Birgit T. Priest

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Voltage-gated sodium (NaV1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to NaV1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding NaV1.7, 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 NaV1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires NaV1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with NaV1.7 channels, inhibiting NaV1.7 with an IC50 value of 0.3 nM, compared with IC50 values of 30 to 150 nM for other heterologously expressed NaV1 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 Aβ-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 (125I-ProTx-II) binds with high affinity (Kd = 0.3 nM) to recombinant hNaV1.7 channels. Binding of 125I-ProTx-II is insensitive to the presence of other well characterized NaV1 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 125I-ProTx-II binding assay, described here, offers a new tool in the search for novel NaV1.7-selective blockers.

Ion Channels as Drug Targets: The Next GPCRs

Ion channels are well recognized as important therapeutic targets for treating a number of different pathophysiologies. Historically, however, development of drugs targeting this protein class has been difficult. Several challenges associated with molecular-based drug discovery include validation of new channel targets and identification of acceptable medicinal chemistry leads. Proof of concept approaches, focusing on combined molecular biological/pharmacological studies, have been successful. New, functional, high throughput screening (HTS) strategies developed to identify tractable lead structures, which typically are not abundant in small molecule libraries, have also yielded promising results. Automated cell-based HTS assays can be configured for many different types of ion channels using fluorescence methods to monitor either changes in membrane potential or intracellular calcium with high density format plate readers. New automated patch clamp technologies provide secondary screens to confirm the activity of hits at the channel level, to determine selectivity across ion channel superfamilies, and to provide insight into mechanism of action. The same primary and secondary assays effectively support medicinal chemistry lead development. Together, these methodologies, along with classical drug development practices, provide an opportunity to discover and optimize the activity of ion channel drug development candidates. A case study with voltage-gated sodium channels is presented to illustrate these principles.

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

Blocking sodium channels to treat neuropathic pain

Neuropathic pain remains a large unmet medical need. A number of therapeutic options exist, but efficacy and tolerability are less than satisfactory. Based on animal models and limited data from human patients, the pain and hypersensitivity that characterize neuropathic pain are associated with spontaneous discharges of normally quiescent nociceptors. Sodium channel blockers inhibit this spontaneous activity, reverse nerve injury-induced pain behavior in animals and alleviate neuropathic pain in humans. Several sodium channel subtypes are expressed primarily in sensory neurons and may contribute to the efficacy of sodium channel blockers. In this report, the authors review the current understanding of the role of sodium channels and of specific sodium channel subtypes in neuropathic pain signaling.

Role of hERG potassium channel assays in drug development.

Numerous structurally and functionally unrelated drugs block the hERG potassium channel. HERG channels are involved in cardiac action potential repolarization, and reduced function of hERG lengthens ventricular action potentials, prolongs the QT interval in an electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. In order to reduce the risk of investing resources in a drug candidate that fails preclinical safety studies because of QT prolongation, it is important to screen compounds for activity on hERG channels early in the lead optimization process. A number of hERG assays are available, ranging from high throughput binding assays on stably expressed recombinant channels to very time consuming electrophysiological examinations in cardiac myocytes. Depending on the number of compounds to be tested, binding assays or functional assays measuring membrane potential or Rb+ flux, combined with electrophysiology on a few compounds, can be used to efficiently develop the structure-function relationship of hERG interactions.

Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic β-cells

Voltage‐gated potassium (Kv) currents of human pancreatic islet cells were studied by whole‐cell patch clamp recording. On average, 75% of the cells tested were identified as β‐cells by single cell, post‐recording RT‐PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above −20 mV and had a V½ for activation of −5.3 mV. Onset of inactivation was slow for a major component (τ= 3.2 s at +20 mV) observed in all cells; a smaller component (τ= 0.30 s) with an amplitude of ∼25% was seen in some cells. Recovery from inactivation had a τ of 2.5 s at −80 mV and steady‐state inactivation had a V½ of −39 mV. In 12% of cells (21/182) a low‐threshold, transient Kv current (A‐current) was present. The A‐current activated at membrane potentials above −40 mV, inactivated with a time constant of 18.5 ms at −20 mV, and had a V½ for steady‐state inactivation of −52 mV. TEA inhibited total Kv current with an IC50= 0.54 mm and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an IC50= 0.57 μm. The total Kv current was insensitive to margatoxin (100 nm), agitoxin‐2 (50 nm), kaliotoxin (50 nm) and ShK (50 nm). Hanatoxin (100 nm) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β‐cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose‐dependent insulin secretion.

Imidazopyridines: a novel class of hNav1.7 channel blockers.

A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.

Discovery of Selective Small Molecule ROMK Inhibitors as Potential New Mechanism Diuretics.

The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.

Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-Tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain.

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.