qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): Hepg2 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: 17
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.
Phosphopantetheinyl transfer is a process essential to cellular viability and maintenance in all forms of life. Cell-permeable and stable compounds acting as PPTase inhibitors are anticipated to be growth inhibitory agents. This assay sought to assess the toxicity of test compounds in the HepG2 hepatocarcinoma cells; a human cell line whose use additionally determines the toxicity of test article metabolites. Growth inhibition or death of this cell line was is considered unattractive characteristic and compounds possessing such activity were deprioritized in this project.
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
Test compounds' toxicity was assessed by measuring cellular ATP content using a luciferase-coupled ATP quantitation assay (CellTiter-Glo; Promega, Madison, WI). In this assay, luminescent signal is proportional to amount of ATP, and thus to the number of metabolically competent cells. Briefly, Hepg2 cell suspension (5 uL, 4 x 105 cells/mL) was dispensed into 1,536-well tissue-culture treated white/solid bottom assay plates (Greiner Bio-One North America, Monroe, NC) using a Flying Reagent Dispenser (Aurora Discovery, Carlsbad, CA). Cells were incubated at 37 deg C for 6 hr to allow for cell attachment, followed by addition of compounds via pin tool (Kalypsys, San Diego, CA). After compound addition, plates were incubated for 48 hr at 37 deg C. At the end of the incubation period, CellTiter-Glo reagent (5 uL) was added, plates were incubated at room temperature in the dark for 30 min, and the luminescence intensity of each well was determined using a ViewLux plate reader (PerkinElmer, Shelton, CT). The positive control was 92 uM and 41 uM of tetra-N-Octylammonium bromide, and the negative control was DMSO.
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)