A naturally inspired antibiotic to target multidrug-resistant pathogens

Abstract

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections^1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin³. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens⁴. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.

Extended Fig. 1 |. Proposed macolacin biosynthetic pathway.

The predicted biosynthetic scheme for macolacin based on detailed bioinformatic analysis of the mac BGC is depicted.

Extended Fig. 2 |. Phylogenetic trees constructed from A-domain sequences associated with complete colistin and macolacin BGC.

Phylogenetic trees constructed from A domain sequences associated with complete colistin and macolacin A BGC. a) A1 domain; b) A3 domain; c) A7 domain and d) A10 domain. Each A-domain sequence was extracted from the polymyxin-like BGCs was then aligned together with known characterized polymyxin BGCs (e.g., MIBIG IDs: BGC0000408, BGC0001192, BGC0001153) using the MUSCLE alignment software. The resulting phylogenetic tree was visualized using iTOLv5 software. Red color represents hits in polymyxin clade. Blue color represents hits in macolacin clade.

Extended Fig. 3 |. Structures of all synthetic macolacin derivates.

Structural differences compared to macolacin are depicted in blue.

Extended Fig. 4 |. Cytotoxicity and pharmacokinetic evaluation of macolacin and biphenyl-macolacin.

a) Cytotoxicity of macolacin and biphenyl-macolacin against HEK293. Data are presented as means ± SD. n=3 technical replicates. b) Pharmacokinetic assessment of macolacin and biphenyl-macolacin. Total plasma concentrations of macolacin, biphenyl-macolacin or colistin versus time after administration of a single subcutaneous dose (10 mg/kg) to neutropenic mice. n=2 biologically independent mice. Data are presented as mean of two independent assays. c) The level of serum NGAL in colistin or biphenyl-macolacin treated mice. Significant differences between groups were determined by one-way analysis of variance (ANOVA) (*P<0.05) (n=6 biologically independent mice). Data are presented as means ± SD. Vehicle vs. Colistin, P value=0.0069; Vehicle vs. Biphenyl-macolacin, P value=0.0104; Colistin vs. Biphenyl-macolacin, P value=0.9773.

Fig. 1 |. Discovery of macolacin

a) Extensive use of antibiotics has resulted in the increased appearance of antibiotic resistant pathogens in the clinical setting. In nature, a similar phenomenon is likely occurring in response to the natural production of antibiotics by bacteria. Nature however is not static, and the response to the development of resistance by some bacteria will be the selection of BGCs that encode variants of antibiotics that are capable of circumventing common resistance mechanisms. Here we use BGC guided chemical synthesis to identify a naturally occurring analog of colistin that is active against resistance encoded by the recently identified and now globally distributed mcr-1 gene. b) Structure of colistin. c) The mac gene cluster. The domain structure encoded by NRPS genes macA-macE. NRP synthesis is initiated from the Cs (condensation starter) domain. C (condensation), A (adenylation), and T (thiolation) domains make a minimal NPRS module that extends the growing NRP by one amino acid. Inclusion of an epimerization (E) domain in the module alters the stereochemistry of the T domain bound amino acid. The TE (thioesterase) domain releases the mature NRP from the final T domain. d) Comparison of the predicted macolacin decapeptide to decapeptides found in characterized polymyxin (poly) structures. The number of amino acids that each peptide differs from the consensus peptide derived from all known polymyxin structure is shown (Delta). e) Chemical synthesis of macolacin.

Fig. 2 |. Antibacterial activity of macolacin

a) Fold increase in MIC for polymyxin, colistin and macolacin upon introduction of the mcr-1 resistance gene into K. pneumoniae or A. baumannii. b) Disc diffusion assays (10 μg of antibiotic/disk) against K. pneumoniae and A. baumannii with or without the mcr-1 resistance gene. c) MIC of colistin or macolacin against K. pneumoniae and A. baumannii (n=2) upon addition of different cell wall components to the culture media. d) Growth curves (n=3) for cultures of A. baumannii (blue) as well as A. baumannii in the presence of either the LpxC inhibitor CHIR-090 (green), one of three different antibiotics (colistin, macolacin or kanamycin) (red) or both CHIR-090 and an additional antibiotic (purple).

Fig. 3 |. In vitro and in vivo activity of biphenyl-macolacin.

a) Among the macolacin analogs we synthesized with different lipid substituents, biphenyl-macolacin was the most potent analog against mcr-1 containing pathogens. b) MIC values of biphenyl-macolacin, macolacin and colistin against colistin sensitive and resistant pathogens (n=3 in duplicate). CFU counts from a neutropenic thigh infection model using A. bauamnnii-SM1536-mcr-1 (c) or A. baumannii-0301-mcr-1 (d). Two hours after mice were exposed to the pathogen, mice were subcutaneously given biphenyl-macolacin (20 mg/kg), colistin (20 mg/kg) or vehicle alone (0.9% saline) at 6 hour intervals. After 24 hours colony forming units (CFUs) were determined from homogenized thigh tissue samples. Significant differences between groups were analyzed by one-way analysis of variance (ANOVA) (****P<0.0001) (n=4 mice, n=8 thighs). Mean CFU counts and SD are shown.