Selective, direct activation of high-conductance, calcium-activated potassium channels causes smooth muscle relaxation.

High-conductance calcium-activated potassium (Maxi-K) channels are present in smooth muscle where they regulate tone. Activation of Maxi-K channels causes smooth muscle hyperpolarization and shortening of action-potential duration, which would limit calcium entry through voltage-dependent calcium channels leading to relaxation. Although Maxi-K channels appear to indirectly mediate the relaxant effects of a number of agents, activators that bind directly to the channel with appropriate potency and pharmacological properties useful for proof-of-concept studies are not available. Most agents identified to date display significant polypharmacy that severely compromises interpretation of experimental data. In the present study, a high-throughput, functional, cell-based assay for identifying Maxi-K channel agonists was established and used to screen a large sample collection (>1.6 million compounds). On the basis of potency and selectivity, a family of tetrahydroquinolines was further characterized. Medicinal chemistry efforts afforded identification of compound X, from which its two enantiomers, Y and Z, were resolved. In in vitro assays, Z is more potent than Y as a channel activator. The same profile is observed in tissues where the ability of either agent to relax precontracted smooth muscles, via a potassium channel-dependent mechanism, is demonstrated. These data, taken together, suggest that direct activation of Maxi-K channels represents a mechanism to be explored for the potential treatment of a number of diseases associated with smooth muscle hyperexcitability.

A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-à-go-go related gene potassium channel.

The voltage-gated potassium channel, human Ether-à-go-go related gene (hERG), represents the molecular component of IKr, one of the potassium currents involved in cardiac action potential repolarization. Inhibition of IKr increases the duration of the ventricular action potential, reflected as a prolongation of the QT interval in the electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. Because hERG is an appropriate surrogate for IKr, hERG assays that can identify potential safety liabilities of compounds during lead identification and optimization have been implemented. Although the gold standard for hERG evaluation is electrophysiology, this technique, even with the medium capacity, automated instruments that are currently available, does not meet the throughput demands for supporting typical medicinal chemistry efforts in the pharmaceutical environment. Assays that could provide reliable molecular pharmacology data, while operating in high capacity mode, are therefore desirable. In the present study, we describe a high-capacity, 384- and 1,536-well plate, functional thallium flux assay for the hERG channel that fulfills these criteria. This assay was optimized and validated using different structural classes of hERG inhibitors. An excellent correlation was found between the potency of these agents in the thallium flux assay and in electrophysiological recordings of channel activity using the QPatch automated patch platform. Extension of this study to include 991 medicinal chemistry compounds from different internal drug development programs indicated that the thallium flux assay was a good predictor of in vitro hERG activity. These data suggest that the hERG thallium flux assay can play an important role in supporting drug development efforts.

A KV2.1 gating modifier binding assay suitable for high throughput screening.

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.

ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors.

Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.

Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin.

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.

Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function.

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.

Discovery of potent and use-dependent sodium channel blockers for treatment of chronic pain.

A new series of voltage-gated sodium channel blockers with potential for treatment of chronic pain is reported. Systematic structure-activity relationship studies, starting with compound 1, led to identification of potent analogs that displayed use-dependent block of sodium channels, were efficacious in pain models in vivo, and most importantly, were devoid of activity against the cardiac potassium channel hERG.

A disubstituted succinamide is a potent sodium channel blocker with efficacy in a rat pain model.

Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In a high-throughput functional assay, a novel sodium channel (Na(V)) blocker, N-[[2'-(aminosulfonyl)biphenyl-4-yl]methyl]-N'-(2,2'-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of Na(V)1.2, Na(V)1.5, and Na(V)1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of Na(V)1.2 was 1.2 and 0.14 microM, respectively. BPBTS blocked Na(V)1.7 and Na(V)1.2 with similar potency, whereas block of Na(V)1.5 was slightly more potent. The slow tetrodotoxin-resistant Na(+) current in small-diameter DRG neurons was also potently blocked by BPBTS. [(3)H]BPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K(d) = 6.1 nM), and in membranes derived from HEK cells stably expressing Na(V)1.5 (K(d) = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.

Di-substituted cyclohexyl derivatives bind to two identical sites with positive cooperativity on the voltage-gated potassium channel, K(v)1.3.

Di-substituted cyclohexyl (DSC) derivatives inhibit the voltage-gated potassium channel, K(v)1.3, and have immunosuppressant activity (Schmalhofer et al. (2002) Biochemistry 41, 7781-7794). This class of inhibitors displays Hill coefficients of near 2 in functional assays, and trans DSC analogues appear to selectively interact with K(v)1.3 channel conformations related to C-type inactivation. To further understand the details of the DSC inhibitor interaction with potassium channels, trans-1-(N-n-propylcarbamoyloxy)-4-phenyl-4-(3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl)cyclo-hexane (trans-NPCO-DSC) was radiolabeled with tritium, and its binding characteristics to K(v)1.3 channels were determined. Specific binding of [(3)H]-trans-NPCO-DSC to K(v)1.3 channels is a saturable, time-dependent, and fully reversible process. Saturation binding isotherms and competition binding experiments are consistent with the presence of two receptor sites for DSC derivatives on the K(v)1.3 channel that display positive allosteric cooperativity. The high affinity interaction of [(3)H]-trans-NPCO-DSC with K(v)1.3 channels appears to correlate with the rates of C-type inactivation of the channel. These data, taken together, mark the first demonstration of the existence of multiple binding sites for an inhibitor of an ion channel and suggest that the high affinity interaction of trans-NPCO-DSC and similar inhibitors with K(v)1.3 channels could be exploited for the development of selective molecules that target this protein.

Benzamide derivatives as blockers of Kv1.3 ion channel.

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.

Identification of a new class of inhibitors of the voltage-gated potassium channel, Kv1.3, with immunosuppressant properties.

The voltage-gated potassium channel, K(v)1.3, is a novel target for development of immunosuppressants. Using a functional (86)Rb(+) efflux assay, a new class of high-affinity K(v)1.3 inhibitors has been identified. The initial active in this series, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone (PAC), which is representative of a disubstituted cyclohexyl (DSC) template, displays a K(i) of ca. 300 nM and a Hill coefficient near 2 in the flux assay and in voltage clamp recordings of K(v)1.3 channels in human T-lymphocytes. PAC displays excellent specificity as it only blocks members of the K(v)1 family of potassium channels but does not affect many other types of ion channels, receptors, or enzyme systems. Block of K(v)1.3 by DSC analogues occurs with a well-defined structure-activity relationship. Substitution at the C-1 ketone of PAC generates trans (down) and cis (up) isomer pairs. Whereas many DSC derivatives do not display selectivity in their interaction with different K(v)1.x channels, trans DSC derivatives distinguish between K(v)1.x channels based on their rates of C-type inactivation. DSC analogues reversibly inhibit the Ca(2+)-dependent pathway of T cell activation in in vitro assays. Together, these data suggest that DSC derivatives represent a new class of immunosuppressant agents and that specific interactions of trans DSC analogues with channel conformations related to C-type inactivation may permit development of selective K(v)1.3 channel inhibitors useful for the safe treatment of autoimmune diseases.