Compounds for modulating TRPV3 function
A compound, unsubstituted technology, applied in the field of compounds that modulate the function of TRPV3, can address issues such as increased intracellular calcium levels or concentrations
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Embodiment 1
[0531] Example 1: High throughput screening analysis
[0532] This assay relies on the detection of intracellular calcium concentrations ([Ca 2+ ] i ) rises after channel activation. Ca 2+ Rising utilization is loaded into cells and subsequently directed to the [Ca 2+ ] i Fluorescent Ca 2+ indicator for quantification. Ca 2+ Influx follows activation of TRPV3 channels. Inhibit the [Ca 2+ ] i Rising compounds were viewed as targets for further research.
[0533] A commercially available HEK293 / TREx line (Invitrogen) was stably transfected with the TRPV3 construct and screened by immunostaining for clones with TRPV3 expression after stimulation with 1 μg / ml tetracycline. Clonal TRPV3-expressing cells were maintained in growth medium recommended by the manufacturer supplemented with 100 μg / ml hygromycin to facilitate maintenance of the TRPV3 construct. After growing to nearly confluent sheets, cells were plated in 384-well plates at a density of ~25,000 cells / well i...
Embodiment 2
[0534] Embodiment 2. Patch clamp experiment
[0535] Whole-cell patch-clamp experiments allow detection of currents through TRPV3 channels in the above cell lines. A glass electrode is brought into contact with a single cell, and the cell membrane is then disrupted, allowing the voltage across the cell membrane to be controlled and the current flowing through the membrane to be measured using an amplifier connected to the electrode. The perfusion system allows control of the extracellular solution, including the addition of current blockers and activators. The current can be activated by heating the solution to 28°C or hotter, or by adding 20 μM 2-APB to the solution.
[0536] TRPV3 cells were induced for 20-48 hours, removed from growth plates, and replated on coverslips at low density (to obtain good physical separation of single cells) for measurements. In some instances, cells were grown overnight on coverslips at low density. Patch-clamp recordings were performed in ...
Embodiment 3
[0539] Example 3: Other Screening Assays
[0540] While the exemplary TRPV3 inhibitors provided herein were identified using the assays described in Examples 1 and 2, other cell-based assays can also be used to identify and / or characterize TRPV3 inhibitors. One such assay is described in US Application 11,078,188, filed March 11, 2005, the contents of which are hereby incorporated by reference in their entirety. TRPV3 protein can be expressed in the prokaryotic cell system described in Application No. 11,078,188, and this system can be used to screen for compounds that modulate the activity of TRPV3 protein. Alternatively, ion channels other than TRPV3 can be expressed in a prokaryotic cell system, and this system can be used to evaluate the activity profile of identified TRPV3 inhibitors on other ion channels.
[0541] Assays to identify and / or characterize compounds that inhibit TRPV3 activity can be performed in a high-throughput fashion, or can be performed on a smaller...
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