Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport

Abstract

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.

Constructing and deconstructing the bacterial cell wall

“…Intimate structural synergy is now recognized between the peptidoglycan and the teichoic acids of the Gram‐positive bacteria, and between the peptidoglycan and the outer membrane of the Gram‐negative bacteria . Indeed, new opportunities for antibiotic discovery have been identified that interfere with teichoic acid integration into the cell wall of Gram‐positive bacteria and with respect to lipopolysaccharide transport in Gram‐negative bacteria . Within this universe of opportunity, we exemplify our own efforts toward the mechanistic and structural understanding of key enzymes of the bacteria with roles in cell envelope biosynthesis and antibiotic resistance.…”

“…77,78 Indeed, new opportunities for antibiotic discovery have been identified that interfere with teichoic acid integration into the cell wall of Gram-positive bacteria 69,[79][80][81][82][83][84] and with respect to lipopolysaccharide transport in Gram-negative bacteria. [85][86][87][88][89][90] Within this universe of opportunity, we exemplify our own efforts toward the mechanistic and structural understanding of key enzymes of the bacteria with roles in cell envelope biosynthesis and antibiotic resistance.…”

Rapid Intrabacterial Hydrolysis for Activating Antibiotics against Gram-negative Bacteria

“…After synthesized the analogs ( Figure 3B), we examined their activities against E. coli ( Figure 3C). Although the replacement of succinate by cyclohexane-1,2-dicarboxylic acid produces a prodrug (6) with rather low activity (MIC > 200 M), the conjugation of GG to 6 significantly increases the activity of the prodrug (7, MIC = 50 M). Because the ciprofloxacin derivative, with the diglycine conjugated at the carboxylic acid of ciprofloxacin, hardly shows activity against E. coli (Scheme S5 and Figure S7), we made a ciprofloxacin derivative (8), which has 2-hydroxyacetic acid attach to the piperazine end of ciprofloxacin, for conjugating GG to ciprofloxacin.…”

“…4 Thus, there is an urgent need for developing new antimicrobial approaches against MDR bacterial pathogens. Besides investigating novel agents against new targets in bacteria [6][7][8][9] via previously unexplored mechanisms, another strategy is to enhance the efficacy and safety of the existing antibiotics via drug combinations or increasing specificity. For example, Kahne et al demonstrated that the use of novobiocin analogs greatly lower the dose of polymyxins against Gram-negative bacteria via inhibiting DNA gyrase, binding LptB, and disrupting outer membrane.…”

“…For example, Kahne et al demonstrated that the use of novobiocin analogs greatly lower the dose of polymyxins against Gram-negative bacteria via inhibiting DNA gyrase, binding LptB, and disrupting outer membrane. 6 Nolan et al reported that the conjugate of a synthetic siderophore and ciprofloxacin affords excellent selectivity against a pathogenic E. coli strain. 10 Yan et al reported nanoaggregates of ciprofloxacin for inhibiting E. coli.…”