Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

Abstract

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyltRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC 50 s) ranging from 3 to 14 M. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five-or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 g/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

“…An aminoacylation/translation (A/T) system was developed which contained all the components required for translation of poly(U) mRNA from P. aeruginosa: ribosomes, EF-Tu, EF-Ts, EF-G, and PheRS. The coupled A/T reaction was adapted from separate aminoacylation and translation assays as previously described (21). The assay was optimized for the detection of inhibition of any component of the A/T system, and compounds were screened in 96-well microtiter plates.…”

“…Reactions to determine the inhibitory effect of compounds on eukaryotic protein synthesis were performed using wheat germ cell extracts as described previously (21). The concentrations of the compounds in these assays ranged from 0.8 to 100 M, and the concentration of the cycloheximide in the control reactions ranged from 0.3 to 300 M.…”

“…The mixture was incubated for 15 min with various concentrations of the compound (0.8 to 200 M), and the reaction was then initiated by the addition of E. coli tRNA Phe (2 M). The reaction was stopped by dilution into 3 ml of ice-cold 5% trichloroacetic acid and filtration through glass fiber filters as described previously (21).…”

“…Macromolecular synthesis (MMS) assays were performed in cultures of E. faecalis as previously described (21). Levofloxacin, rifampin, tetracycline and vancomycin were used as quality controls in assays to measure inhibition of DNA synthesis, RNA synthesis, protein synthesis and cell wall synthesis, respectively.…”

Identification of Chemical Compounds That Inhibit Protein Synthesis in Pseudomonas aeruginosa

“…4 The concentrations of the compounds in these assays ranged from 0.4 to 200 µM, and the concentration of the cycloheximide in the control reactions was from 0.3 to 300 µM.…”