological Characterization of Human Inwardly Rectifying Kir2.1 Channels on an Automated Patch-Clamp Platform.

Inwardly rectifying IK1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding Kir2.1 channels, which primarily conduct ventricular IK1, are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of Kir2.1-mediated IK1 currents is a cardiosafety concern during new drug discovery and development. Kir2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of Kir2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human Kir2.1 channels. We reproduced key electrophysiological and pharmacological features known for native IK1, including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the Kir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of this assay authorizes medium throughput screening for cardiosafety purposes and could help to enrich the currently limited Kir2.1 pharmacology.

From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei.

BACKGROUND AND PURPOSE: The NaV 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. EXPERIMENTAL APPROACH: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models. KEY RESULTS: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNaV 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel.

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.

Expression of human FE65 in amyloid precursor protein transgenic mice is associated with a reduction in beta-amyloid load.

FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non-neuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and beta-amyloid (Abeta) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Abeta deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Abeta accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP-CTFs. The reduced accumulation of Abeta in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Abeta42 level observed at 4 months of age and to the lower APP-CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.