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BioAssay: AID 2120

qHTS Assay for Identification of Small Molecule Inducers of Hypoxia Response

Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that controls cellular responses to low oxygen concentration. HIF-1 is composed of two subunits: hypoxia responsive HIF-1a and constitutively expressed HIF-1b, which is also known as aryl hydrocarbon receptor nuclear translocator. During hypoxic conditions, HIF-1a heterodimerizes with HIF-1b and translocates more ..
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 Tested Compounds
 Tested Compounds
All(1335)
 
 
Active(6)
 
 
Inactive(1225)
 
 
Inconclusive(105)
 
 
 Tested Substances
 Tested Substances
All(1408)
 
 
Active(6)
 
 
Inactive(1293)
 
 
Inconclusive(109)
 
 
 Related BioAssays
 Related BioAssays
AID: 2120
Data Source: NCGC (HRE953)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-11-09

Data Table ( Complete ):           Active    All
Target
Sequence: hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) [Homo sapiens]
Description ..   
Protein Family: HIF-1 alpha C terminal transactivation domain

Gene:HIF1A     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 6
Depositor Specified Assays
AIDNameTypeComment
914qHTS Assay for Identification of Small Molecule Agonists for Hypoxia Response Element Signaling PathwayconfirmatoryPrimary screen.
915qHTS Assay for Identification of Small Molecule Antagonists for Hypoxia Response Element Signaling PathwayconfirmatoryPrimary screen.
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

MLSCN Grant: None

NCGC Assay Overview

Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that controls cellular responses to low oxygen concentration. HIF-1 is composed of two subunits: hypoxia responsive HIF-1a and constitutively expressed HIF-1b, which is also known as aryl hydrocarbon receptor nuclear translocator. During hypoxic conditions, HIF-1a heterodimerizes with HIF-1b and translocates into the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes such as VEGF (vascular endothelial growth factor). The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia.

Using a beta-lactamase reporter-gene under control of the hypoxia response element, a cell-based assay [CellSensor(TM) HRE-bla ME-180 developed by Invitrogen Corp.] was used to measure hypoxia-induced signaling. Library compounds were measured for their ability to induce the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The inducers were identified based on the AC50 values derived from the screening data.
Protocol
NCGC Assay Protocol Summary

2500 cells/5uL in Opti-MEM medium containing 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM sodium pyruvate and 10 mM HEPES was dispensed into 1536-well plates and cells were cultured at 37 degrees Celsius for 4-5 hours and then 23 nL of compounds or DMSO were delivered to each well using a pin tool. The plates were incubated at 37 degrees Celsius for 17 hours. 1 uL of CCF4-dye mixture was added to each well. After incubated at room temperature for 2.5 hours the plates were measured on an EnVision plate reader at Excitation 405nm, Emission1=460nm and Emission 2=530nm. The %Activity was determined from the ratio of 460nm/530nm.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5W460-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6W460-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7W460-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8W460-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9W460-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10W460-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11W460-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12W460-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13W460-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14W460-Activity at 0.0006000000 uM (0.0006μM**)% Activity at given concentration.Float%
15W460-Activity at 0.00290 uM (0.0029μM**)% Activity at given concentration.Float%
16W460-Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
17W460-Activity at 0.033 uM (0.0328μM**)% Activity at given concentration.Float%
18W460-Activity at 0.073 uM (0.0734μM**)% Activity at given concentration.Float%
19W460-Activity at 0.164 uM (0.1642μM**)% Activity at given concentration.Float%
20W460-Activity at 0.367 uM (0.367μM**)% Activity at given concentration.Float%
21W460-Activity at 0.821 uM (0.8207μM**)% Activity at given concentration.Float%
22W460-Activity at 1.835 uM (1.8351μM**)% Activity at given concentration.Float%
23W460-Activity at 4.103 uM (4.1033μM**)% Activity at given concentration.Float%
24W460-Activity at 9.175 uM (9.1749μM**)% Activity at given concentration.Float%
25W460-Activity at 20.51 uM (20.515μM**)% Activity at given concentration.Float%
26W460-Activity at 45.87 uM (45.8716μM**)% Activity at given concentration.Float%
27W460-Activity at 91.74 uM (91.7431μM**)% Activity at given concentration.Float%
28Ratio-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
29Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
30Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
31Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
32Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
33Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
34Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
35Ratio-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
36Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
37Ratio-Activity at 0.0005876147 uM (0.000587615μM**)% Activity at given concentration.Float%
38Ratio-Activity at 0.00294 uM (0.0029367μM**)% Activity at given concentration.Float%
39Ratio-Activity at 0.015 uM (0.0146835μM**)% Activity at given concentration.Float%
40Ratio-Activity at 0.033 uM (0.0328303μM**)% Activity at given concentration.Float%
41Ratio-Activity at 0.073 uM (0.0733945μM**)% Activity at given concentration.Float%
42Ratio-Activity at 0.164 uM (0.164128μM**)% Activity at given concentration.Float%
43Ratio-Activity at 0.367 uM (0.367018μM**)% Activity at given concentration.Float%
44Ratio-Activity at 0.866 uM (0.86581μM**)% Activity at given concentration.Float%
45Ratio-Activity at 1.586 uM (1.5856μM**)% Activity at given concentration.Float%
46Ratio-Activity at 2.517 uM (2.51721μM**)% Activity at given concentration.Float%
47Ratio-Activity at 4.356 uM (4.35602μM**)% Activity at given concentration.Float%
48Ratio-Activity at 7.977 uM (7.97738μM**)% Activity at given concentration.Float%
49Ratio-Activity at 12.74 uM (12.7434μM**)% Activity at given concentration.Float%
50Ratio-Activity at 21.91 uM (21.9146μM**)% Activity at given concentration.Float%
51Ratio-Activity at 40.14 uM (40.1354μM**)% Activity at given concentration.Float%
52Ratio-Activity at 64.51 uM (64.5133μM**)% Activity at given concentration.Float%
53Ratio-Activity at 104.3 uM (104.275μM**)% Activity at given concentration.Float%
54Ratio-Activity at 177.8 uM (177.778μM**)% Activity at given concentration.Float%
55Ratio-Activity at 266.7 uM (266.667μM**)% Activity at given concentration.Float%
56Ratio-Activity at 400.0 uM (400μM**)% Activity at given concentration.Float%
57W530-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
58W530-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
59W530-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
60W530-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
61W530-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
62W530-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
63W530-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
64W530-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
65W530-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
66W530-Activity at 0.0006000000 uM (0.0006μM**)% Activity at given concentration.Float%
67W530-Activity at 0.00290 uM (0.0029μM**)% Activity at given concentration.Float%
68W530-Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
69W530-Activity at 0.033 uM (0.0328μM**)% Activity at given concentration.Float%
70W530-Activity at 0.073 uM (0.0734μM**)% Activity at given concentration.Float%
71W530-Activity at 0.164 uM (0.1642μM**)% Activity at given concentration.Float%
72W530-Activity at 0.367 uM (0.367μM**)% Activity at given concentration.Float%
73W530-Activity at 0.821 uM (0.8207μM**)% Activity at given concentration.Float%
74W530-Activity at 1.835 uM (1.8351μM**)% Activity at given concentration.Float%
75W530-Activity at 4.103 uM (4.1033μM**)% Activity at given concentration.Float%
76W530-Activity at 9.175 uM (9.1749μM**)% Activity at given concentration.Float%
77W530-Activity at 20.51 uM (20.515μM**)% Activity at given concentration.Float%
78W530-Activity at 45.87 uM (45.8716μM**)% Activity at given concentration.Float%
79W530-Activity at 91.74 uM (91.7431μM**)% Activity at given concentration.Float%
80Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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