Biological characterization of novel inhibitors of the gram-positive DNA polymerase IIIC enzyme

Abstract

Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 microg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired.

FIG. 1.

Important pharmacophoric elements of a generic AU.

FIG. 2.

SDS-PAGE (7.5% polyacrylamide gel) analysis of the purification of His-tagged Pol IIIC from S. aureus 133 with Coomassie blue staining. Fractions that eluted from a Ni-NTA-agarose column by using 100 mM imidazole are shown. Lane 1, uninduced control; lanes 2 and 3, expressed Pol IIIC (∼166-kDa) protein in the presence of 1 mM IPTG; lane 4, molecular mass marker.

FIG. 3.

Time course of S. aureus Pol IIIC activity. The assay was performed as described in Materials and Methods in the absence of dGTP, and bioluminescence is shown in relative light units (RLU) per minute.

FIG. 4.

Inhibition of the S. aureus Pol IIIC enzyme by TMAU with an allyl side chain substitution at N-3. An IC₅₀ of 0.6 μM was determined. Bioluminescence of ∼187,000 relative light units/min was measured for the uninhibited Pol IIIC enzyme.

FIG. 5.

Killing curve of EMAIPU (compound 1) (MIC, 8 μg/ml) for S. aureus 133. The compound was tested, as indicated, at half (0.5×), two times (2×), and eight times (8×) its MIC.

FIG. 6.

Mode-of-action studies for EMAIPU (compound 1) by the incorporation of radiolabeled metabolic precursors (as indicated) into whole cells of B. subtilis 168 (MIC, 2 μg/ml). Labeling was performed at one-fourth the MIC, the MIC, and four times the MIC. —, control; ▴, EMAIPU at 0.5 μg/ml; •, EMAIPU at 2 μg/ml; ▪, EMAIPU at 8 μg/ml.

FIG. 7.

Development of S. aureus 133 resistance to EMAIPU (compound 1; ▪), determined as described in Material and Methods, in comparison to the development of resistance to rifampin (•) and moxifloxacin (▴).