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BioAssay: AID 540253

qHTS Assay for Inhibitors of RanGTP induced Rango (Ran-regulated importin-beta cargo) - Importin beta complex dissociation

The small GTPase Ran regulates transport of macromolecules between the nucleus and the cytoplasm (nuclear transport) and has important functions in mitotic spindle assembly, nuclear envelope (NE) formation and nuclear pore complex (NPC) assembly [1]. RanGTP gradient is thought to promote spindle assembly by providing a spatial clue to microtubule nucleation and Aurora A activation [1]. Because more ..
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 Tested Compounds
 Tested Compounds
All(340708)
 
 
Active(242)
 
 
Inactive(324832)
 
 
Inconclusive(15660)
 
 
 Tested Substances
 Tested Substances
All(340951)
 
 
Active(242)
 
 
Inactive(325034)
 
 
Inconclusive(15675)
 
 
AID: 540253
Data Source: NCGC (ranb001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-14

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 242
Related Experiments
AIDNameTypeComment
540262Inhibitors of RanGTP induced Rango (Ran-regulated importin-beta cargo) - Importin beta complex dissociation: SummarySummarydepositor-specified cross reference
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 MH090808-01A1
Assay Submitter (PI): Petr Kalab

NCGC Assay Overview:

The small GTPase Ran regulates transport of macromolecules between the nucleus and the cytoplasm (nuclear transport) and has important functions in mitotic spindle assembly, nuclear envelope (NE) formation and nuclear pore complex (NPC) assembly [1]. RanGTP gradient is thought to promote spindle assembly by providing a spatial clue to microtubule nucleation and Aurora A activation [1]. Because the localized release and binding of importin alpha cargos relays this spatial clue to SAFs, inhibitors of the Rango - importin beta complex can then be used in further understanding their role in cell cycle specifically in mitosis progression. In addition RanGTP, importin beta, and importin alpha has been shown to regulate the activation of Aurora A though its binding to TPX2 [2-4]. Activation of Aurora A has been linked to different types of cancer and a number of Aurora kinase inhibitors are being developed as potential canter therapeutics [5-6]; therefore, identified inhibitors of this complex could be developed into potential anti-mitotic probes for cancer treatment.

Purified recombinant proteins Rango - importin beta and RanGTP-RCC1 stock premixes were provided by the assay submitter. The assay used a fluorescence resonance energy transfer (FRET) Rango biosensor that contains the IBB of human snurportin 1 flanked by yellow fluorescent protein (EYFP) at the amino terminus and cerulean CFP10 at the carboxy terminus. Rango assumes extended conformation when bound to importin beta and displays low FRET. RanGTP binding to the complex (in the presence of RCC1) releases the Rango from the complex. The flexibility of the IBB in the freed Rango allows the donor - acceptor fluorophores to interact, resulting in a high FRET signal [7]. Inhibitors will therefore be identified based on the ability to interfere with the FRET signal increase. DMSO treated and no RanGTP-RCC1 (buffer only) wells were used as negative and positive controls respectively. FRET readings were obtained at two time points. FRET reading prior to importin beta addition (pre-read) were used flag potential compound interference; FRET reading after importin beta addition (post-read) were used to identify inhibitors.
Protocol
NCGC Assay Protocol Summary:
Two and a half uL/well of Rango- importin beta premix solution (0.1 uM Rango, 0.25 uM importin beta, 1x PBS, 1mM DTT, 0.02% Tween-20, 0.1% BSA final concentrations) was dispensed into 1536-well assay plates (Greiner, solid black medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using Kalypsys pin tool equipped with a 1536-pin tool and incubated at room temperature for 5 min followed by a FRET pre-read using the PerkinElmer Envision plate reader. Two and a half uL/well of RanGTP-RCC1 premix solution (0.6 uM Ran, 2mM GTP, 0.2 uM RCC1, 1x PBS, 1mM DTT, 0.02% Tween-20, 0.1% BSA final concentrations) were then added and incubated at room temperature giving a final reaction volume of 5uL. After 45min FRET post-read was obtained via the Envision plate reader using the same optics and settings as the pre-read (CFP/YFP optimized dual optical module D450/D505; excitation mirror CFP 430/24 nm; and emission filters CFP 486/10 nm and YFP 530/10 nm).
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 inhibitors are ranked higher than compounds that showed apparent activation.
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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
5CFP-preread-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
6CFP-preread-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7CFP-preread-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8CFP-preread-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9CFP-preread-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10CFP-preread-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11CFP-preread-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12CFP-preread-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
13CFP-preread-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14CFP-preread-Activity at 0.00593 uM (0.00593μM**)% Activity at given concentration.Float%
15CFP-preread-Activity at 0.030 uM (0.0296μM**)% Activity at given concentration.Float%
16CFP-preread-Activity at 0.741 uM (0.741μM**)% Activity at given concentration.Float%
17CFP-preread-Activity at 3.700 uM (3.7μM**)% Activity at given concentration.Float%
18CFP-preread-Activity at 18.50 uM (18.5μM**)% Activity at given concentration.Float%
19CFP-preread-Activity at 92.60 uM (92.6μM**)% Activity at given concentration.Float%
20YFP-preread-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
21YFP-preread-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
22YFP-preread-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
23YFP-preread-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
24YFP-preread-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
25YFP-preread-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
26YFP-preread-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
27YFP-preread-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
28YFP-preread-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
29YFP-preread-Activity at 0.00593 uM (0.00593μM**)% Activity at given concentration.Float%
30YFP-preread-Activity at 0.030 uM (0.0296μM**)% Activity at given concentration.Float%
31YFP-preread-Activity at 0.741 uM (0.741μM**)% Activity at given concentration.Float%
32YFP-preread-Activity at 3.700 uM (3.7μM**)% Activity at given concentration.Float%
33YFP-preread-Activity at 18.50 uM (18.5μM**)% Activity at given concentration.Float%
34YFP-preread-Activity at 92.60 uM (92.6μM**)% Activity at given concentration.Float%
35ratio-postread-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
36ratio-postread-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
37ratio-postread-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
38ratio-postread-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
39ratio-postread-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
40ratio-postread-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
41ratio-postread-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
42ratio-postread-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
43ratio-postread-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
44ratio-postread-Activity at 0.00295 uM (0.00295μM**)% Activity at given concentration.Float%
45ratio-postread-Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
46ratio-postread-Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
47ratio-postread-Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
48ratio-postread-Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
49ratio-postread-Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
50Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH090808-01A1

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