qHTS Assay for Inhibitors of Bacillus subtilis AcpS phosphopantetheinyl transferase (PPTase): 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 ..
BioActive Compounds: 4
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 project seeks to identify Sfp-PPTase inhibitors. This assay sought to determine the activity of these compounds with holo-acyl carrier protein synthase (AcpS) from E. coli, the canonical representative of the AcpS-PPTase subgrouping (vide supra). The evaluation utilized the qHTS assay format, with the only change being direct substitution of the enzyme.
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 assay was performed in a buffer consisting of 50mM HEPES-Na pH 7.6, 10mM MgCl2, 0.01% Nonidet P-40, and 0.01% BSA. Reagents (3 uL) were dispensed into a 1536-well black solid bottom plate (100 nM AcpS PPTase incolumns 1, 2, 5 through 48; buffer sans enzyme in columns 3 &4). DMSO solution of test articles (23 nL) were transferred via Kalypsys pintool equipped with 1,536-pin array. Following a room temperature incubation (15 min), enzyme reactions were initiated by the addition of substrate (1 uL , final concentration in the plte for rhodamine-CoA and BHQ-2-YbbR 5 uM and 12.5 uM, respectively). The plate was then centrifuged (15 s, 1000 rpm), and the fluorescence intensity recorded on a ViewLux High-throughput CCD imager (Perkin-Elmer, 525 nm excitation and 598 nm emission). The plate was then incubated at room temperature (30 min), after which a second read on the ViewLux was recorded. The fluorescence intensity difference over the 30-minute period was used to calculate the respective reaction rate for each well.
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)