qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase): SAR in B subtilis HM489
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: 40
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. Cellular permeable and stable compounds acting as PPTase inhibitors are anticipated to be antimicrobial agents in organisms with complimenting genotypes. The primary test organism in these experiments, B. subtilis, possesses two PPTase loci in the genome, acpS and sfp. This experiment evaluated a series of test compounds for antimicrobial activity with B. subtilis HM489 (genotype: acpS- and sfp+), a strain that contains a targeted lesion in the acpS locus and thus requires a functional Sfp gene product for viability. [Mootz HD, 2001] This strain is expected to be sensitized to cell-permeable Sfp inhibitors.
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
Bacillus subtilis HM489 is maintained on lysogeny broth (LB) solidified by the addition of 1.5% w/v agar. Single colonies were used to LB medium (2 mL) and shaken overnight at 30 deg C. In the morning, this culture (100 uL) was used to seed a fresh LB medium (10 mL) and was shaken at 30 deg C until the culture OD600 reached 0.5. This culture was diluted 1:100 in fresh LB medium to provide the inoculum below.
LB medium (2 uL) is dispensed into wells of a sterile white 1536-well plate. Test compounds (23 nL) prepared as serial dilutions in DMSO are added to the plate by pintool transfer. Innoculum (2 uL) is added; the plates are covered with a vented Kalypsys assay lid and incubated at 30 deg C. After 5 h, Bac-Titer Glo (4 uL; Promega Corp, Madison, WI) is added to the plates. They are incubated 10 minutes at room temperature and then the luminescence detected in a ViewLux multimodal plate reader.
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)