A screen for compounds that inhibit the activity of LtaS in Staphylococcus aureus
The current epidemic of infections caused by antibiotic-resistant Gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of Gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction more ..
BioActive Compounds: 878
The current epidemic of infections caused by antibiotic-resistant Gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of Gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of Gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of Gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant Gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.
Cation-adjusted Mueller Hinton II broth supplemented with 0.005% Tween-80 was inoculated with a single colony of S. aureus RN4220. Following overnight incubation, the culture was diluted 1:100 into fresh medium, and incubation was continued until the bacterial suspension reached an optical density at 600 nm (OD600) of 0.6, corresponding to a bacterial density of 5 x 10E8 colony forming units (CFU)/mL. An aliquot of this culture was diluted 1:400 with fresh medium and stored on ice until use. Assay plates were prepared for compound transfer in quadruplicate to enable screening of compounds at two temperatures in duplicate. The plate layout was as follows: wells in columns 1-22 were loaded with culture medium at 25 microL/well; wells in column 23 contained the screening negative control (medium only); wells in column 24 contained the screening positive control (medium containing 25 microg/mL chloramphenicol). 300 nL of experimental compounds were transferred by stainless steel pin array from library plate to assay plates.
25 microL of the bacterial suspension was added to each assay well. Assay plates were incubated at 42 degrees C in humidified chambers (humidity > 85%). After 20 h, the OD600 was measured using a plate reader in absorbance mode.
Data analysis description:
Absorbance values at 42 degrees C and 30 degrees C for each replicate were normalized to positive and negative control plate average absorbance, and replicate normalized values were averaged to calculate an average relative absorbance at both temperatures. A substance was considered a temperature-dependent positive at 42 degrees C if average relative absorbance <= 50 and the difference between relative absorbance at 30 degrees C and 42 degrees C >= 20 (relative absorbance 30 degrees C - relative absorbance 42 degrees C >= 20). A substance was considered a temperature-independent positive if relative absorbance at both 30 degrees C and 42 degrees C < 10.
Activity scores were calculated using average relative absorbance at both temperatures. Average relative absorbance <= 0 was scored as 100 for activity; average relative absorbance >= 100 was scored as 0 for activityy. Average relative absorbance between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average relative absorbance = 40 corresponds to an activity score of 60).
Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active at both temperatures. The final Pubchem activity score is the activity score at 42 degrees C, and compounds scored as active (PUBCHEM_ACTIVITY_OUTCOME = 2) include both temperature-dependent and temperature-independent active substances.
Data Table (Concise)