Identification and characterization of an inhibitor specific to bacterial NAD⁺‐dependent DNA ligases

Abstract

DNA ligase is an enzyme essential for DNA replication, repair and recombination in all organisms [1][2][3]. The enzyme catalyzes the formation of phosphodiester bonds at single-strand breaks in duplex DNA during these physiological processes [1][2][3]. There are two classes of DNA ligases, identified on the basis of their cofactor requirements. Bacterial DNA ligases involved in DNA replication utilize NAD + as a cofactor, whereas mammalian and viral DNA ligases require ATP for activity [1][2][3]. Biochemical studies of bacterial DNA ligase (NAD + -dependent), and viral and human DNA ligases (ATP-dependent) have established that the mechanism of the reactions catalyzed by the two different groups of DNA ligases is similar [1][2][3]. The reaction mechanism consists of three reversible steps: (a) adenylation of the enzyme by transferring the adenylate group from NAD + or ATP to the e-NH 2 group of Lys in the enzyme with the release of nicotinamide + and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD + -dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.Abbreviations DDPP, 2,4-diamino-7-dimethylamino-pyrimido [4,5-d]pyrimidine; FRET, fluorescence resonance energy transfer; NMN, nicotinamide mononucleotide.

Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

“…LigA is well conserved among eubacterial species, is architecturally and biochemically distinct from the ATP-dependent DNA ligases of eukaryotic cells, and has been found to be essential for bacterial viability wherever examined (13,14,15,17,31). Moreover, the DNA ligation reaction has been dissected mechanistically, mutationally, and structurally (8,20,25,26,33,34,35), and screening assays have been reported for the complete reaction cycle and for individual component steps (2,11,18).…”

“…Numerous LigA inhibitors have been reported to date, including arylamino acids, such as chloroquine (4), glycosyl ureides and glycosylamines (27,28), tetracyclic indoles (29), a pyrimidopyrimidine inhibitor (17), substituted adenosine analogs (19,30), and the pyridochromanones (1). Pyridochromanones were identified by high-throughput screening as potent competitive inhibitors of DNA ligation by LigA from Staphylococcus aureus (50% inhibitory concentration [IC 50 ] Յ 0.9 M) (1).…”

Discovery of a compound that acts as a bacterial PyrG (CTP synthase) inhibitor

“…In fact, a number of studies in pharmaceutical companies and in academia have developed screening platforms based on enzymic assays and structure-based drug design. As a result, inhibitors that target enzymes such as N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), NAD + -dependent DNA ligases (LigA), enoyl-(acyl-carrier-protein) reductase (FabI) and met tRNA synthase, as well as those that inhibit cell-wall synthesis, have been discovered and validated for their antimicrobial activity (Silver, 2003;Payne et al, 2007;Meier et al, 2008;Pereira et al, 2009).…”

Novel Bacterial NAD + -Dependent DNA Ligase Inhibitors with Broad-Spectrum Activity and Antibacterial Efficacy In Vivo

“…In contrast, the ATP-dependent DNA ligases of mammals, fungi, and viruses form a loosely defined cluster of associated enzymes (32,41). The bacterial NAD ϩ -dependent DNA ligases are essential for viability in all Gram-positive and Gram-negative bacteria tested to date, including Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella enterica serovar Typhimurium, and Escherichia coli (12,18,23,31,33). NAD ϩ -dependent DNA ligase has attracted interest as a prospective broad-spectrum antibacterial target because it is indispensable for DNA replication, conserved among bacterial pathogens, and distinctly different from the eukaryotic DNA ligases (5,6,9,10,15,23,25).…”

“…The bacterial NAD ϩ -dependent DNA ligases are essential for viability in all Gram-positive and Gram-negative bacteria tested to date, including Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella enterica serovar Typhimurium, and Escherichia coli (12,18,23,31,33). NAD ϩ -dependent DNA ligase has attracted interest as a prospective broad-spectrum antibacterial target because it is indispensable for DNA replication, conserved among bacterial pathogens, and distinctly different from the eukaryotic DNA ligases (5,6,9,10,15,23,25). X-ray crystal structures have facilitated a better understanding of substrate binding and the catalytic mechanism of DNA ligases (14,16,28; S. Lahiri and S. D. Mills, U.S. patent application 2008/0262811).…”