qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR in hGST A1-1
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 ..
BioActive Compounds: 5
Depositor Specified Assays
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 assay sought to assess the promiscuity of test articles with an unrelated target enzyme. The appropriate target for this work was determined to be human glutathione-S-transferase, an enzyme responsible for detoxifying modifying electrophilic xenobiotics to enhance their excretion. Inhibitory activity with this enzyme in the contexts of this project is indicative of nonspecific behavior.
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
Reaction buffer consisted of 50 mM HEPES pH 7.5 with 0.01% Tween 20, and 133 uM glutathione (GSH, final assay concentration 100 uM). Reagent solution was transferred to a 1536-well solid black Greiner plate ( 3 uL, 6.67 nM human glutathione-S-transferase (hGST) A1-1 in reaction buffer to columns 1 - 2, 5 to 48, or buffer sans enzyme to columns 3 and 4). Control (Reactive Blue 2) and test compounds (46 nL) were transferred to the plate via Kalypsys pintool and incubated at room temperature (15 min). Reactions were initiated by the addition substrate PBI 3773 (1 uL, providing a final assay concentration of 40 uM), the plates centrifuged (15 sec, 1,000 rpm) and then incubated at room temperature (40 minutes). Luciferase Detection Reagent was then added (4 uL, Promega Corporation), and the plate read in Luminescence mode on a ViewLux High-throughput CCD imager following a short room temperature incubation (15 min).
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)