Synthetic resin acid derivatives selectively open the hKV 7.2/7.3 channel and prevent epileptic seizures.

OBJECTIVE: About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKV ) channel hKV 7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hKV 7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hKV 7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. METHODS: Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. RESULTS: Fourteen resin acid derivatives were tested on hKV 7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hKV 7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hKV 7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hKV 11.1, hNaV 1.5, or hCaV 1.2, or on the amplitude of hKV 11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. SIGNIFICANCE: The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel.

Voltage-gated potassium (KV) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.

Atom-by-atom tuning of the electrostatic potassium-channel modulator dehydroabietic acid.

Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel's positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel's voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug.

A drug pocket at the lipid bilayer-potassium channel interface.

Many pharmaceutical drugs against neurological and cardiovascular disorders exert their therapeutic effects by binding to specific sites on voltage-gated ion channels of neurons or cardiomyocytes. To date, all molecules targeting known ion channel sites bind to protein pockets that are mainly surrounded by water. We describe a lipid-protein drug-binding pocket of a potassium channel. We synthesized and electrophysiologically tested 125 derivatives, analogs, and related compounds to dehydroabietic acid. Functional data in combination with docking and molecular dynamics simulations mapped a binding site for small-molecule compounds at the interface between the lipid bilayer and the transmembrane segments S3 and S4 of the voltage-sensor domain. This fundamentally new binding site for small-molecule compounds paves the way for the design of new types of drugs against diseases caused by altered excitability.

Resin-acid derivatives as potent electrostatic openers of voltage-gated K channels and suppressors of neuronal excitability.

Voltage-gated ion channels generate cellular excitability, cause diseases when mutated, and act as drug targets in hyperexcitability diseases, such as epilepsy, cardiac arrhythmia and pain. Unfortunately, many patients do not satisfactorily respond to the present-day drugs. We found that the naturally occurring resin acid dehydroabietic acid (DHAA) is a potent opener of a voltage-gated K channel and thereby a potential suppressor of cellular excitability. DHAA acts via a non-traditional mechanism, by electrostatically activating the voltage-sensor domain, rather than directly targeting the ion-conducting pore domain. By systematic iterative modifications of DHAA we synthesized 71 derivatives and found 32 compounds more potent than DHAA. The most potent compound, Compound 77, is 240 times more efficient than DHAA in opening a K channel. This and other potent compounds reduced excitability in dorsal root ganglion neurons, suggesting that resin-acid derivatives can become the first members of a new family of drugs with the potential for treatment of hyperexcitability diseases.

Drug-induced ion channel opening tuned by the voltage sensor charge profile.

Polyunsaturated fatty acids modulate the voltage dependence of several voltage-gated ion channels, thereby being potent modifiers of cellular excitability. Detailed knowledge of this molecular mechanism can be used in designing a new class of small-molecule compounds against hyperexcitability diseases. Here, we show that arginines on one side of the helical K-channel voltage sensor S4 increased the sensitivity to docosahexaenoic acid (DHA), whereas arginines on the opposing side decreased this sensitivity. Glutamates had opposite effects. In addition, a positively charged DHA-like molecule, arachidonyl amine, had opposite effects to the negatively charged DHA. This suggests that S4 rotates to open the channel and that DHA electrostatically affects this rotation. A channel with arginines in positions 356, 359, and 362 was extremely sensitive to DHA: 70 µM DHA at pH 9.0 increased the current >500 times at negative voltages compared with wild type (WT). The small-molecule compound pimaric acid, a novel Shaker channel opener, opened the WT channel. The 356R/359R/362R channel drastically increased this effect, suggesting it to be instrumental in future drug screening.