Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound

Abstract

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ "hybrids" offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, "251D," were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of Staphylococcus aureus resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens.

FIG. 1.

Structures of the hybrid compound, 251D, its anilinouracil component, 3-(4-hydroxybutyl)-6-(3-ethyl-4-methylanilino)uracil (HB-EMAU), and its fluoroquinolone component, {1-cyclopropyl-6,8-difluoro-7-[1-(3-methyl)piperazinyl]-4-oxo-1,4-dihydroquinoline}-3-carboxylic acid (340E).

FIG. 2.

Double reciprocal plots demonstrating the effect of varying dGTP concentrations on inhibition of pol IIIC. (A) 251D; (B) HB-EMAU. Inhibitor concentrations: ♦, no inhibitor; ▪, 3× K_i; ▴, 10× K_i; ×, 30× K_i.

FIG. 3.

Effect of compounds on macromolecule synthesis in B. subtilis. Assays were performed using radiolabeled precursor ([³H]adenine for DNA and RNA and [³H]leucine for protein synthesis measurements) and B. subtilis strain BD54 as described in Materials and Methods using compounds at 5× or 1× their MICs as indicated. (A) DNA synthesis using compounds at 5× their respective MICs; (B) DNA synthesis using compounds at 1× their respective MICs; (C) RNA synthesis using compounds at 5× their respective MICs; (D) protein synthesis using compounds at 5× their respective MICs. DMSO control, filled diamonds; HB-EMAU, open diamonds; 340E, filled squares; 251D, open squares; rifampin, filled triangles; chloramphenicol, open triangles.

FIG. 4.

Time kill assays showing the bactericidal activities of 251D (A), HB-EMAU (B), and 340E (C). S. aureus (ATCC 13709), filled symbols; E. faecium (ATCC 19434), open symbols; control, diamonds; 8× MIC, squares; 4× MIC, triangles; 2× MIC, circles.