AID	Name	Type	Comment
1819	Probe Development Summary of Inhibitors of Bacillus subtilis Sfp phosphopantetheinyl transferase (PPTase)	summary	Summary AID

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

Innoculum preparation. 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 masculineC. In the morning, this culture (100 uL) was used to seed a fresh LB medium (10 mL) and was shaken at 30 masculineC until the culture OD600 reached 0.5. This culture was diluted 1:100 in fresh LB medium to provide the inoculum below.

Bacterial susceptibility. 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 masculineC. After 5 hrs, 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.

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