Brett Antonio

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.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Nav1.8 sodium channel with efficacy in a model of neuropathic pain

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

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.

N-Pyridyl and Pyrimidine Benzamides as KCNQ2/Q3 Potassium Channel Openers for the Treatment of Epilepsy

A series of N-pyridyl benzamide KCNQ2/Q3 potassium channel openers were identified and found to be active in animal models of epilepsy and pain. The best compound 12 [ICA-027243, N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide] has an EC50 of 0.38 μM and is selective for KCNQ2/Q3 channels. This compound was active in several rodent models of epilepsy and pain but upon repeated dosing had a number of unacceptable toxicities that prevented further development. On the basis of the structure-activity relationships developed around 12, a second compound, 51, [N-(2-chloro-pyrimidin-5-yl)-3,4-difluoro-benzamide, ICA-069673], was prepared and advanced into a phase 1 clinical study. Herein, we describe the structure-activity relationships that led to the identification of compound 12 and to the corresponding pyrimidine 51.

Subtype-selective Nav1.8 sodium channel blockers: Identification of potent, orally active nicotinamide derivatives

A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. (Journal of Biomolecular Screening 2003:332-339)

The effect of kappa-opioid receptor agonists on tetrodotoxin-resistant sodium channels in primary sensory neurons.

BACKGROUND: A non-opioid receptor-mediated inhibition of sodium channels in dorsal root ganglia (DRGs) by &kgr;-opioid receptor agonists (&kgr;-ORAs) has been reported to contribute to the antinociceptive actions in animals and humans. In this study, we examined structurally diverse &kgr;-ORAs for their abilities to inhibit tetrodotoxin-resistant (TTX-r) sodium channels in adult rat DRGs. METHODS: Whole-cell recordings of TTX-r sodium currents were performed on cultured adult rat DRGs. Structurally diverse &kgr;-ORAs were studied for their abilities to inhibit TTX-r sodium channels. RESULTS: The racemic &kgr;-ORA, (±)U50,488, inhibited TTX-r sodium currents in a voltage-dependent manner, yielding IC50 values of 49 and 8 &mgr;M, at prepulse potentials of −100 and −40 mV, respectively. Furthermore, we found that both the &kgr;-ORA U50,488 active enantiomer 1S,2S U50,488 and the inactive enantiomer 1R,2R U50,488 were equally potent inhibitors of TTX-r sodium currents. Structurally related &kgr;-ORAs, such as BRL 52537 and ICI 199,441 also inhibited TTX-r sodium currents. However, sodium channel inhibition and &kgr;-opioid receptor agonism have a distinct structure-activity relationship because another &kgr;-ORA (ICI 204,488) was inactive versus TTX-r sodium channels. We further investigated the sodium channel block of this class of compounds by studying (±)U50,488. (±)U50,488 was found to preferentially interact with the slow inactivated state of TTX-r sodium channels and to retard recovery from inactivation. CONCLUSION: Our results suggest that TTX-r sodium channels can be inhibited by many &kgr;-ORAs via an opioid receptor-independent mechanism. Although the potency for sodium channel inhibition is typically much less than apparent affinity for opioid receptors, sodium channel block may still contribute to the antinociceptive effects of this class of compounds.

GI‐530159, a novel, selective, mechanosensitive two‐pore‐domain potassium (K2P) channel opener, reduces rat dorsal root ganglion neuron excitability

TREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.

Validation of ion channel targets.

A prerequisite for a successful target-based drug discovery program is a robust data set that increases confidence in the validation of the molecular target and the therapeutic approach. Given the significant time and resource investment required to carry a drug to market, early selection of targets that can be modulated safely and effectively forms the basis for a strong portfolio and pipeline. In this article we present some of the more useful scientific approaches that can be applied toward the validation of ion channel targets, a molecular family with a history of clinical success in therapeutic areas such as cardiovascular, respiratory, pain and neuroscience.

GI-530159, a novel, selective, mechano-sensitive K2P channel opener, reduces rat dorsal root ganglion (DRG) neuron excitability.

TREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.