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

Abstract

We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD ؉ -dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [K i ] ‫؍‬ 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC 50 ] ‫؍‬ 28 nM) or its isolated adenylation domain (IC 50 ‫؍‬ 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistant S. aureus (MSSA and MRSA) strains (MIC ‫؍‬ 1.0 g/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 ؋ 10 ؊7 ) of high-level resistant mutants (MIC > 64) with associated ligA lesions. There were no observable effects on growth rate in these mutants. N ADϩ -dependent DNA ligase (LigA) has been identified by numerous authors as an attractive potential target for broadspectrum antibacterial chemotherapy (7,23). 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).DNA ligation activities are essential for multiple DNA processes in replication and repair, including the joining of Okazaki fragments into a continuous strand during chromosomal DNA replication. Enzymatically, DNA ligation proceeds via three successive adenylyl transfer steps (Fig. 1) (32): first, DNA-independent covalent adenylation of the catalytic lysine by the NAD ϩ substrate; second, adenylyl transfer to the free 5= phosphate at the nicked DNA ligation site; and third, the covalent sealing of the DNA nick with concomitant AMP release. Biochemical functions of distinct domains in the modular enzyme structure have been assigned to particular reaction steps. The DNA-independent adenylyl transfer activity resides within the amino-terminal adenylation domain, which comprises an amino-terminal Ia region that is specific to NAD ϩ -dependent DNA ligases and a nucleotidyl transferase (NTase) region that is universal among DNA and RNA ligases. The subsequent coupling of adenylation to DNA ligation depends upon downstream DNA-binding domains, which include an oligonucleotide-binding fold (OB fold) and a helix-hairpin-helix (HhH) domain. Structural studies of the adenylation domain have revealed conformational transitions that accompany the adenylation cycle (8), and structural study of the full-length enzyme bound to DNA-adenylate has identified specific contacts between the DNA-binding domains and the DNA duplex substrate near the nicked ligation site (20).Numerous LigA inhibitors have been reported to date, including ary...

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

“…6.5.1.2), although additional ATP-dependent DNA ligases are found in some species of eubacteria. Because of this distinction, the essentiality of the enzyme for DNA replication, and the low sequence conservation between bacterial and mammalian DNA ligases, NAD + -dependent DNA ligase has recently been investigated as a target for novel antibacterial drug discovery [ 4 - 6 ].…”

Biochemical and structural characterization of DNA ligases from bacteria and archaea

“…Biochemical and structural studies have made significant progress in identifying compounds that inhibit NDLs. However, the prospects of developing such compounds as antimicrobial drugs remains challenging, particularly since it is likely that effective drugs will have to inhibit NDLs very efficiently, with very good discrimination against ADLs [ 21 , 96 ]. Recent comparative analysis of co-factor and inhibition experiments raises the possibility that useful inhibition may be obtained due to targeting of the significant conformational changes that occur within DNA ligases during their catalytic cycle.…”

“…These compounds were then refined to generate analogues that had improved activity, selectivity, solubility and other factors that make the compounds more ‘druggable’ [ 53 , 74 – 76 , 92 – 98 ]. Importantly, some of these studies also highlighted the challenges associated with developing such compounds as antimicrobial drugs [ 96 ].…”

Synthesis, optical and photoelectrical characterizations of the novel 10-chloro-6H,8H-dichromeno[2,3-b:3′,4′-e]pyridine-6,8-dione (CDPD) and its photodiode application

“…Among these materials, chromeno [2,3-b]pyridines is one of the most promising organic structure for possible applications because they exhibit various biological activities including pharmacological [1], antiplatelet [2], antiallergic [3], antiangiogenic [4], antirheumatic [5], antibacterial [6], anti-inflammatory and analgesic [7]. Friedländer condensation of 2-aminochromone-3-carboxaldehyde is a well-known reaction for preparation of chromeno [2,3-b]pyridine derivatives [8][9][10].…”