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BioAssay: AID 602373

qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Hepg2 Assay

The covalent attachment of a phosphopantetheinyl (4'-PP) arm to a variety of synthases and other proteins is a key posttranslational protein modification. The 4'-PP is installed on the proteins post-translationally from coenzyme A (CoA) on a conserved serine residue by action of phosphopantetheinyl transferase (PPTase) enzymes. Phosphopantetheinylation is essential for synthase activity, and more ..
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 Tested Compounds
 Tested Compounds
All(213)
 
 
Active(17)
 
 
Inactive(104)
 
 
Inconclusive(98)
 
 
 Tested Substances
 Tested Substances
All(229)
 
 
Active(19)
 
 
Inactive(109)
 
 
Inconclusive(101)
 
 
AID: 602373
Data Source: NCGC (PPTA905)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-13
Hold-until Date: 2013-03-11
Modify Date: 2013-03-11

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 17
Related Experiments
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AIDNameTypeComment
1819Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Summarydepositor-specified cross reference: Summary AID
1490qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Confirmatorysame project related to Summary assay
2701Confirmation qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Confirmatorysame project related to Summary assay
2707Gel-Based Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Confirmatorysame project related to Summary assay
493214Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Dry Powder FollowupConfirmatorysame project related to Summary assay
540350qHTS Assay for Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Chemistry Optimization FollowupConfirmatorysame project related to Summary assay
540352qHTS for Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Chemistry Optimization Followup in B. Subtilis 168Confirmatorysame project related to Summary assay
540353qHTS for Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Chemistry Optimization Followup in B. Subtilis HM489Confirmatorysame project related to Summary assay
540355Gel-Based Assay for Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Chemistry Optimization FollowupConfirmatorysame project related to Summary assay
602360qHTS Assay for Inhibitors of Bacillus subtilis AcpS phosphopantetheinyl transferase (PPTase): SARConfirmatorysame project related to Summary assay
602362qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Gel Based AssayConfirmatorysame project related to Summary assay
602366qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR in B subtilis HM489Confirmatorysame project related to Summary assay
602370qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR Round 2Confirmatorysame project related to Summary assay
602371qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR in B subtilis 168Confirmatorysame project related to Summary assay
602391qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR in hGST A1-1Confirmatorysame project related to Summary assay
602392qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Label Free Assay for SAROthersame project related to Summary assay
602479qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Mouse Liver Microsome Stability (Half Time)Othersame project related to Summary assay
602480qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Aqueous Solubility in PBSOthersame project related to Summary assay
624112qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Aqueous Solubility in PBSOthersame project related to Summary assay
624113qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Mouse Plasma StabilityOthersame project related to Summary assay
624118qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): CYP 3A4Othersame project related to Summary assay
624119qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): CYP 2D6Othersame project related to Summary assay
Description:
The covalent attachment of a phosphopantetheinyl (4'-PP) arm to a variety of synthases and other proteins is a key posttranslational protein modification. The 4'-PP is installed on the proteins post-translationally from coenzyme A (CoA) on a conserved serine residue by action of phosphopantetheinyl transferase (PPTase) enzymes. Phosphopantetheinylation is essential for synthase activity, and removal of the PPTase gene precludes natural product synthesis in microorganisms, or in the case of fatty acid biosynthesis, renders the organism unviable. PPTase enzymes belong to a distinct structural superfamily. Within bacteria, these enzymes are grouped into two classes based upon primary structure, the AcpS-Type and Sfp-Type PPTases.

Sfp-type PPTases, corresponding to an activator of surfactin production in Bacillus subtilis, are responsible for modifying type I polyketide and nonribosomal peptide synthases of prokaryotes. Sfp-type PPTases are responsible for the activation of a variety of pathogen-associated virulence factors. Among these compounds are toxins such as mycolactone from Mycobacterium ulcerans, siderophores such as vibriobactin from Vibrio cholerae or mycobactin from Mycobacterium tuberculosis, as well as the mycolic acids which form the waxy cell wall of Mycobacteria. The biosyntheses of these natural products are considered attractive targets for drug design.

In search of small molecule Sfp-PPTase inhibitors, a fluorescence quenching assay was developed for detection of Bacillus subtilis Sfp-PPTase enzymatic activity in a miniaturized high-throughput format. The consensus ybbr acceptor peptide DALEFIASKLA was N-terminally labeled with Black Hole Quencher-2 (BHQ-2) and used in combination with rhodamine-labeled coenzyme A as a co-substrate. The PPTase-catalyzed reaction leads to a product containing both the rhodamine fluorophore and the BHQ-2 quencher covalently attached to the ybbr scaffold; thus, the rhodamine fluorescence, which in the starting state is unperturbed, is dramatically reduced upon its incorporation into the BHQ-2-tagged peptide.

Phosphopantetheinyl transfer is a process essential to cellular viability and maintenance in all forms of life. Cell-permeable and stable compounds acting as PPTase inhibitors are anticipated to be growth inhibitory agents. This assay sought to assess the toxicity of test compounds in the HepG2 hepatocarcinoma cells; a human cell line whose use additionally determines the toxicity of test article metabolites. Growth inhibition or death of this cell line was is considered unattractive characteristic and compounds possessing such activity were deprioritized in this project.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH083226
Assay Submitter (PI): Michale Burkart, University of California, San Diego
Protocol
Test compounds' toxicity was assessed by measuring cellular ATP content using a luciferase-coupled ATP quantitation assay (CellTiter-Glo; Promega, Madison, WI). In this assay, luminescent signal is proportional to amount of ATP, and thus to the number of metabolically competent cells. Briefly, Hepg2 cell suspension (5 uL, 4 x 105 cells/mL) was dispensed into 1,536-well tissue-culture treated white/solid bottom assay plates (Greiner Bio-One North America, Monroe, NC) using a Flying Reagent Dispenser (Aurora Discovery, Carlsbad, CA). Cells were incubated at 37 deg C for 6 hr to allow for cell attachment, followed by addition of compounds via pin tool (Kalypsys, San Diego, CA). After compound addition, plates were incubated for 48 hr at 37 deg C. At the end of the incubation period, CellTiter-Glo reagent (5 uL) was added, plates were incubated at room temperature in the dark for 30 min, and the luminescence intensity of each well was determined using a ViewLux plate reader (PerkinElmer, Shelton, CT). The positive control was 92 uM and 41 uM of tetra-N-Octylammonium bromide, and the negative control was DMSO.
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 PubChem:
Assay Cell Type: HEPG2
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0002601382 uM (0.000260138μM**)% Activity at given concentration.Float%
16Activity at 0.0007804147 uM (0.000780415μM**)% Activity at given concentration.Float%
17Activity at 0.00234 uM (0.00234124μM**)% Activity at given concentration.Float%
18Activity at 0.00702 uM (0.00702378μM**)% Activity at given concentration.Float%
19Activity at 0.021 uM (0.0210713μM**)% Activity at given concentration.Float%
20Activity at 0.063 uM (0.0632139μM**)% Activity at given concentration.Float%
21Activity at 0.190 uM (0.189642μM**)% Activity at given concentration.Float%
22Activity at 0.569 uM (0.568925μM**)% Activity at given concentration.Float%
23Activity at 1.707 uM (1.70678μM**)% Activity at given concentration.Float%
24Activity at 5.120 uM (5.12033μM**)% Activity at given concentration.Float%
25Activity at 15.36 uM (15.361μM**)% Activity at given concentration.Float%
26Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
27Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH083226

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