David Printzenhoff

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels

Significance Voltage-gated sodium (Nav) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Nav channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Nav channel modulators are unable to distinguish between Nav channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Nav 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-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1–S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the “extracellular” facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.

Sodium Channel Inhibitor Drug Discovery Using Automated High Throughput Electrophysiology Platforms

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 and IonWorks, 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 and IonWorks 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 has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpress 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.

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release

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.

A novel selective and orally bioavailable Nav1.8 channel blocker, PF‐01247324, attenuates nociception and sensory neuron excitability

NaV1.8 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 Nav1.8 channel blocker of novel chemotype.

Tenidap, a novel anti-inflammatory agent, is an opener of the inwardly rectifying K+ channel hKir2.3.

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 microM.), 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 micro1 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.

Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

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.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7

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 vitro and in vivo characterization of a novel naphthylamide ATP‐sensitive K+ channel opener, A‐151892

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.

Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical probes

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.

Addition of a single methyl group to a small molecule sodium channel inhibitor introduces a new mode of gating modulation.

Aryl sulfonamide Nav1.3 or Nav1.7 voltage‐gated sodium (Nav) 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.