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BioAssay: AID 602128

Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 2)

Name: Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 2). ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
AID: 602128
Data Source: The Scripps Research Institute Molecular Screening Center (TRPML3_AG_FLUO8_1536_3XEC50 MDRUN2 SELECTIVE)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-10
Hold-until Date: 2012-12-08
Modify Date: 2012-12-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
1424Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)Screening depositor-specified cross reference: Primary screen (TRPN1 agonists in singlicate)
1448Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel ML3 (TRPML3)Screening depositor-specified cross reference: Primary screen (TRPML3 agonists in singlicate)
1525Counterscreen assay for TRPML3 agonists: cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)Screening depositor-specified cross reference: Counterscreen (TRPN1 agonists in triplicate)
1526Confirmation cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)Screening depositor-specified cross reference: Confirmation screen (TRPML3 agonists in triplicate)
1562Dose response cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)Confirmatory depositor-specified cross reference: Dose response (TRPML3 agonists in singlicate)
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 depositor-specified cross reference: Dose response counterscreen (TRPN1 agonists in triplicate)
1809Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)Summary2 depositor-specified cross reference: Summary (TRPML3 agonists)
2116Late stage results from the probe development efforts to identify agonists of the Transient Receptor Potential Channels 3 and 2 (TRPML3 and TRPML2).Screening same project related to Summary assay
2510Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonistsConfirmatory same project related to Summary assay
2583Late 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.Confirmatory same project related to Summary assay
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 same project related to Summary assay
2694Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPML3 patch clamp assayScreening same project related to Summary assay
2719Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): Fura-2 profiling assayScreening same project related to Summary assay
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 same project related to Summary assay
602129Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 3)Confirmatory same project related to Summary assay
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: TRPML3_AG_FLUO8_1536_3XEC50 MDRUN2 SELECTIVE

Name: Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 2).

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. Qian F, Noben-Trauth K. Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2). Pflugers Arch. 2005 Oct;451(1):277-85.
6. Atiba-Davies M, Noben-Trauth K. TRPML3 and hearing loss in the varitint-waddler mouse.
Biochim Biophys Acta. 2007 Aug;1772(8):1028-31.
7. 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.
8. Di Palma, F.; Belyantseva, I. A.; Kim, H. J.; Vogt, T. F.; Kachar, B.; Noben-Trauth, K. Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice. Proc. Nat. Acad. Sci. 99: 14994-14999, 2002.
9. Nagata K, Zheng L, Madathany T, Castiglioni AJ, Bartles JR, Garcia-Anoveros J. The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration. Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):353-8.
10. van Aken AF, Atiba-Davies M, Marcotti W, Goodyear RJ, Bryant JE, Richardson GP, Noben-Trauth K, Kros CJ. J Physiol. 2008 Sep 18. TRPML3 mutations cause impaired mechano-electrical transduction and depolarization by an inward-rectifier cation current in auditory hair cells of varitint-waddler mice.
11. Small molecule activators reveal that TRPML3 integrates multiple stimulation modes. Grimm C, Jors S, Saldanha SA, Obukhov AG, Pan B, Oshima K, Cuajungco MP, Chase P, Hodder P, and Heller S. Chemistry & Biology.

Keywords:

Late stage, probes, selective, powders, synthesized, TRPML3, TRP, cation channel, mucolipin 3, MCOLN3, deafness, HEK 293, agonist, agonism, activator, activation, activate, fluorescence, calcium, Fluo-8 dye, FLUO8, cells, cell-based, dose response, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine dose response curves for powder samples of compounds identified as possible selective TRPML3 agonist probe candidates. This assay may also have tested analogs of the above compounds. This assay employs a HEK293 cell line that stably expresses the human TRPML3-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 TRPML3 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye and increased well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 29.9 uM.

Protcol Summary:

The TRPML3 HEK293 cell line was routinely cultured in T-175 sq cm flasks at 37 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 ug/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1500 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 2 uL 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 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 uM 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.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

The 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 with carbachol.
Low_Control is defined as wells with 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 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 29.9 uM.

PubChem Activity Outcome and Score:

Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.

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 active compounds is 100-100, and for inactive compounds 0-0.

List of Reagents:

TRPML3 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.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: lead optimization

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: receptor: transmembrane receptor: ion channel

BAO: meta target: biological process target: regulation of molecular function

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: assay design: membrane potential reporter: dye binding

BAO: detection technology: fluorescence: fluorescence intensity

BAO: bioassay specification: bioassay type: binding

BAO: bioassay specification: assay footprint: microplate: 1536 well plate

BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates

BAO: assay format detail: assay phase characteristic: homogeneous assay

BAO: detection technology detail: detection instrumentation: flipr tetra

BAO: detection technology detail: detection instrumentation manufacturer: molecular devices

BAO: endpoint detail: perturbagen concentration: concentration unit: micromolar

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
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
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Response RangeThe range of Y.Float
9Chi 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
10RsquareThis 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
11Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
12Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
13Activation at 0.002 uM [1] (0.002μM**)Value of percent activation at 0.002 uM activator concentration; replicate [1]Float%
14Activation at 0.002 uM [2] (0.002μM**)Value of percent activation at 0.002 uM activator concentration; replicate [2]Float%
15Activation at 0.002 uM [3] (0.002μM**)Value of percent activation at 0.002 uM activator concentration; replicate [3]Float%
16Activation at 0.005 uM [1] (0.005μM**)Value of percent activation at 0.005 uM activator concentration; replicate [1]Float%
17Activation at 0.005 uM [2] (0.005μM**)Value of percent activation at 0.005 uM activator concentration; replicate [2]Float%
18Activation at 0.005 uM [3] (0.005μM**)Value of percent activation at 0.005 uM activator concentration; replicate [3]Float%
19Activation at 0.014 uM [1] (0.014μM**)Value of percent activation at 0.014 uM activator concentration; replicate [1]Float%
20Activation at 0.014 uM [2] (0.014μM**)Value of percent activation at 0.014 uM activator concentration; replicate [2]Float%
21Activation at 0.014 uM [3] (0.014μM**)Value of percent activation at 0.014 uM activator concentration; replicate [3]Float%
22Activation at 0.041 uM [1] (0.041μM**)Value of percent activation at 0.041 uM activator concentration; replicate [1]Float%
23Activation at 0.041 uM [2] (0.041μM**)Value of percent activation at 0.041 uM activator concentration; replicate [2]Float%
24Activation at 0.041 uM [3] (0.041μM**)Value of percent activation at 0.041 uM activator concentration; replicate [3]Float%
25Activation at 0.123 uM [1] (0.123μM**)Value of percent activation at 0.123 uM activator concentration; replicate [1]Float%
26Activation at 0.123 uM [2] (0.123μM**)Value of percent activation at 0.123 uM activator concentration; replicate [2]Float%
27Activation at 0.123 uM [3] (0.123μM**)Value of percent activation at 0.123 uM activator concentration; replicate [3]Float%
28Activation at 0.369 uM [1] (0.369μM**)Value of percent activation at 0.369 uM activator concentration; replicate [1]Float%
29Activation at 0.369 uM [2] (0.369μM**)Value of percent activation at 0.369 uM activator concentration; replicate [2]Float%
30Activation at 0.369 uM [3] (0.369μM**)Value of percent activation at 0.369 uM activator concentration; replicate [3]Float%
31Activation at 1.1 uM [1] (1.1μM**)Value of percent activation at 1.1 uM activator concentration; replicate [1]Float%
32Activation at 1.1 uM [2] (1.1μM**)Value of percent activation at 1.1 uM activator concentration; replicate [2]Float%
33Activation at 1.1 uM [3] (1.1μM**)Value of percent activation at 1.1 uM activator concentration; replicate [3]Float%
34Activation at 3.3 uM [1] (3.3μM**)Value of percent activation at 3.3 uM activator concentration; replicate [1]Float%
35Activation at 3.3 uM [2] (3.3μM**)Value of percent activation at 3.3 uM activator concentration; replicate [2]Float%
36Activation at 3.3 uM [3] (3.3μM**)Value of percent activation at 3.3 uM activator concentration; replicate [3]Float%
37Activation at 10.0 uM [1] (10μM**)Value of percent activation at 10.0 uM activator concentration; replicate [1]Float%
38Activation at 10.0 uM [2] (10μM**)Value of percent activation at 10.0 uM activator concentration; replicate [2]Float%
39Activation at 10.0 uM [3] (10μM**)Value of percent activation at 10.0 uM activator concentration; replicate [3]Float%
40Activation at 29.9 uM [1] (29.9μM**)Value of percent activation at 29.9 uM activator concentration; replicate [1]Float%
41Activation at 29.9 uM [2] (29.9μM**)Value of percent activation at 29.9 uM activator concentration; replicate [2]Float%
42Activation at 29.9 uM [3] (29.9μM**)Value of percent activation at 29.9 uM activator concentration; replicate [3]Float%

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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