High-Throughput Screening for N-Type Calcium Channel Blockers Using a Scintillation Proximity Assay

Zhang, Sui‐Po; Kauffman, Jack A.; Yagel, Susan K.; Codd, Ellen E.

doi:10.1177/1087057106289210

Cited by 11 publications

(8 citation statements)

References 43 publications

(48 reference statements)

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“…On the other hand, if the toxin allosterically affects gating, it may be possible to generate small organic compounds that bind to the toxin site to mimic the gating effects without blocking N-channel conductance. There are hundreds of such compounds that have been screened for the ability to displace CTX binding (Zhang et al 2006). The most promising of these compounds should be examined to determine if they modulate N-channel gating as predicted by our results.…”

Section: Implications For the Treatment Of Neuropathic Painmentioning

confidence: 98%

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

Yarotskyy¹,

Elmslie²

2009

Journal of Neurophysiology

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Yarotskyy V, Elmslie KS. -conotoxin GVIA alters gating charge movement of N-type (CaV2.2) calcium channels. J Neurophysiol 101: 332-340, 2009. First published October 29, 2008 doi:10.1152/jn.91064.2008. -conotoxin GVIA (CTX) is a specific blocker of N-type calcium (CaV2.2) channels that inhibits neuropathic pain. While the toxin appears to be an open channel blocker, we show that N-channel gating charge movement is modulated. Gating currents were recorded from N-channels expressed along with ß 2a and ␣ 2 ␦ subunits in HEK293 cells in external solutions containing either lanthanum and magnesium (La-Mg) or 5 mM Ca 2ϩ plus CTX (CTX-Ca). A comparison showed that CTX induced a 10-mV right shift in the gating charge versus voltage (Q-V) relationship, smaller off-gating current time constant ( Q Off ), a lower Q Off voltage dependence, and smaller on-gating current (Q On ) . We also examined gating current in La-Mg plus CTX and found no significant difference from that in CTX-Ca; this demonstrates that the modulation was induced by the toxin. A model with strongly reduced open-state occupancy reproduced the CTX effect on gating current and showed that the gating modulation alone would inhibit N-current by 50%. This mechanism of N-channel inhibition could be exploited to develop novel analgesics that induce only a partial block of N-current, which may limit some of the side effects associated with the toxin analgesic currently approved for human use (i.e., Prialt).

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Section: Implications For the Treatment Of Neuropathic Painmentioning

confidence: 98%

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

Yarotskyy¹,

Elmslie²

2009

Journal of Neurophysiology

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“…Prior screening efforts to identify Cav2.2 antagonists included either the displacement of ziconotide binding 11 or the influx of calcium using cells either recombinantly or natively expressing the channel. 12,13 A more recent study employed the coexpression of an inwardly rectifying potassium channel with the voltage-gated calcium channel subtype of interest in heterologous cells to control the resting level of channel activation via external potassium concentration.…”

Section: Introductionmentioning

confidence: 99%

An Integrated Multiassay Approach to the Discovery of Small-Molecule N-Type Voltage-Gated Calcium Channel Antagonists

Finley

Lubin

Neeper

et al. 2010

ASSAY and Drug Development Technologies

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The N-type voltage-gated calcium channel (Cav2.2) has been intensively explored as a target for novel, small-molecule analgesic drugs because of its distribution in the pain pathway and its role in nociceptive processing. For example, Cav2.2 is localized at presynaptic terminals of pain fibers in the dorsal horn, and it serves as a downstream effector of μ-opioid receptors. Most importantly, antagonism of the channel by the highly specific and potent Cav2.2 blocker ω-conotoxin MVIIA (ziconotide) produces clinical efficacy in the treatment of severe, intractable pain. To identify novel small-molecule Cav2.2 inhibitors, we developed new tools and screening methods critical to enhance the efficiency and probability of success. First, we established and characterized a new cell line stably expressing the three subunits of the Cav2.2, including an α-subunit splice variant that is uniquely expressed by dorsal root ganglion neurons. Second, using this cell line, we validated and employed a fluorescence-based calcium flux assay. Third, we developed a new "medium-throughput" electrophysiology assay using QPatch-HT to provide faster turnaround on high-content electrophysiology data that are critical for studying ion channel targets. Lastly, we used a therapeutically relevant, ex vivo spinal cord calcitonin gene-related peptide-release assay to confirm activities in the other assays. Using this approach we have identified compounds exhibiting single-digit nM IC₅₀ values and with a positive correlation across assay methods. This integrated approach provides a more comprehensive evaluation of small-molecule N-type inhibitors that may lead to improved therapeutic pharmacology.

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“…The effect of ligand depletion on the measured IC 50 ligand depletion as has been documented for other SPAs (Carter et al, 2007). SPAs for other ion channels have been based on membrane preparations rather than purified protein (Hui et al, 2005;Zhang et al, 2006). One precaution common to any chimera approach is the need to test any hits identified from a screen for activity against the wild-type Na V 1.7 in a functional assay.…”

Section: Discussionmentioning

confidence: 99%

A Functional Na_V1.7-Na_VAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site

Rajamani

Rodrigo

et al. 2017

Mol Pharmacol

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The Na1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in Na1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of Na1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high Na1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of Na1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the Na1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a Na1.7-NaAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel Na1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

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High-Throughput Screening for N-Type Calcium Channel Blockers Using a Scintillation Proximity Assay

Cited by 11 publications

References 43 publications

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

An Integrated Multiassay Approach to the Discovery of Small-Molecule N-Type Voltage-Gated Calcium Channel Antagonists

A Functional Na_V1.7-Na_VAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site

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High-Throughput Screening for N-Type Calcium Channel Blockers Using a Scintillation Proximity Assay

Cited by 11 publications

References 43 publications

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

ω-Conotoxin GVIA Alters Gating Charge Movement of N-Type (CaV2.2) Calcium Channels

An Integrated Multiassay Approach to the Discovery of Small-Molecule N-Type Voltage-Gated Calcium Channel Antagonists

A Functional NaV1.7-NaVAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site

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Product

Resources

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A Functional Na_V1.7-Na_VAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site