New compound effective against bacterial species resistant to multiple classes of existing antibiotics

By Dr. Chinta SidharthanReviewed by Danielle Ellis, B.Sc.Jan 4 2024

Recent studies by Zampaloni et al. and Pahil et al. published in the journal Nature describe a novel method of inhibiting the growth of Gram-negative bacteria such as Acinetobacter using antibiotics consisting of macrocyclic peptides that target the bacterial protein bridge machinery that transports lipopolysaccharides from the cytoplasm to the outer membrane.

Background

The amphipathic lipopolysaccharides in the outer leaflet of the asymmetric outer membrane bilayer of Gram-negative bacteria block antibiotic entry, making the treatment of bacterial infections involving Gram-negative bacteria difficult. Furthermore, the development of antibiotic resistance in bacteria, especially Gram-negative bacteria such as Acinetobacter baumannii, is a rapidly increasing global health concern since antibiotic-resistant bacterial infections are becoming increasingly common among hospitalized and critically ill patients.

The lipopolysaccharide is synthesized inside the bacterial cell in the inner membrane, transported across the cell membrane, and assembled in the outer leaflet. The transportation of lipopolysaccharides occurs with the help of LptB₂FGC, a subcomplex in the inner membrane that enlists adenosine triphosphate (ATP) hydrolysis and a protein bridge to extract lipopolysaccharides from the inner membrane and transport it to the outer membrane. Targeting this transportation complex could effectively inhibit the lipopolysaccharide biosynthesis, making the Gram-negative bacteria susceptible to antibacterial activity.

About the studies

The two studies described the use of macrocyclic peptides that target the LptB₂FGC transportation machinery in A. baumannii and A. baylyi to prevent the transport of bacterial lipopolysaccharide from the inner membrane and, consequently, its assembly on the outer membrane. In the study by Zampaloni et al., the team of researchers identified and optimized a novel class of antibiotics called tethered macrocyclic peptides that inhibit the transport of lipopolysaccharide across the bacterial membrane. They also selected an antibiotic candidate, Zosurabalpin, for clinical testing by conducting in vitro experiments and in vivo testing in animal models.

The study used whole-cell phenotyping screening to test close to 45,000 macrocyclic peptides against various strains of Gram-positive and Gram-negative pathogens that infect humans. Additionally, mouse models were used to assess whether selected macrocyclic peptides could treat infections caused by multidrug-resistant and Carbapenem-resistant A. baumannii strains. Furthermore, based on the tolerability results from the in vivo experiments in the mouse models, zwitterionic tethered macrocyclic peptides were explored to improve the potency and tolerability of the treatment.

The second study by Pahil et al. aimed to understand the molecular mechanisms through which Zosurabalpin inhibits the extraction of lipopolysaccharide across the membrane bilayer. For this, they used cryo-electron microscopy to determine the structure of the LptB₂FGC transporter of A. baylyi binding with three macrocyclic peptides, including Zosurabalpin. The study also used point mutants to understand whether modifications to the lipopolysaccharide structure would have an impact on the efficacy of Zosurabalpin in inhibiting the transport of the lipopolysaccharide.

The cryo-electron microscope structures of the three macrocyclic peptides bound to the complex containing LptB₂FGC transporter and lipopolysaccharide were also used to understand how effectively the three macrocyclic peptides — Zosurabalpin, RO7196472, and RO7075573 — bound to the lipopolysaccharide- LptB₂FGC complex. They also attempted to understand what structural differences between the three macrocyclic peptides contributed to the difference in binding efficacy.

Results

The results from the Zampaloni et al. study showed that the novel antibiotic class consisting of macrocyclic peptides was able to treat infections caused by Carbapenem-resistant A. baumannii in both in vitro and in vivo settings, exhibiting the ability to overcome the drug-resistance mechanisms developed by the bacteria. The drug also showed efficacy against pan-drug-resistant strains of Acinetobacter. A serum precipitation assay also helped optimize the physico-chemical properties of macrocyclic peptides, resulting in the development of the clinically testable drug Zosurabalpin.

Zosurabalpin exhibited favorable safety and pharmacokinetic profiles in the in vivo studies involving mouse models of infection, including efficacy against lung and thing infections and sepsis caused by Carbapenem-resistant A. baumannii strains. The scientists believe that Zosurabalpin is effective and safe enough to be tested in humans and reported that human clinical trials were underway to develop the drug for human use.

The Pahil et al. study further elucidated the molecular mechanism through which macrocyclic peptides such as Zosurabalpin successfully inhibit the transport of the lipopolysaccharide into the outer membrane. A combination of genetic, biochemical, and structural analyses showed that these macrocyclic peptides trap the lipopolysaccharide-LptB₂FGC transporter complex by recognizing and binding to a composite binding site consisting of the lipopolysaccharide and the LptB₂FGC transporter.

Conclusions

Overall, the findings from these studies reported that a novel class of antibiotics consisting of macrocyclic peptides has been shown through in vivo and in vitro experiments to effectively treat infections caused by multidrug-resistant and Carbapenem-resistant A. baumannii strains.

Furthermore, the results have laid the basis for human clinical trials to test the safety and efficacy of Zosurabalpin, a macrocyclic peptide, for the treatment of drug-resistant Acinetobacter infections in humans. These antibiotics treat the infection by trapping the lipopolysaccharide-LptB₂FGC transporter complex and preventing it from exporting the lipopolysaccharide to the outer membrane.