Gel-Based Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)
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: 34
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 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).
A DMSO solution of confirmed hits (0.5 microL) was added to a 1.33X enzyme solution (15 microL, containing 26.6 nM Sfp, 66 mM HEPES, 13.3 mM MgCl2, 0.0133% NP-40, 0.133% BSA, pH 7.6). After a 10 minute incubation, the enzymatic reaction was initiated by the addition of 4 X substrate solution (4 microL, containing 50 microM Rhodamine CoA and 50 microM apo-Actinorhodin-ACP). The reactions were terminated after a 30 minute incubation at room temperature by the addition of 2X quench solution (20 microL, containing 4 M Urea, 25 mM EDTA, 0.004% phenol red, pH 8.0). Samples were separated under native conditions on a 20% polyacrylamide gel using standard Laemmli conditions. Following the run, gels were imaged with a Chemi-Doc Plus imager (Bio-Rad, Hercules, CA) and band intensity quantified using the ImageJ software package. Pixel density values were normalized to control wells (SCH202676) and fit with the 4-parameter Hill equation using in-house tools .
 Yasgar, et al. (PMID: 20094656) for complete protocol details.
Compounds were ranked by taking the floor of the -Log_IC50*10. Compounds were 'active' if Potency < 100uM. If compound gave dose-dependent effect but had Potency > 100uM, it was considered 'inconclusive'. All other compounds were considered 'inactive'.
* Activity Concentration.
Data Table (Concise)