Background and Purpose Pharmacological agents that either inhibit or enhance flux of ions through voltage-gated sodium (Na-v) channels may provide opportunities for treatment of human health disorders. During studies to characterize agents that modulate Na(v)1.3 function, we identified a compound that appears to exhibit both enhancement and inhibition of sodium ion conduction that appeared to be dependent on the gating state that the channel was in. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel. Experimental Approach Electrophysiology and site-directed mutation were used to investigate the effects of PF-06526290 on Na-v channel function. Key Results PF-06526290 greatly slows inactivation of Na-v channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Na-v subtype-selective inhibitor. Mutation of the domain 4 voltage sensor modulates inhibition of Na(v)1.3 or Na(v)1.7 channels by PF-06526290 but has no effect on PF-06526290 mediated slowing of inactivation. Conclusions and Implications These findings suggest that distinct interactions may underlie the two modes of Na-v channel modulation by PF-06526290 and that a single compound can affect sodium channel function in several ways.

The discovery and selection of a highly potent and selective NaV1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein–ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

In this article, we describe the discovery of an aryl ether series of potent and selective Nav1.3 inhibitors. Based on structural analogy to a similar series of compounds we have previously shown bind to the domain IV voltage sensor region of Nav channels, we propose this series binds in the same location. We describe the development of this series from a published starting point, highlighting key selectivity and potency data, and several studies designed to validate Nav1.3 as a target for pain.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7’s role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

Background and PurposeAryl sulfonamide Na(v)1.3 or Na(v)1.7 voltage-gated sodium (Na-v) channel inhibitors interact with the Domain 4 voltage sensor domain (D4 VSD). During studies to better understand the structure-activity relationship of this interaction, an additional mode of channel modulation, specifically slowing of inactivation, was revealed by addition of a single methyl moiety. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel.Experimental ApproachElectrophysiology and site-directed mutation were used to compare the effects of PF-06526290 and its desmethyl analogue PF-05661014 on Na-v channel function.Key ResultsPF-05661014 selectively inhibits Na(v)1.3 versus Na(v)1.7 currents by stabilizing inactivated channels via interaction with D4 VSD. In contrast, PF-06526290, which differs from PF-05661014 by a single methyl group, exhibits a dual effect. It greatly slows inactivation of Na-v channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Na-v subtype selective inhibitor similar to PF-05661014. Mutation of the D4 VSD modulates inhibition of Na(v)1.3 or Na(v)1.7 by both PF-05661014 and PF-06526290, but has no effect on the inactivation slowing produced by PF-06526290. This finding, along with the absence of functional inhibition of PF-06526290-induced inactivation slowing by PF-05661014, suggests that distinct interactions underlie the two modes of Na-v channel modulation.Conclusions and ImplicationsAddition of a methyl group to a Na-v channel inhibitor introduces an additional mode of gating modulation, implying that a single compound can affect sodium channel function in multiple ways.

BACKGROUND AND PURPOSE:NaV 1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH:The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS:PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : ∼10 μM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : ∼10-18 μM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS:Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Significance Voltage-gated sodium (Na v ) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Na v channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Na v channel modulators are unable to distinguish between Na v channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Na v channel inhibitors that interact with a region of the channel that controls voltage sensitivity. This interaction site may enable development of selective therapeutic interventions with reduced potential for toxicity.

Voltage dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders such as epilepsy and pain as well as cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic and antiarrhythmic sodium channel blockers. While patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery in which there is often a need to evaluate many thousands to hundreds of thousands of compounds. Fortunately over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms like the PatchXpress (TM) and IonWorks (TM), which are now widely used in drug discovery. This review highlights the strengths and weaknesses of these two high throughput devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpress (TM) and IonWorks (TM) electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state dependent modulation of sodium channels. IonWorks (TM) has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpress (TM) has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery.

Openers of ATP‐sensitive K+ channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N‐[2‐(2,2,2‐trifluoro‐1‐hydroxy‐1‐trifluoromethyl‐ethyl)‐naphthalen‐1‐yl]‐acetamide (A‐151892), as an opener of the ATP‐sensitive potassium channels. A‐151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide‐sensitive whole‐cell current and fluorescence‐based membrane potential responses (−log EC50=7.63) in guinea‐pig bladder smooth muscle cells. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4‐dihydropyridine opener [125I]A‐312110. A‐151892 displaced [125I]A‐312110 binding to bladder membranes with a −log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. In pig bladder strips, A‐151892 suppressed phasic, carbachol‐evoked and electrical field stimulus‐evoked contractility in a glibenclamide‐reversible manner with −log IC50 values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (−log IC50=7.81) and portal vein (−log IC50=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. In vivo, A‐151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED35% values of 8.05 and 7.43, respectively. These results demonstrate that naphthylamide analogs exemplified by A‐151892 are novel KATP channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms. British Journal of Pharmacology (2004) 143, 81–90. doi:10.1038/sj.bjp.0705908

We studied the effect of a novel anti-inflammatory agent, tenidap, on a cloned inwardly rectifying K+ channel, hKir2.3. Tenidap (a) potently potentiated 86Rb+ efflux through hKir2.3 channels expressed in Chinese hamster ovary cells (EC50=402 nM), (b) reversibly and dose-dependently increased whole-cell and macro-patch hKir2.3 currents (maximum whole-cell current response to tenidap was 230±27% of control; EC50=1.3 μM.), and (c) caused dose-dependent and Ba2+-sensitive membrane hyperpolarizations and concurrent decreases in input resistance. Potentiation of hKir2.3 by tenidap was unaffected by inhibitors of phospholipase A2, protein kinase C, or arachidonic acid metabolic pathways. The action of tenidap was not intracellular. Tenidap also had little or no effect on currents flowing through hKir2.1, Kv1.5, and μ1 Na+ channels. Our results demonstrate that tenidap is a potent opener of hKir2.3 and suggest that it can serve as a valuable pharmacological tool for studying physiological and pathological processes involving Kir2.3.

With the emergence of combinatorial chemistry, whether based on parallel, mixture, solution, or solid phase chemistry, it is now possible to generate large numbers of diverse or focused compound libraries. In this paper we aim to demonstrate that it is possible to design targeted libraries by applying nonparametric statistical methods, recursive partitioning in particular, to large data sets containing thousands of compounds and their associated biological data. Moreover, when applied to an experimental high-throughput screening (HTS) data set, our data strongly suggest that this method can improve the hit rate of our primary screens (about 4- to 5-fold) while increasing screening efficiency: less than one-fifth of the complete selection needs to be screened in order to identify about 75% of all actives present.