Modifications to the tetracaine scaffold produce cyclic nucleotide-gated channel blockers with widely varying efficacies

Five new tetracaine analogues were synthesized and evaluated for potency of blockade of cyclic nucleotide-gated channels relative to a multiply charged tetracaine analogue described previously (4). Increased positive charge at the tertiary amine end of tetracaine results in higher potency and voltage dependence of block. Modifications that reduce the hydrophobic character at the butyl tail are deleterious to block. The tetracaine analogues described here have apparent affinities for CNGA1 channels that vary over nearly 8 orders of magnitude

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice

<div><p>Background</p><p>Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects.</p><p>HC-070 <i>in vitro</i></p><p>To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases.</p><p>HC-070 <i>in vivo</i></p><p>Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC₅₀) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.</p></div

HC-070 reduces marble burying behavior.

<p>(A) Mice were administered vehicle or 1, 3 or 10 mg/kg HC-070 orally, 60 minutes prior to testing. The positive control, 10 mg/kg zimelidine, was administered IP 45 minutes prior to testing. At 1, 3, and 10 mg/kg, HC-070 significantly decreased the number of buried marbles compared to vehicle (p <0.05 *, p<0.01**, Dunnett’s post-hoc test following one-way ANOVA, n = 10/group). Zimelidine also decreased the number of buried marbles (p < 0.05, t-test, n = 10/group). Each animal is represented on the graph and the horizontal lines represent the mean. (B) Average plasma exposures from animals that completed the test. Error bars are the standard deviation.</p

Effects of HC-070 on CSD-induced fear hyper-reactivity.

<p>HC-070 reduces the increased capacity for fear memory in mice exposed to chronic social stress on days 1–15. (A) Day 16: Without drug administration, when placed in a relatively unfamiliar arena (context) without tone or electroshock, mice that had been exposed to chronic social defeat (CSD) tended to show more freezing—a fear behavior—than did control mice (CON). (B) Day 17: Without drug administration, mice were placed back in the same context and exposed to 6 pairings of a 20 s tone conditioned stimulus (CS) that announced a 2 s electroshock unconditioned stimulus (US). CSD mice acquired more freezing to the CS than did CON mice. The left panel presents the fear learning curve using the average freezing during CS-US trials 1–2, 3–4 and 5–6, and the right panel presents the average freezing across all six CS-US trials. Immediately after this CS-US conditioning session, CSD and CON mice received either vehicle or 1 mg/kg HC-070 orally. (C) Day 18: Mice received vehicle or 1 mg/kg HC-070 orally and 1 hour later were placed in the same context in which CS-US conditioning took place the previous day, and a 21-min test of context fear memory was conducted, i.e. in the absence of the CS (and US). The left panel presents the context-fear expression curve using the average freezing per 3-min block. The right panel presents the average context-fear expression across all 21 min. HC-070 significantly reduced the context fear memory in CSD mice, as indicated by these mice showing freezing levels similar to those of CON mice and lower than those of CSD-VEH mice. (D) Day 18: Immediately after the context fear memory test, the tone-CS fear memory test was conducted, comprising 12 trials of 30-s CS separated by 90-s inter-trial intervals. The fear expression curve to the CS is presented using the average freezing per pair of consecutive CS trials. HC-070 tended to reduce the CS fear memory in CSD mice, as indicated by the increased rate at which their freezing level during the CS attenuated compared with CSD-VEH mice, i.e. faster extinction learning. (E) Day 18: In the tone CS memory test presented in (D), freezing was also measured in the inter-trial intervals (ITIs) between CS presentations. The left panel presents the fear expression curve in ITIs between CSs using the average freezing per pair of consecutive ITIs. The right panel presents the average freezing across all 12 ITIs. Here also, HC-070 reduced fear memory in CSD mice, as indicated by the increased rate at which their freezing level during the ITIs attenuated compared with CSD-VEH mice, i.e. faster extinction learning.</p

HC-070 attenuates CCK-4 induced EPSCs recorded from the basolateral amygdala in a slice preparation.

<p>(A) Representative traces before and after CCK-4 application in the presence of vehicle. (B) Representative traces before and after CCK-4 application in the presence or absence of HC-070. (C) Quantitation of the results (n = 8). HC-070 was pre-incubated with the slice. CCK-4 was applied at the time indicated. Error bars represent the SEM (* p < 0.05, ***p <0.001, Tukey’s multiple comparisons test following two-way analysis of variance (ANOVA)). The holding potential was -70 mV.</p