Screening Technologies for Ion Channel Drug Discovery

Terstappen, Georg C.; Roncarati, Renza; Dunlop, John; Peri, Ravikumar

doi:10.4155/fmc.10.180

Cited by 51 publications

(36 citation statements)

References 86 publications

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“…These shifts in potency were observed in both electrophysiology and fluorescent imaging assays, despite the significant differences in the IC 50 values obtained with the approaches. Such differences in electrophysiology and fluorescent imaging assays for ion channels have been reported previously (Mathes et al, 2009;Terstappen et al, 2010), but the underlying cause remains to be elucidated.…”

Section: Discussionsupporting

confidence: 57%

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

et al. 2015

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Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtypeselective inhibitors of voltage-gated sodium (Na V ) channels, we screened spider venoms for inhibitors of human Na V 1.7 (hNa V 1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na V 1.7 inhibitor, m-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa V 1.7 . hNa V 1.6 . hNa V 1.2 . hNa V 1.1 . hNa V 1.3 channels in fluorescent assays. Na V 1.7 inhibition was diminished (IC 50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC 50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNa V 1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mM. Unlike most spider toxins that modulate Na V channels, Tp1a inhibited hNa V 1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa V 1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa V 1.7 inhibitors for treatment of chronic pain.

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Section: Discussionsupporting

confidence: 57%

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

et al. 2015

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“…A modern safe alternative for the radioligand binding assay is the fluorescence detection analysis. Kv1 channels were studied using flow cytometry (60), single molecule fluorescent microscopy (61), and the analysis of membrane potential changes using fluorescent dyes and fluorescence readers (62). Recently, a convenient fluorescent screening system based on KcsA-Kv1.3 hybrid receptors was developed and applied successfully to screen Orthochirus scrobiculosus venom for peptide toxins, which specifically target the pore region of Kv1.3 (36,38).…”

Section: Discussionmentioning

confidence: 99%

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

Kuzmenkov

Vassilevski

Kudryashova

et al. 2015

Journal of Biological Chemistry

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Background: Scorpion venoms are an ample source of toxins targeting potassium channels. Results: A comprehensive search for new toxins was performed by combining transcriptomics and peptidomics with a fluorescent test system. Conclusion: We identified five new high affinity potassium channel blockers in the venom of Mesobuthus eupeus. Significance: The proposed integrated approach is of general utility for potassium channel pharmacology.

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“…At the 10 min mark, 10 μL/well of a Tl + stimulus buffer was added and data collection continued for an additional 5 min. The Tl + stimulus buffer consisted of 125 mM KHCO 3 . For Tl + -flux assays, stock compound solutions in 384-well plates were serially diluted in DMSO using a Bravo liquid handler (Agilent, Technologies, Santa Clara, CA) and then transferred to another 384-well plate using an Echo 555 plate reformatter (Labcyte, Sunnyvale, CA).…”

Section: ■ Methodsmentioning

confidence: 99%

Development and Validation of a Thallium Flux-Based Functional Assay for the Sodium Channel NaV1.7 and Its Utility for Lead Discovery and Compound Profiling

Du¹,

Days²,

Romaine³

et al. 2015

ACS Chem. Neurosci.

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Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.

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Screening Technologies for Ion Channel Drug Discovery

Cited by 51 publications

References 86 publications

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

Development and Validation of a Thallium Flux-Based Functional Assay for the Sodium Channel NaV1.7 and Its Utility for Lead Discovery and Compound Profiling

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