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BioAssay: AID 2707

Gel-Based Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)

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(209)
 
 
Active(34)
 
 
Inactive(114)
 
 
Inconclusive(62)
 
 
 Tested Substances
 Tested Substances
All(210)
 
 
Active(34)
 
 
Inactive(114)
 
 
Inconclusive(62)
 
 
AID: 2707
Data Source: NCGC (PPTA238)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-26
Modify Date: 2013-01-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 34
Related Experiments
Show more
AIDNameTypeComment
1490qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)
1819Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Summarydepositor-specified cross reference: Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PP
493214Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Dry Powder FollowupConfirmatorydepositor-specified cross reference
540355Gel-Based Assay for Inhibitos of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Chemistry Optimization FollowupConfirmatorydepositor-specified cross reference
2701Confirmation qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)Confirmatorysame 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
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
602373qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Hepg2 AssayConfirmatorysame 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 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).
Protocol
A DMSO solution of confirmed hits (0.5 microL) was added to a 1.33X enzyme solution (15 microL, containing 26.6 nM Sfp, 66 mM HEPES, 13.3 mM MgCl2, 0.0133% NP-40, 0.133% BSA, pH 7.6). After a 10 minute incubation, the enzymatic reaction was initiated by the addition of 4 X substrate solution (4 microL, containing 50 microM Rhodamine CoA and 50 microM apo-Actinorhodin-ACP). The reactions were terminated after a 30 minute incubation at room temperature by the addition of 2X quench solution (20 microL, containing 4 M Urea, 25 mM EDTA, 0.004% phenol red, pH 8.0). Samples were separated under native conditions on a 20% polyacrylamide gel using standard Laemmli conditions. Following the run, gels were imaged with a Chemi-Doc Plus imager (Bio-Rad, Hercules, CA) and band intensity quantified using the ImageJ software package. Pixel density values were normalized to control wells (SCH202676) and fit with the 4-parameter Hill equation using in-house tools [1].

[1] Yasgar, et al. (PMID: 20094656) for complete protocol details.
Comment
Compounds were ranked by taking the floor of the -Log_IC50*10. Compounds were 'active' if Potency < 100uM. If compound gave dose-dependent effect but had Potency > 100uM, it was considered 'inconclusive'. All other compounds were considered 'inactive'.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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, IC50. Extrapolated IC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3Log_IC50The logarithm of the IC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
4Compound QCSource of compound QCString

* Activity Concentration.
Additional Information
Grant Number: MH083266-01

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