In Vitro Antimicrobial Activities of Novel Anilinouracils Which Selectively Inhibit DNA Polymerase III of Gram-Positive Bacteria

Abstract

The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3-iodo-4-methylanilino]uracil) and EMAU (6-[3-ethyl-4-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.New antibacterial agents are needed to combat the multiply resistant gram-positive bacteria endemic in modern health care facilities (15). The 6-anilinouracils (AUs) illustrated in Fig. 1 are selective inhibitors of DNA polymerase III-c2 (pol III), an enzyme product of the polC gene (2,8,12). This enzyme is essential for the replication of the chromosome in gram-positive bacteria (7,9,17) and is found in low-GϩC-content eubacteria, including staphylococci, enterococci, streptococci, Listeria species, Bacillus species, and clostridia (1, 8). Briefly, the AUs, inhibitors of the DNA pol III enzyme, act through their capacity to mimic the guanine moiety of dGTP by forming three hydrogen bonds with cytosine at one of the two active domains (6,7,14). This leaves the second active site on the inhibitor, the aryl domain, available to bind to DNA pol III, which sequesters the enzyme into a nonproductive complex with template primer DNA (19).Structure-activity relationships of these AUs have been described previously (18, 19). The prototypic AUs, which have either weak antimicrobial activities or unacceptably low aqueous solubility (3-6, 13, 17), have now been substituted in their N3 positions and aryl rings to produce a series of more potent and more soluble molecules (13,16,19). The latest generation of these soluble forms (19) includes the N 3 -hydroxybutyl (HB) and N 3 -methoxybutyl (MB) derivatives of 6-[3Ј-ethyl-4Ј-methylanilino]uracil (EMAU) and 6-[3Ј-iodo-4Ј-methylanilino]uracil (IMAU) shown in Fig. 1. In this study, we describe the in vitro activities of HB-IMAU, HB-EMAU, MB-IMAU, and MB-EMAU against staphylococci and enterococci, bacteria that are pathogenic in humans and are difficult to treat with currently available and investigational antimicrobial agents.(This work was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 1999 [J. S. Daly, T. Giehl, N. C. Brown, C.Zhi, G. E. Wright, and R. T. Ellison III, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1808, 1999].) Materials and methods. Bacterial strains used in this study were un...

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N 3 -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

“…The AU compounds are effective against many pathogenic strains of S. aureus and Enterococcus species, including antibioticresistant organisms (9,20). In addition, they are protective against lethal staphylococcal infections in vivo (20).…”

“…First, they are highly specific inhibitors of Pol IIIC but inhibit DNA polymerase IIIE from Gr ϩ organisms only at a low level and do not inhibit Pol IIIE from Gr Ϫ organisms or polymerase alpha from mammalian sources (20). Second, they are potent antibacterial agents against all Gr ϩ organisms tested, including a number of antibiotic-resistant clinical isolates such as MRSA strains and VRE (9,20). Third, they are bactericidal at concentrations one to four times their MICs (9).…”

“…Second, they are potent antibacterial agents against all Gr ϩ organisms tested, including a number of antibiotic-resistant clinical isolates such as MRSA strains and VRE (9,20). Third, they are bactericidal at concentrations one to four times their MICs (9). Finally, they exhibit selectivity for growth of Gr ϩ pathogens versus mammalian cells, with 50% inhibitory concentrations for a variety of mammalian lines be-ing from 8-to 64-fold greater than the MICs for the pathogens (Microbiotix, Inc., unpublished observations).…”

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

“…As a consequence, the inhibitor sequesters the enzyme into a nonproductive, reversible ternary complex with the template primer (25). The increased inhibitory activities of AU compounds against Pol IIIC and the growth of gram-positive bacteria have been described for N-3-substituted anilinouracils (9,25). The enhanced antibacterial activities of the compounds with the N-3 side chain alkyl substituents compared to those of N-3-unsubstituted molecules have been described (9,25).…”

“…The identification of novel classes of antibacterial substances that inhibit a new or unexploited target is expected to facilitate novel therapeutic approaches for the treatment of infections caused by multiresistant pathogens. 6-Anilinouracils (AUs) have been described in the literature (1,9,24,25,27,28,31) and in patent applications (3,7) as a novel antibacterial class showing selective inhibition of DNA polymerase IIIC (Pol IIIC; formerly Pol III), the enzyme required for the replication of chromosomal DNA in gram-positive organisms with low GϩC contents (29).…”