Structural and biological studies on bacterial nitric oxide synthase inhibitors

Abstract

Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

Keywords: antibiotics; crystallography.

Fig. 1.

NOS inhibitors and their effect on B. subtilis survival. Bacterial survival of B. subtilis WT and Δnos strains decreases in the presence of 1.25 mM ACR and NOS inhibitors. The concentrations were 500 μM for l-NNA and 1 and 250 μM for 2, indicating that 2+ACR is more effective at inhibiting growth at 250 μM inhibitor than l-NNA+ACR at 500 μM inhibitor. Error bars represent the mean ± the SEM of at least three replicates. Student t test gives ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 2.

The effect of ACR and compounds 1 and 2 on bacterial growth in both WT and Δnos B. subtilis: (A) WT + compound 1, (B) WT + compound 2, (C) Δnos + compound 1, and (D) Δnos + compound 2. Error bars represent the mean ± the SEM of three replicates.

Fig. 3.

Active site structure of (A) bsNOS-1 complex with 2F₀-F_c electron density map contoured at 1.0σ, (B) bsNOS-2 complex with 2F₀-F_c electron density contoured at 1.0σ, (C) Rattus norvegicus nNOS-1 complex with the 2Fo-Fc electron density map contoured at 1.0σ, and (D) nNOS-2 complex.