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BioAssay: AID 602362

qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Gel Based 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(45)
 
 
Active(36)
 
 
Inactive(6)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(47)
 
 
Active(38)
 
 
Inactive(6)
 
 
Inconclusive(3)
 
 
AID: 602362
Data Source: NCGC (PPTA904)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-12
Hold-until Date: 2013-03-11
Modify Date: 2013-03-11

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 36
Depositor Specified Assays
AIDNameTypeComment
1490qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)confirmatory
1819Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)summarySummary AID
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.

In this assay, compounds were evaluated by monitoring of fluorescence transfer to whole carrier protein substrates via polyacrylamide gel electrophoresis. See Yasgar, et al. (PMID: 20094656) for further details on assay. This assay was used to evaluate follow-up compounds from the primary screen (AID: 1490).

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
The SDS-PAGE based assay detects the direct incorporation of a fluorescent label into a carrier protein substrate. To perform this assay, serial dilutions of the compounds will be prepared and treated with Sfp (20 nM) for a preincubation time of 30 minutes (100 uL total volume). Reactions are initiated by the addition of 100 uL substrate solution to give final conditions with 2 uM ACP (3.5 ug) and 50 uM Rhodamine CoA. The reactions are allowed to progress for 30 minutes and then quenched by the addition of 100 uL 50 mM EDTA. 50 uL of the quenched reaction is aliquoted and treated with 10 uL 5x SDS-PAGE buffer and boiled for 5 minutes. The samples are then separated for 1h by SDS-PAGE on a 20% gel, which is subsequently scanned with a Typhoon scanner. The images are analysed by fluorescent densometry using the ImageJ software package available from the NIH.
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.
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 0.00141 uM (0.00141126μM**)% Activity at given concentration.Float%
16Activity at 0.00423 uM (0.00423377μM**)% Activity at given concentration.Float%
17Activity at 0.013 uM (0.0127013μM**)% Activity at given concentration.Float%
18Activity at 0.038 uM (0.0381039μM**)% Activity at given concentration.Float%
19Activity at 0.114 uM (0.114312μM**)% Activity at given concentration.Float%
20Activity at 0.343 uM (0.342936μM**)% Activity at given concentration.Float%
21Activity at 1.029 uM (1.02881μM**)% Activity at given concentration.Float%
22Activity at 3.086 uM (3.08642μM**)% Activity at given concentration.Float%
23Activity at 9.259 uM (9.25926μM**)% Activity at given concentration.Float%
24Activity at 27.78 uM (27.7778μM**)% Activity at given concentration.Float%
25Activity at 83.33 uM (83.3333μM**)% Activity at given concentration.Float%
26Activity at 250.0 uM (250μM**)% 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)
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