title: 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 μg/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. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains. In vitro characterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrate K(m) and a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy.
authors.affiliation: Achillion Pharmaceuticals, New Haven, Connecticut, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Podos
authors.forename: Jane A
authors.initials: JA
authors.lastname: Thanassi
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pucci
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D011088:DNA Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011088:DNA Ligases; C120640:DNA ligase (NAD)