Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility.

Gene dosage has frequently been exploited to select for genetic interactions between a particular mutant and clones from a random genomic library at high copy. We report here the first use of multicopy suppression as a forward genetic method to determine cellular targets and potential resistance mechanisms for novel antibacterial compounds identified through high-throughput screening. A screen of 8640 small molecules for growth inhibition of a hyperpermeable strain of Escherichia coli led to the identification of 49 leads for suppressor selection from clones harboring an E. coli genomic library. The majority of suppressors were found to encode the multidrug efflux pump AcrB, indicating that those compounds were substrates for efflux. Two leads, which produced clones containing the gene folA, encoding dihydrofolate reductase (DHFR), proved to target DHFR in vivo and were competitive inhibitors in vitro.

High throughput screening identifies novel inhibitors of Escherichia coli dihydrofolate reductase that are competitive with dihydrofolate.

This communication describes the high-throughput screen of a diverse library of 50,000 small molecules against Escherichia coli dihydrofolate reductase to detect inhibitors. Sixty-two compounds were identified as having significant inhibitory activity against the enzyme. Secondary screening of these revealed twelve molecules that were competitive with dihydrofolate, nine of which have not been previously characterized as inhibitors of dihydrofolate reductase. These novel molecules ranged in potency (K(i)) from 26 nM to 11 microM and may represent fresh starting points for new small molecule therapeutics directed against dihydrofolate reductase.

CTP:glycerol 3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the cytidylyl transfer via an ordered Bi-Bi reaction mechanism with micromolar K(m) values.

CTP:glycerol 3-phosphate cytidylyltransferase catalyzes the formation of CDP-glycerol, an activated form of glycerol 3-phosphate and key precursor to wall teichoic acid biogenesis in Gram-positive bacteria. There is high sequence identity (69%) between the CTP:glycerol 3-phosphate cytidylyltransferases from Bacillus subtilis 168 (TagD) and Staphylococcus aureus (TarD). The B. subtilis TagD protein was shown to catalyze cytidylyltransferase via a random mechanism with millimolar K(m) values for both CTP and glycerol 3-phosphate [J. Biol. Chem. 268, (1993) 16648] and exhibited negative cooperativity in the binding of substrates but not in catalysis [J. Biol. Chem. 276, (2001) 37922]. In the work described here on the S. aureus TarD protein, we have elucidated a steady state kinetic mechanism that is markedly different from that determined for B. subtilis TagD. Steady state kinetic experiments with recombinant, purified TarD employed a high-performance liquid chromatography assay developed in this work. The data were consistent with a ternary complex model. The K(m) values for CTP and glycerol 3-phosphate were 36 and 21 microM, respectively, and the k(cat) was 2.6 s(-1). Steady state kinetic analysis of the reverse (pyrophosphorylase) reaction was also consistent with a ternary complex model. Product inhibition studies indicated an ordered Bi-Bi reaction mechanism where glycerol 3-phosphate was the leading substrate and the release of CDP-glycerol preceded that of pyrophosphate. Finally, we investigated the capacity of S. aureus tarD to substitute for tagD in B. subtilis. The tarD gene was placed under control of the xylose promoter in a B. subtilis 168 mutant defective in tagD (temperature-sensitive, tag-12). Growth of the resulting strain at the restrictive temperature (47 degrees C) was shown to be xylose-dependent.