Reporting sodium channel activity using calcium flux: pharmacological promiscuity of cardiac Nav1.5.

Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 µM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions.

Integrated analysis of drug-induced gene expression profiles predicts novel hERG inhibitors.

Growing evidence suggests that drugs interact with diverse molecular targets mediating both therapeutic and toxic effects. Prediction of these complex interactions from chemical structures alone remains challenging, as compounds with different structures may possess similar toxicity profiles. In contrast, predictions based on systems-level measurements of drug effect may reveal pharmacologic similarities not evident from structure or known therapeutic indications. Here we utilized drug-induced transcriptional responses in the Connectivity Map (CMap) to discover such similarities among diverse antagonists of the human ether-à-go-go related (hERG) potassium channel, a common target of promiscuous inhibition by small molecules. Analysis of transcriptional profiles generated in three independent cell lines revealed clusters enriched for hERG inhibitors annotated using a database of experimental measurements (hERGcentral) and clinical indications. As a validation, we experimentally identified novel hERG inhibitors among the unannotated drugs in these enriched clusters, suggesting transcriptional responses may serve as predictive surrogates of cardiotoxicity complementing existing functional assays.

Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator.

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.

Discovery of a series of 2-phenyl-N-(2-(pyrrolidin-1-yl)phenyl)acetamides as novel molecular switches that modulate modes of K(v)7.2 (KCNQ2) channel pharmacology: identification of (S)-2-phenyl-N-(2-(pyrrolidin-1-yl)phenyl)butanamide (ML252) as a potent, brain penetrant K(v)7.2 channel inhibitor.

A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC(50) = 69 nM), was discovered after a high-throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to hydrogen) caused a functional shift from antagonist to agonist activity (37, EC(50) = 170 nM), suggesting an interaction at a critical site for controlling gating of KCNQ2 channels.

Knockout of Zn transporters Zip-1 and Zip-3 attenuates seizure-induced CA1 neurodegeneration.

CA1 pyramidal neurons are the final integrators of information flow leaving the hippocampus, yet are singularly vulnerable to activity-dependent cell death. Zinc (Zn) entry into cells may add to this vulnerability. Here, we find that Slc39a1 and Slc39a3, members of the Zip (Zrt/Irt-like protein) plasmalemmal Zn transporter family, are predominantly expressed in the hippocampus. We examined Zip-1,3-deficient mice to investigate their role in neurodegeneration following intense synaptic activation. When isolated by blockade of NMDA receptors and voltage-gated calcium channels, the absence of both transporters slowed passive Zn uptake into CA1 neurons measured with intracellular fluorescent Zn dyes. In vivo CA1 cell damage following kainic acid exposure was greatly attenuated. Consistent with the hypothesis that Zn entry contributes to neurodegeneration, Znt-3-deficient mice lacking synaptic Zn also show less hippocampal cell damage following kainic acid injection. Zip transporters may provide selective therapeutic targets to protect these neurons from early Zn-induced neurodegeneration following injury.

Possible mechanisms for the anticonvulsant activity of fructose-1,6-diphosphate.

Fructose-1,6-diphosphate (FDP), an intracellular metabolite of glucose, has anticonvulsant activity in several models of acute seizures in laboratory animals. The anticonvulsant effect of FDP is most likely due to a direct effect since intraperitoneal and oral administration results in significant increases in brain levels. A number of mechanisms have been proposed for this action of FDP. One possibility is that peripheral administration of FDP results in changes in brain metabolism that are anticonvulsant. Glucose can be metabolized through the glycolytic or pentose phosphate pathway. There is evidence that the pentose phosphate pathway is more active in the brain than in other tissues, and that, in the presence of elevated levels of FDP, the majority of glucose is metabolized by the pentose phosphate pathway. The pentose phosphate pathway generates NADPH, which is used to reduce glutathione. The reduced form of endogenous glutathione has been shown to have anticonvulsant activity. Taken together, the data suggest a hypothesis that exogenously administered FDP gets into the brain and astrocytes where it increases the flux of glucose through the pentose phosphate pathway, generating additional NADPH for the reduction of glutathione.

Oral administration of fructose-1,6-diphosphate has anticonvulsant activity.

Recently it has been shown that fructose-1,6-diphosphate (FDP) has dose-dependent anticonvulsant activity in rat models of acute generalized motor seizures induced with chemical convulsants. The present study asked whether FDP also has activity in an epileptic brain after oral administration and activity against non-convulsive seizures. Animals (n = 14) were administered pilocarpine to induce status epilepticus. Several weeks later, these animals had spontaneous seizures and a baseline rate of seizure frequency was determined over a 6-day period. Animals were then continued without treatment (n = 8) or 0.5% FDP was added to the drinking water (n = 6). In animals treated with FDP the seizures completely stopped after 7 days. Removal of FDP from the water resulted in the return of seizure activity in 4 of the 6 animals by 16 days of observation. To induce non-convulsive seizures, animals (n = 6) received a single injection of gamma-butyrolactone (GBL, 100 mg/kg i.p.). All animals had spike-wave activity recorded in the cortex within minutes after GBL administration. Administration of a single injection of FDP (500 g/kg i.p.) had no effect on the baseline cortical activity, nor on the spike-wave activity induced by GBL (n = 5). These experiments suggest that oral administration of FDP may have utility in the treatment of partial or generalized convulsive seizure disorders, but not absence seizures.

Antioxidants and free radical scavengers do not consistently delay seizure onset in animal models of acute seizures.

A number of herbal compounds with direct antioxidant activity slow the onset, or completely block, the occurrence of seizures. This increase in latency has been proposed to be due to the antioxidant activity. This hypothesis was directly tested by determining the effects of Trolox, a vitamin E analog, vitamin C, melatonin, and alpha-lipoic acid on the latency to acute seizures induced with pilocarpine, kainic acid, or subcutaneous pentylenetetrazol (PTZ) in adult rats. Trolox, vitamin C, and alpha-lipoic acid had significant anticonvulsant activity against pilocarpine, but there were no acute changes in reduced glutathione levels at 15 or 120 minutes. Other than reduced mortality with vitamin C in the PTZ model, none of the antioxidants had a significant effect against PTZ- or kainic acid-induced seizures. The lack of consistent anticonvulsant effect suggests that the antioxidant activity of the herbal preparations cannot account for the delay in seizure onset.

Pharmacokinetics of fructose-1,6-diphosphate after intraperitoneal and oral administration to adult rats.

Exogenously administered fructose-1,6-diphosphate (FDP) has been studied for its ability to protect tissue during hypoxia or ischemia. Recently, a clear effect of FDP on the central nervous system has raised the question whether FDP can get into the brain. FDP levels were measured in blood, brain, liver, kidney, muscle and fat after intraperitoneal administration of a single 0.5gkg(-1) dose of FDP to adult male Sprague-Dawley rats. A complete time course of the levels in blood and brain was determined. The levels of FDP in the blood and brain increase simultaneously, i.e. there is no lag in the increase in the brain. The levels of FDP fall to baseline in liver, kidney, muscle and fat by 12h, but remain elevated in blood and brain. However, levels in the blood at 12h are significantly decreased from the peak levels, while those in brain are not different from the peak levels, suggesting that the kinetics of FDP in blood and brain are quite different. Stripping the endothelial cells from the brain tissue sample did not change the levels of FDP indicating that FDP is not trapped in the capillary cells. Incubation of brain slices in a solution of FDP, followed by washing, raised tissue levels of FDP indicating that FDP is taken up into cells within the brain. Finally, the experiments demonstrate a significant increase in brain levels of FDP after oral administration. These data suggest that an oral formulation of FDP might be developed for treatment of neurological disease.

Regulation of extracellular calcium in the hippocampus in vivo during epileptiform activity--role of astrocytes.

Astrocytes have been suggested to regulate the extracellular calcium concentration ([Ca(2+)](o)), but this has not been thoroughly investigated. Adult, male Sprague-Dawley rats were used to record changes in [Ca(2+)](o) in the hippocampus during epileptiform activity. Maximal decreases in [Ca(2+)](o) in CA1 were measured in the pyramidal cell layer during 20 Hz, 20s stimulus trains to the contralateral CA3 region. Maximal decreases in [Ca(2+)](o) in the dentate gyrus were measured when maximal dentate activation had appeared-irrespective of the location, frequency or duration of the stimulation. Maximal decreases were 36% greater in the dentate gyrus than in CA1. During prolonged discharges, [Ca(2+)](o) recovered partially towards the baseline in both hippocampal regions. To investigate the role of astrocytes, local injections of fluorocitrate (FC), a metabolic toxin selectively taken up by astrocytes, were used. FC (0.1, 0.25 or 0.5mM FC), but not vehicle (2 microl), caused a small, but significant decrease in the maximal changes in CA1, but an increase in the dentate gyrus. The results suggest that maximal decreases in [Ca(2+)](o) occur in the hippocampus in response to burst firing of neurons and that astrocytes play a minimal role in the regulation of [Ca(2+)](o) during epileptiform activity.

Myasthenia gravis induced in mice by immunization with the recombinant extracellular domain of rat muscle-specific kinase (MuSK).

UNLABELLED: Myasthenia gravis (MG) is mostly caused by anti-acetylcholine receptor (AChR) auto-antibodies (Abs). Such Abs are undetectable in 10-15% of MG patients, but many have anti-muscle-specific kinase (MuSK) Abs. We injected recombinant rat-MuSK extracellular domain in H-2(a), H-2(b), H-2(bm12) and H-2(d) mice. Certain strains exhibited exercise-induced fatigue, tremors, weight loss, and some died after 2-3 injections. Compound muscle action potentials showed decrement with low-frequency repetitive nerve stimulation. Miniature endplate potentials decreased, suggesting lower numbers of endplates functional AChRs. Myasthenic sera inhibited agrin-induced AChR aggregation in C2C12 myotubes. CONCLUSION: Anti-MuSK Abs induce MG, which might also result from blocking the agrin-signaling pathway.

Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph.

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert.