Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U)

Abstract

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or,most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: (i) AZT was a substrate for E. coli thymidine kinase; (ii) spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and (iii) intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.Nucleoside antibiotics have been under investigation for many years (27). Some of the most clinically effective antiviral agents currently in use are purine or pyrimidine nucleoside analogs (24). For example, ribavirin, a synthetic nucleoside similar in structure to guanosine and inosine, has potent in vitro activity against a broad spectrum of viruses, including the epidemic respiratory viruses (3,25). Two effective inhibitors of bacteria are 9-,B-D-arabinofuranosyladenine and 2',3'-dideoxyadenosine. reported the lethality of the former to a purinerequiring strain of Escherichia coli B. In this organism, 9-4-D-arabinofuranosyladenine markedly inhibited DNA synthesis and had virtually no effect upon RNA synthesis. In addition, 2',3'-dideoxyadenosine was shown to be lethal to selected strains of E. coli by irreversibly inhibiting DNA synthesis in susceptible microorganisms (5, 28).As a result of screening synthetic compounds for potential antimicrobial activity, we have observed that compound BW A509U (3'-azido-3'-deoxythymidine, referred to as AZT in this paper; Fig. 1) has potent, bactericidal in vitro activity against various members of the family Enterobacteriaceae. This report describes the extent of the in vitro growthinhibiting activity of AZT and proposes a mechanism to explain its lethal properties. In addition, the antibacterial activity of AZT is discussed in light of the recent finding that this compound inhibits human T-cell lymphotropic virus type III/lymphadenopathy-assoc...

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

“…We assayed colony formation in the presence of low levels of the replication inhibitor AZT, which causes DNA chain termination during DNA synthesis (6) and is only expected to damage replicating cells. Null mutants in iraD exhibited dramatic hypersensitivity to AZT (Fig.…”

“…Phleomycin induces random single-or double-strand breaks in the backbone of DNA (5), whereas AZT blocks DNA synthesis, leading to single-strand gaps in the replication fork (6). One insertion mutant in iraD (previously an unknown gene, yjiD) was hypersensitive to phleomycin and AZT.…”

Validation of a High-Throughput Screening Assay for Identification of Adjunctive and Directly Acting Antimicrobials Targeting Carbapenem-Resistant Enterobacteriaceae

“…44 Incorporation of AZT into DNA results in chain termination and ultimately cell death. 44 Therefore, AZT also acts as an antibacterial agent, and previous studies have reported activity against Escherichia, Enterobacter, Klebsiella, Salmonella, and Shigella. [44][45][46][47] Our findings are therefore consistent with prior studies performed with a small number of carbapenem-susceptible Enterobacteriaceae strains.…”

“…42,43 However, AZT is also a substrate for bacterial thymidine kinase (TK). 44 TK, a critical part of the thymidine salvage pathway, catalyzes the first of three phosphorylation events that make thymidine or AZT competent for incorporation into DNA. 44 Incorporation of AZT into DNA results in chain termination and ultimately cell death.…”

“…44 TK, a critical part of the thymidine salvage pathway, catalyzes the first of three phosphorylation events that make thymidine or AZT competent for incorporation into DNA. 44 Incorporation of AZT into DNA results in chain termination and ultimately cell death. 44 Therefore, AZT also acts as an antibacterial agent, and previous studies have reported activity against Escherichia, Enterobacter, Klebsiella, Salmonella, and Shigella.…”