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BioAssay: AID 602392

qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Label Free Assay for SAR

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 ..
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 602392
Data Source: NCGC (PPTA906)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-14
Hold-until Date: 2013-03-11
Modify Date: 2013-03-11

Data Table ( Complete ):           Active    All
BioActive Compounds: 14
Depositor Specified Assays
1819Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)summarySummary AID
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.

This project seeks to identify inhibitors of Sfp phosphopantetheinyl transferase. The primary and secondary screens both utilized a rhodamine-modified CoA substrate. This experiment sought to confirm compound activity in a biochemical assay using the natural nucleotide substrate in conjunction with a whole-protein acceptor. We employed polyacrylamide gel electrophoresis in the presence of modest urea concentrations to achieve a conformationally sensitive separation of the apo- and holo- states of the acyl carrier protein (ACP) from E. coli [Rock, 1981, Cronan 1982]. Under conditions of limiting Sfp concentrations, quantitative determinations of enzyme activity can be made from images of the gels after treatment with an appropriately sensitive protein stain.

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
Preparation of apo-ACP substrate.

A derivative of the AcpP locus from E. coli K12 containing a C-terminal fusion to a hexahistidine tag is expressed and purified from E.coli BL21(DE3) following standard procedures, (27) providing a mixture of the apo- and holo- forms of ACP in a ratio 3:2; as judged by UREA-PAGE. The resulting mixture (10 mg/ml) is reduced overnight in PBS buffer containing 10 mM DTT, and excess reducing agent removed by passage over a PD6 desalting column (Bio-Rad Laboratories, Hercules, CA). The sample is immediately pooled with an equal volume of activated thiol sepharose 4B (cat #17-0640-01, GE Healthcare, Piscataway, NJ) prepared according to the manufacturers recommendations; and the slurry agitated gently overnight at 4 deg C. The slurry was decanted into a disposable chromatography column and washed with 3 volumes of PBS. The filtrate and washes were pooled, concentrated, and the process iterated once to provide a solution of ACP that was >98% apo-form as judged by conformationally sensitive UREA-PAGE.

Label-free gel assay for phosphopantetheinylation.

Test compound (0.5 ul) dissolved in DMSO was added to 1.33 X enzyme solution (15 ul) containing 66 nM Sfp, 66 mM HEPES*Na pH 7.6, 13.3 mM MgCl2, 0.0133% NP40, and 1.33 mg/ml BSA. These solutions were incubated at room temperature for 10 minutes, at which point the phosphopantetheinylation reaction was initiated by the addition of 5x substrate solution (4 ul) containing 50 uM apo-ACP [vide supra] and 50microM coenzyme A in 10 mM HEPES*Na pH 7.6. After incubation at room temperature for 30 minutes, quench/load solution (5 ul) containing 50mM EDTA pH 8.0, 50% glycerol and 0.005% phenol red was added. The samples were electrophoretically separated on a discontinuous polyacrylamide gel using the Laemmli buffers sans SDS; with urea (2M final) included in the resolving gel (15% total acrylamide/bisacrylamide concentration).

After separation, the gels were fixed (50% MeOH, 7% AcOH, 30 min) and washed thrice with deionized water (200 ml, 5 min per wash). The gels were stained with Sypro Ruby (R) according to the manufacturer's recommendations, and imaged with a Bio-Rad ChemiDocTM XRS Gel Imager using standard ethidium bromide settings. Protein bands were quantified via densitometry using the Image software package,
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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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2PotencyConcentration 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%
15IC50 (visual scoring) (0.000322571μM**)FloatμM
16Compound QC (0.000967714μM**)String

** Test Concentration.
Additional Information
Grant Number: MH083226

Data Table (Concise)