Simocyclinone D8, an inhibitor of DNA gyrase with a novel mode of action

Abstract

We have characterized the interaction of a new class of antibiotics, simocyclinones, with bacterial DNA gyrase. Even though their structures include an aminocoumarin moiety, a key feature of novobiocin, coumermycin A(1), and clorobiocin, which also target gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins. Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both Ca(2+)- and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Taken together, our results demonstrate that simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA. This is a novel mechanism for a gyrase inhibitor and presents new possibilities for antibacterial drug development.

FIG. 1.

Structures of simocyclinones and aminocoumarin drugs; the structure of ciprofloxacin is also shown for comparison.

FIG. 2.

Effects of simocyclinones D4 and D8 on DNA supercoiling by gyrase. (Top panel) Supercoiling in the presence of increasing concentrations of novobiocin (Novo), simocyclinones D4 and D8, and ciprofloxacin (CFX). NC, nicked circle; R, relaxed; SC, supercoiled. (Middle panel) Estimation of IC₅₀s (in parentheses) of simocyclinones D4 and D8 compared with those of novobiocin and ciprofloxacin. (Bottom panel) Effect of ATP concentration on the extent of inhibition. Samples containing the estimated IC₅₀s of the drugs were incubated in the presence of a range of ATP concentrations.

FIG. 3.

Effects of simocyclinones D4 and D8 on gyrase ATPase activity. (Top panel) The N-terminal ATPase domain of GyrB (GyrB43; 20 μM) was incubated with increasing concentrations of novobiocin and simocyclinones D4 and D8, and the rates of hydrolysis were determined and plotted as the residual ATPase activity against the drug concentration. (Bottom panel) Gyrase (A₂B₂; 70 nM) was incubated with increasing concentrations of simocyclinone D8 in the presence of linear pBR322 DNA (2.8 nM).

FIG. 4.

Effect of simocyclinone D8 on gyrase relaxation. Gyrase (60 nM) and supercoiled pBR322 DNA (6 nM) were incubated with increasing concentrations of novobiocin (Novo), simocyclinone D8, and ciprofloxacin (CFX) for 1 h at 37°C. NC, nicked circle; R, relaxed; SC, supercoiled.

FIG. 5.

Effect of simocyclinone D8 on DNA supercoiling by quinolone-resistant gyrase. The supercoiling activities of gyrase containing GyrA Trp⁸³ in the presence of increasing concentrations of ciprofloxacin (CFX), simocyclinone D8, and novobiocin (Novo) are indicated. NC, nicked circle; R, relaxed; SC, supercoiled.

FIG. 6.

Effect of simocyclinone D8 on Ca²⁺- and ciprofloxacin (CFX)-induced DNA cleavage by gyrase. Increasing concentrations of simocyclinone D8 (left-hand panels) were incubated in the presence of fixed concentrations of Ca²⁺ (4 mM) or ciprofloxacin (5 μM). Controls with novobiocin (Novo) are shown on the right. Gels were run in the presence of 1 μg of ethidium bromide per ml. C, control (no enzyme or drug); E, enzyme only; NC, nicked circle; L, linear; R, relaxed.

FIG. 7.

SPR sensorgram showing the interaction between gyrase and DNA in the presence of simocyclinone D8. A 5′-biotinylated 147-bp linear DNA fragment was immobilized onto the chip, and gyrase (20 nM) was allowed to flow across the chip in the absence and presence of 50 and 500 nM simocyclinone D8. Spikes at the beginning and end of the injection are due to the slight difference in the DMSO concentration between the sample and the running buffer. RU, resonance units

FIG. 8.

Binding of simocyclinone D8 to GyrA59 by isothermal titration calorimetry. The integrated data, after correction for the heat of ligand dilution as a function of the molar ratio, is presented as kilojoules per mole of injectant versus the molar ratio of drug/protein. The solid line shows the best fit obtained by least-squares regression by use of a one-site model. This analysis gave an approximate stoichiometry of 1:1.