TP0586532, a non-hydroxamate LpxC inhibitor, reduces LPS release and IL-6 production both in vitro and in vivo

Abstract

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an essential enzyme in the biosynthesis of Lipid A, an active component of lipopolysaccharide (LPS), from UDP-3-O-acyl-N-acetylglicosamine. LPS is a major component of the cell surface of Gram-negative bacteria. LPS is known to be one of causative factors of sepsis and has been associated with high mortality in septic shock. TP0586532 is a novel non-hydroxamate LpxC enzyme inhibitor. In this study, we examined the inhibitory effect of TP0586532 on the LPS release from Klebsiella pneumoniae both in vitro and in vivo. Our results confirmed the inhibitory effect of TP0586532 on LPS release from the pathogenic bacterial species. On the other hand, meropenem and ciprofloxacin increase the level of LPS release. Furthermore, the effects of TP0586532 on LPS release and interleukin (IL)-6 production in the lung were determined using a murine model of pneumonia caused by K. pneumoniae. As observed in the in vitro study, TP0586532 showed the marked inhibitory effect on LPS release in the lungs, whereas meropenem- and ciprofloxacin-treated mice showed higher levels of LPS release and IL-6 production in the lungs as compared to those in the lungs of vehicle-treated mice. Moreover, TP0586532 used in combination with meropenem and ciprofloxacin attenuated the LPS release and IL-6 production induced by meropenem and ciprofloxacin in the lung. These results indicate that the inhibitory effect of TP0586532 on LPS release from pathogenic bacteria might be of benefit in patients with sepsis.

Fig. 1

The chemical structure of TP0586532

Fig. 2

Efficacy of bacterial killing and inhibitory effects on LPS release in the cultures of K. pneumoniae 4124. Viable cell counts in the cultures treated with TP0586532 (a), meropenem (b), ciprofloxacin (c), and ceftazidime (d). LPS levels in the cultures treated with TP0586532 (e), meropenem (f), ciprofloxacin (g), and ceftazidime (h). Each symbol represents the mean and SEM (n = 3–4). 0.5 MIC (□), 1 MIC (♦), 2 MIC (○), and no agent (–)

Fig. 3

Efficacy of bacterial killing and inhibitory effects on LPS release and IL-6 production in the lungs in a murine model of pneumonia caused by K. pneumoniae 4124. Viable cell counts (a), LPS levels (b), and IL-6 levels (c) are shown. Each symbol represents the mean and SEM (n = 6). TP0586532 (100 mg kg⁻¹ dose⁻¹); MEM, meropenem/cilastatin (25 mg kg⁻¹ dose⁻¹); CIP, ciprofloxacin (3 mg kg⁻¹ dose⁻¹). Statistical comparisons of LPS release and IL-6 production were performed by the Wilcoxon test (vs. vehicle in the same time point, *: p < 0.05, **: p < 0.01)

Fig. 4

Efficacy of bacterial killing and inhibitory effects of TP0586532 in combination on LPS release induced by other antibiotics in the cultures of K. pneumoniae 4124. Viable cell counts in the cultures treated with meropenem plus TP0586532 (a), ciprofloxacin plus TP0586532 (b), ceftazidime plus TP0586532 (c), and meropenem plus ciprofloxacin (d). LPS levels in the cultures treated with meropenem plus TP0586532 (e), ciprofloxacin plus TP0586532 (f), ceftazidime plus TP0586532 (g), and meropenem plus ciprofloxacin (h). Each symbol represents the mean and SEM (n = 3). Meropenem, ciprofloxacin, or ceftazidime alone (□), combined with 0.25 MIC TP0586532 or ciprofloxacin (♦), combined with 0.5 MIC TP0586532 or ciprofloxacin (○), combined with 1 MIC TP0586532 or ciprofloxacin (▲), and no agent (–)

Fig. 5

Efficacy of bacterial killing and inhibitory effects of TP0586532 in combination on LPS release and IL-6 production induced by other antibiotics in the lungs of a murine model of pneumonia caused by K. pneumoniae 4124 at 6 h after inoculation. Viable cell counts (a), LPS levels (b), and IL-6 levels (c) are shown. Each symbol represents the mean and SEM (n = 6). MEM, meropenem/cilastatin; CIP, ciprofloxacin. Statistical comparisons of LPS release and IL-6 production were performed by the Steel test (*: p < 0.05, TP0586532 vs. vehicle, meropenem/cilastatin plus TP0586532 vs. meropenem/cilastatin alone, ciprofloxacin plus TP0586532 vs. ciprofloxacin alone)