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BioAssay: AID 2583

Late stage counterscreen for the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPN1 agonists.

Name: Late stage counterscreen for the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPN1 agonists. ..more
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 Tested Compounds
 Tested Compounds
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Inactive(6)
 
 
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 Tested Substances
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Inactive(6)
 
 
AID: 2583
Data Source: The Scripps Research Institute Molecular Screening Center (TRPN1_AG_FLUO8_1536_3XEC50_LATE STAGE CS DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-17
Hold-until Date: 2010-03-19
Modify Date: 2010-03-18

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
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AIDNameTypeProbeComment
1424Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)screening Primary Assay (TRPML3 agonists, 1X%ACT)
1448Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel ML3 (TRPML3)screening Counterscreen Assay (TRPN1 agonists, 1X%ACT)
1525Counterscreen assay for TRPML3 agonists: cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)screening Counterscreen Assay (TRPN1 agonists, 3X%ACT)
1526Confirmation cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)screening Confirmation Assay (TRPML3 agonists, 3X %ACT)
1562Dose response cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)confirmatory Dose Response (TRPML3 agonists, 3X EC50)
1682Fluorescence counterscreen assay for TRPML3 agonists: dose response cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)confirmatory Dose Response Counterscreen (TRPN1 agonists, 3X EC50)
1809Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)summary2 Summary (TRPML3 agonists)
2694Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPML3 patch clamp assayscreening
2719Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): Fura-2 profiling assayscreening
2692Late stage counterscreen results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPN1 patch clamp assayscreening
2770Late stage counterscreen results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): other ion channel Fura-2 profiling assayscreening
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Stefan Heller, Stanford University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R03 MH083077-01
Grant Proposal PI: Stefan Heller, Stanford University
External Assay ID: TRPN1_AG_FLUO8_1536_3XEC50_LATE STAGE CS DRUN

Name: Late stage counterscreen for the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPN1 agonists.

Description:

Cell signaling pathways that mediate osmosensation, photosensation, and thermosensation depend on a family of diverse transient receptor potential (TRP) cation channels, which are activated by agonist-receptor coupling (1-5). A role for these channels in inner ear hair cell mechanotransduction was gleaned from TRP channel mutations identified in flies, worms, and lower vertebrates with defective balance and impaired sensitivity to touch (1-5). TRPML3 (mucolipin 3; MCOLN3) is a TRP channel expressed in inner ear hair cells and stereocilia (5-7), suggesting it may play a role in hearing and mechanotransduction. Reports that mice with mutations in TRPML3 (known as varitint-waddler mutants) exhibit early-onset hearing loss accompanied by head-bobbing and circling behaviors (8-10), provide further support for a role of TRPML3 in hearing and vestibular function. As a result, the identification of selective probes for TRPML3 would be useful to investigate the function of TRPML3 in inner ear mechanotransduction and hearing biology (11).

References:

1. Clapham, D.E., TRP channels as cellular sensors. Nature. 2003. 426(6966): p. 517-24.
2. Cuajungco, M.P., C. Grimm, and S. Heller, TRP channels as candidates for hearing and balance abnormalities in vertebrates. Biochim Biophys Acta. 2007. 1772(8): p. 1022-7.
3. Gillespie, P.G. and R.G. Walker. Molecular basis of mechanosensory transduction. Nature. 2001. 413(6852): p. 194-202.
4. Eberl, D.F., R.W. Hardy, and M.J. Kernan. Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci. 2000. 20(16): p. 5981-8.
5. Gong, Z., W. Son, Y.D. Chung, J. Kim, D.W. Shin, C.A. McClung, Y. Lee, H.W. Lee, D.J. Chang, B.K. Kaang, H. Cho, U. Oh, J. Hirsh, M.J. Kernan, and C. Kim. Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila. J Neurosci. 2004. 24(41): p. 9059-66.
6. Kim, J., Y.D. Chung, D.Y. Park, S. Choi, D.W. Shin, H. Soh, H.W. Lee, W. Son, J. Yim, C.S. Park, M.J. Kernan, and C. Kim. A TRPV family ion channel required for hearing in Drosophila. Nature. 2003. 424(6944): p. 81-4.
7. Walker, R.G., A.T. Willingham, and C.S. Zuker. A Drosophila mechanosensory transduction channel. Science. 2000. 287(5461): p. 2229-34.
8. Corey, D.P. What is the hair cell transduction channel? J Physiol. 2006. 576(Pt 1): p. 23-8.
9. Shin, J.B., D. Adams, M. Paukert, M. Siba, S. Sidi, M. Levin, P.G. Gillespie, and S. Grunder. Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells. Proc Natl Acad Sci U S A. 2005. 102(35): p. 12572-7.
10. Sidi, S., R.W. Friedrich, and T. Nicolson. NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science. 2003. 301(5629): p. 96-9.
11. Small molecule activators of TRPML3. Grimm C, Jors S, Saldanha SA, Obukhov AG, Pan B, Oshima K, Cuajungco MP, Chase P, Hodder P, Heller S. Chem Biol. 2010 Feb 26;17(2):135-148.

Keywords:

Late stage, probes, selective, TRPML3, TRP, cation channel, mucolipin 3, MCOLN3, TRPN1, nompC, deafness, HEK 293, agonist, agonism, activator, activation, activate, fluorescence, calcium, Fluo-8 dye, FLUO8, cells, cell-based, dose response, counterscreen, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
The purpose of this assay is to determine dose response curves for powder samples of compounds identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel ML3" (PubChem AID 1448), and "Dose response cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)" (AID 1562), and that were inactive in a set of experiments entitled, "Fluorescence counterscreen assay for TRPML3 agonists: dose response cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)" (AID 1682). This assay serves as a counterscreen to determine whether these compounds were nonselective due to activation of TRPN1. This assay may also have tested analogs of the above compounds. This assay employs a HEK293 cell line that stably expresses the zebrafish TRPN1-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPN1 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 29.9 micromolar.

Protocol Summary:

The TRPN1 HEK293 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium with GlutaMAX and supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1500 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 microliters of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After a 1 hour incubation at 37 degrees C, 5% CO2, and 95 % RH followed by a 30 minute incubation at room temperature, the assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of test compound in DMSO, DMSO alone (0.3% final concentration), or the cholinergic agonist carbachol (87 micromolar final concentration) in DMSO were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 120 seconds of the assay.

A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 125 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

Percent activation was calculated from the median ratio as follows:

%Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_ Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing carbachol.
Low_Control is defined as wells containing DMSO.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 29.9 micromolar) did not result in greater than 50% activation, the EC50 was determined manually as greater than 50 micromolar. Compounds with an EC50 greater than 10 micromolar were considered inactive. Compounds with an EC50 equal to or less than 10 micromolar were considered active.

PubChem Activity Outcome and Score:

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 0-0, there are no actives.

List of Reagents:

TRPN1 HEK293 cell line (provided by Prof. Stefan Heller)
Fluo-8 No Wash Calcium Assay Kit (ABD Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
MEM with GlutaMAX (Invitrogen, part 41090-101)
Geneticin (Invitrogen, part 10131-027)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Carbachol (Sigma, part C4382)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789072)
Comment
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate calcium levels or channel activity, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentrationString
2EC50*The concentration at which 50 percent of the activity in the agonist assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) from the agonist assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent Activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Activation at 1.5 nM (0.0015μM**)Value of %Activation at 1.5 nM activator concentration; average of triplicate measurement.Float%
12Activation at 4.6 nM (0.0046μM**)Value of %Activation at 4.6 nM activator concentration; average of triplicate measurement.Float%
13Activation at 13.7 nM (0.0137μM**)Value of %Activation at 13.7 nM activator concentration; average of triplicate measurement.Float%
14Activation at 41.0 nM (0.041μM**)Value of %Activation at 41.0 nM activator concentration; average of triplicate measurement.Float%
15Activation at 123.1 nM (0.1231μM**)Value of %Activation at 123.1 nM activator concentration; average of triplicate measurement.Float%
16Activation at 369.3 nM (0.3693μM**)Value of %Activation at 369.3 nM activator concentration; average of triplicate measurement.Float%
17Activation at 1.1 uM (1.1μM**)Value of %Activation at 1 uM activator concentration; average of triplicate measurement.Float%
18Activation at 3.3 uM (3.3μM**)Value of %Activation at 3 uM activator concentration; average of triplicate measurement.Float%
19Activation at 10.0 uM (10μM**)Value of %Activation at 10 uM activator concentration; average of triplicate measurement.Float%
20Activation at 29.9 uM (29.9μM**)Value of %Activation at 29.9 uM activator concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH083077-01

Data Table (Concise)
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