Richard M. Brochu

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.

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.

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.

The Inwardly Rectifying Potassium Channel Kir1.1: Development of Functional Assays to Identify and Characterize Channel Inhibitors

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartters syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartters syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization.

Substituted biaryl oxazoles, imidazoles, and thiazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. With a goal to develop potent peripherally active sodium channel blockers, a series of low molecular weight biaryl substituted imidazoles, oxazoles, and thiazole carboxamides were identified with good in vitro and in vivo potency.

Substituted biaryl pyrazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Nav1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Differentiation of ROMK potency from hERG potency in the phenacetyl piperazine series through heterocycle incorporation

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.