Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo

Abstract

DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

FIG. 1.

Chemical structures of the adenosine analogs discussed in this study. The 2-position of the adenine ring and the 5′ position of the ribose are indicated for compound 1.

FIG. 2.

Enzyme mode of inhibition for compound 1. The activity of H. influenzae LigA was measured using a FRET assay in the presence of rising concentrations of compound 1 (0 to 2.6 μM) and NAD⁺ (0.5 to 3.5 μM), with a fixed concentration (60 nM) of the nicked DNA substrate. As represented in the double-reciprocal plot, the resulting data are consistent with competitive binding between compound 1 and NAD⁺. The apparent K_i for compound 1 was determined to be 110 nM by nonlinear regression analysis of the data.

FIG. 3.

(A) X-ray cocrystal structure of compound 1 bound to the AMP-binding site of H. influenzae LigA. Predicted hydrogen bonds between the inhibitor and the enzyme are shown as dashed lines. Enzyme residues proposed to make key interactions with the compound are labeled. (B) Surface representation of the hydrophobic tunnel of the binding pocket containing compound 1. The surface of L82 (corresponding to L75 in S. pneumoniae LigA) is shown in cyan.

FIG. 4.

Time-kill study. Shown are the effects of increasing concentrations of compound 4 (multiples of the MIC) on the viability of S. pneumoniae (measured as CFU/ml).

FIG. 5.

Accumulation of Okazaki fragments (OF) in [methyl-³H]thymidine-labeled DNA isolated from H. influenzae ARM158 cells treated with increasing concentrations of CIP (0, 2, 8, and 31 ng/ml) (A), compound 2 (0, 16, 250, and 1,000 ng/ml) (B), or compound 4 (0, 63, 500, and 2,000 ng/ml) (C). Symbols represent the absence of the compound (open circles) or increasing concentrations from lowest to highest (filled circles, open squares, and filled squares, respectively). The amount of radioactivity (cpm) was measured in 23 fractions (x axis) obtained from alkaline sucrose gradients. Okazaki fragments typically appeared in fraction 4, whereas high-molecular-weight DNA peaked in fractions 9 to 12.

FIG. 6.

In vivo activity of compound 4 on infection. (A) S. aureus ARC516 in the thighs of neutropenic mice; (B) S. pneumoniae D39 in the lungs of immunocompetent mice. Results are plotted as means; the error bars represent the standard errors of the means. Dosing regimens are given in milligrams per kilogram per day. q.i.d., 4 times per day; q3, every 3 h.