AID	Name	Type	Comment
1819	Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)	summary	Summary AID
1490	qHTS Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)	confirmatory	qHTS
2707	Gel-Based Assay for Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)	confirmatory	Hit Validation 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 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).

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH083266-01
Assay Submitter (PI): Michael Burkart, University of California, San Diego

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 [1].

[1] Yasgar, et al. (PMID: 20094656)

Compound Ranking:

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.

Grant Number: MH083266