Title:High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae
VOLUME: 11 ISSUE: 4
Author(s):Yanmei Hu, Stephanie O. Palmer, Hector Munoz and James M. Bullard
Affiliation:Chemistry Department, SCIE. 3.320, The University of Texas-Pan American, 1201 W. University Drive, Edinburg, TX 78541, USA.
Keywords:Drug discovery, natural product, protein synthesis, Pseudomonas, Streptococcus.
Abstract:Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide
range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase
(PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and
the Km’s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNAPhe were
determined to be 48, 200, and 1.2 µM, respectively, while the Km’s for S. pneumoniae PheRS with
respect to phenylalanine, ATP and tRNAPhe were 21, 225 and 0.94 µM, respectively. P. aeruginosa
and S. pneumoniae PheRS were used to screen a natural compound library and a single compound
was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa
and S. pneumoniae PheRS with IC50’s of 2.3 and 4.9 µM, respectively. The compound had a Ki of
0.83 and 0.98 µM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum
inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive
and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against
wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 µg/ml,
respectively. The mechanism of action of the compound was determined to be competitive with the
amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic
protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.
