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Ion Channel Assay Development |
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Ion channels represent an important drug target class that is relatively untapped due to the challenges in assay development, which include difficulty in generating stable cell lines that over express channel targets and low sensitivity. |
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HEK / TRPV3 |
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HD Biosciences uses proprietary knowledge and technologies to overcome the challenges associated with ion channel assay development, and has generated many internal assays with improved signal windows and stability. As a result, HDB can provide comprehensive services ranging from ion channel assay development and screening to hit confirmation, selectivity profiling, and electrophysiology assays. |
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Calcium influx detection |
Membrane potential assays |
For voltage-gated calcium channel |
Utilize fluorescent dyes sensitive to membrane potential |
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Electrophysiology assays |
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HD Biosciences is equipped with state of the art manual patch-clamp technology.
Whole-cell recording in mammalian expression systems can be conducted to test the effects of drugs on voltage-gated (hERG channel). In order to increase throughput of patch-clamp analysis, HD Biosciences has DADVC-8PP: Pinch valve system for fast switching of perfusion solution. The application system can be easily mounted on already running patch clamp setups. |
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Patch clamp hERG safety testing services
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hERG (human Ether-a-go-go-related gene, in italics, or KCNH2 in the new nomenclature) is a gene that encodes the pore-forming alpha subunit of a voltage-gated potassium channel expressed in the heart and in nervous tissue. The term hERG is often used to denote the protein or channel derived from the hERG transcript. In the heart hERG makes up a part, if not all, of the channel that conducts the ¡®rapid¡¯ component of the delayed rectifier current.
IKr is important in determining the timing of the electrical repolarization of the action potential (AP) in ventricular myocytes. Genetic mutation in the hERG channel can result in long QT syndrome (LQTS), a disorder in which the patient has a substantial risk of sudden death due to an arrhymia known Torsades de pointes (TdP). Typically, an LQTS patient will have no clinical signs expect prolongation of the QT interval on the electrocardiogram, and the patient will appear otherwise healthy, having no other symptoms expect some patients will suffer form occasional syncope.
hERG testing for drug candidates is a crucial element of drug development, as an estimated 25-40% of all lead compounds show some level of hERG related toxicity. HD Bio helps our clients solve hERG safety issues at early discovery stages and during preclinical development.
The HEK293-hERG cell line was generated at HD Biosciences. The cell line is currently being used by HD Bioscience¡¯s electrophysiology group. |
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Figure 1. High amplitudes of the hERG peak tail current throughout experiment time course - rundown<20%. |
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Figure 2. Measurement of hERG-inhibitory activity of quindine (C20H24N3O2) (literature IC50: 300-1000 nM). |
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(A) Cells were voltage-clamped at a holding potential of -80 mV, depolarized to +40 mV for 2000 ms, then clamped to -40 mV for 3000 ms, and last down to -80 mV to record tail current. The interpulse interval is 15 s.
(B) Example traces of hERG currents recorded in the presence of increasing concentrations of quindine.
(C) Time course of the hERG channel inhibition achieved by quindine for data shown in (B). Each dot represents the peak tail current amplitude measured in response to stimulation protocol described in (A). (D) The hill equation was fit to the data from the experiment. |
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Literature IC50 (nM) |
HD Bio IC50 (nM) |
Cisapride |
2-45 |
2.8 |
Quinidine |
300-1000 |
472 |
Astemizole |
1-26 |
1.2 |
Dofetilide |
12-60 |
12.5 |
E-4031 |
8-18 |
8 |
Verapamil |
140-800 |
721 |
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P2X2 Assay |
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P2X2, purinergic receptor P2X, ligand-gated ion channel, 2, also known as P2RX2, is a human gene. This receptor functions as a ligand-gated ion channel. Binding to ATP mediates synaptic transmission between neurons and from neurons to smooth muscle. |
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The HEK293-P2X2 cell line was generated at HD Biosciences. |
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Current traces recorded from HEK293 cells expressed with the P2X2. The inward current was elicited by an application of 100 uM ATP for the P2X2 at a holding potential of -60 mV. |
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Current-voltage (IV) relationship for ATP-induced current response from the peak current recorded from HEK293-P2X2 cell line. (The cell line was generated at HDB. 30 uM ATP induced current responses were recorded at various holding potentials from -100 to +20 mV. |
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Dose response curves for ATP-induced current responses in HEK293 cells expressing P2X2 obtained at a holding potential of - 60 mV. Currents were normalized to the current elicited by 100 uM ATP in HEK293-P2X2. each symbol represents mean¡ÀSE. |
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Concentration-response curve for suramin recorded from P2X2-HEK293.
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