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 ..
BioActive Compounds: 36
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.
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
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.
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)