2,6-Disubstituted pyrazines and related analogs as NR2B site antagonists of the NMDA receptor with anti-depressant activity.

Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (s.c. 60 mg/kg).

Discovery and pharmacological characterization of a small-molecule antagonist at neuromedin U receptor NMUR2.

Neuromedin U (NMU), through its cognate receptor NMUR2 in the central nervous system, regulates several important physiological functions, including energy balance, stress response, and nociception. By random screening of our corporate compound collection with a ligand binding assay, we discovered (R)-5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine] (R-PSOP), a highly potent and selective NMUR2 antagonist. R-PSOP is a nonpeptidic small-molecule with the chemical composition C(20)N(4)O(2)H(22). In competition binding experiments, this compound was found to bind to NMUR2 with high affinity; the K(i) values were determined to be 52 and 32 nM for the human and rat NMUR2, respectively. Moreover, in functional assays measuring phosphoinositide turnover or intracellular calcium mobilization, R-PSOP strongly inhibited the responses stimulated by peptide agonists NMU-25, NMU-23, and NMU-8 in human embryonic kidney 293 cells expressing NMUR2. From Schild analyses, the functional K(b) values for R-PSOP were determined to be 92 and 155 nM at human and rat NMUR2, respectively. Highly selective for NMUR2, R-PSOP exhibited low affinity to the other subtype of NMU receptor, NMUR1, with a K(i) value >10 microM. R-PSOP in vivo attenuated NMU-23-evoked nociceptive responses in a rat spinal reflex preparation. To our knowledge, this is the first antagonist ever reported for NMU receptors. This compound could serve as a valuable tool for further understanding the physiological and pathophysiological roles of NMU system, while providing a chemical starting point that may lead to development of new therapeutics for treatment of eating disorders, obesity, pain, and stress-related disorders.

Cellular HTS assays for pharmacological characterization of Na(V)1.7 modulators.

Ion channels are challenging targets in the early phases of the drug discovery process, especially because of the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The electrophysiological patch-clamp technique, which is the gold standard for studying ion channels, has low throughput and is not amenable to screening large numbers of compounds. However, for random high-throughput screening (HTS) of compounds against ion channel targets, a number of functional cellular assays have become available during the last few years. Here we use the sodium channel NaV1.7 stably expressed in human embryonic kidney 293 cells and compare three HTS assays-a Li flux atomic absorption spectroscopy (AAS) assay, a fluorescent imaging plate reader (FLIP, Molecular Devices, Sunnyvale, CA) membrane potential assay, and a fluorescence resonance energy transfer (FRET)-based membrane potential assay-to an automated electrophysiological assay (the Ionworks HT [Molecular Devices] platform) and characterize 11 known NaV inhibitors. Our results show that all three HTS assays are suitable for identification of NaV1.7 inhibitors, but as an HTS assay the Li-AAS assay is more robust with higher Z' values than the FLIPR and FRET-based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li-AAS assay regarding the potency of the NaV inhibitors investigated. This paper describes the first comparison between all the HTS assays available today to study voltage-gated NaVs, and the results suggest that the Li-AAS assay is more suited as a first HTS assay when starting an NaV drug discovery campaign.

Pyridazinoquinolinetriones as NMDA glycine-site antagonists with oral antinociceptive activity in a model of neuropathic pain.

A series of 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4,10(5H)-triones were synthesized and found to have potent activity at the glycine site of the NMDA receptor. In some cases, these compounds possessed poor aqueous solubility that may have contributed to poor rat oral bioavailability. Subsequently, compounds have been identified with improved aqueous solubility and oral bioavailability. Several of these compounds were examined in a rat chronic constrictive injury (CCI) model of neuropathic pain and found to have potent activity when dosed orally.

The use of TrkA-PathHunter assay in high-throughput screening to identify compounds that affect nerve growth factor signaling.

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.

Novel methodology to identify TRPV1 antagonists independent of capsaicin activation.

TRPV1 was originally characterized as an integrator of various noxious stimuli such as capsaicin, heat, and protons. TRPV1-null mice exhibit a deficiency in sensing noxious heat stimuli, suggesting that TRPV1 is one of the main heat sensors on nociceptive primary afferent neurons and a candidate target for heat hypersensitivity in chronic pain. Several different potent and selective TRPV1 antagonists have been developed by more than 50 companies since the characterization of the receptor in 1997. A consequence of this competitive interest is the crowding of patentable chemical space, because very similar in vitro screening assays are used. To circumvent this issue and to expand our understanding of TRPV1 biology, we sought to take advantage of recent advancements in automated patch-clamp technology to design a novel screening cascade. This SAR-driving assay identified novel modulators that blocked the depolarization-induced activation of outwardly-rectifying TRPV1 currents independent of agonist stimulation, and we correlated the pharmacology to three other innovative assays for higher-throughput screening. Ultimately, we have identified a screening paradigm that would have good predictive value for future TRPV1 drug discovery projects and novel chemical space with a higher probability of gaining intellectual property coverage.

Advances in functional assays for high-throughput screening of ion channels targets.

IMPORTANCE OF THE FIELD: Ion channels are important targets for many disease areas but are challenging to screen due to lack of technologies enabling robust high-throughput assays, particularly for state-dependent interactions. AREAS COVERED IN THIS REVIEW: Current assay technologies used to measure ion channel function are reviewed and assessed for use in high-throughput screening (HTS). An iterative approach to screening is evaluated as an alternative to full collection screening in order to take advantage of low-throughput, high cost assays that yield high quality data. WHAT THE READER WILL GAIN: The reader will gain an understanding of the advantages and disadvantages of various assay techniques used to screen ion channels and their suitability for use in HTS. TAKE HOME MESSAGE: Assays that directly measure ion channel function are prone to less artifact and higher hit confirmation in screening than those using an indirect measure but they are usually lower throughput. However, an iterative approach to screening can make the relatively lower throughput techniques amenable for use in interrogating large collections of compounds.

A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry.

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.

Synthesis of 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4,10(5H)-triones as NMDA glycine-site antagonists.

Several members of the 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4,10(5H)-triones (2) have been identified as being potent and selective NMDA glycine-site antagonists. Increasing size of the alkyl substituent on the alpha-carbon led to a progressive decrease in binding affinity. Some of these analogues possess improved drug-like properties such as cellular permeability, solubility and oral absorption.