The rise of multidrug resistant (MDR) pathogens is an alarming trend that is being observed around the world. This has been met with a dwindling number of antibiotic options available to physicians to treat these life-threatening infections. Commonly used “last resort” antibiotics while powerful are becoming ineffective against MDR infections and carry the risk of toxic side-effects. Addressing the need for new antibiotics has been severely lacking and no new antibiotic classes have been developed against Gram-negative pathogens in the last 50 years.

Recently researchers in California modified macrocyclic lipopeptides called arylomycins generating potent activity against Gram-negative organisms. These compounds originally only had activity against Gram-positive bacteria. However, by harnessing the power of structure aided design and understanding some key principals of bacterial membrane permeation, they generated highly potent arylomycin molecules with activity against MDR Gram-negative bacteria, including A. baumannii and P. aeruginosa.

The arylomycin mechanism of action is through inhibition of the bacterial type I signal peptidase (SPase) called LepB. The LepB protein is a membrane bound protease and has been pursued as an antibiotic target for almost 20 years. The authors utilized structure aided design to modify the existing arylomycin molecule so that it would bind with greater affinity to Gram-negative LepB and pass through the Gram-negative outer membrane. This was achieved by shortening the arylomycin aliphatic tail which improved permeation and binding to LepB, improving activity in Gram negative pathogens including A. baumannii and P. aeruginosa, two notoriously difficult to treat hospital pathogens. Further modifications included changing two phenols to ethyl amines and modifying the pharmacophore by adding a 2-aminoacetonitrile. This second modification was potent in that it generates a covalent binding interaction and very tight binding to LepB, KI = 0.44 nM. These changes generated a new compound, G0775 with 32-500 fold improved MIC over ATCC strains. They went on to test a series of MDR clinical isolates, strains had MIC’s of >0.25 µg/mL. In a panel of MDR A. baumannii and P. aeruginosa 90% of strains exhibited MIC’s ≤4 µg/mL and ≤16 µg/mL, respectively.

The authors next generated G0775 resistant mutants and mapped the location of all mutations through whole genome sequencing and protein co-crystallography. The data identified mutations on 8 residues of LepB confirming on-target binding. Interestingly, the authors also identified a minority of mutants with missense mutations in the efflux pump AcrB, but deletions of the efflux components AcrB and TolC did not affect the potency of G0775 suggesting a possible gain of function mutation.

The mechanism of G0775 import was found to be independent of OmpC and OmpF, but mutation to the LPS or addition of outer membrane destabilizing compounds such as EDTA did reduce activity of G0775 by 32-fold. Conversely, modification to the compound by removing primary amines making the chemical more neutral, also reduced potency by 32-fold. This suggested a charge dependent uptake mechanism similar to what is theorized for aminoglycosides.

Finally, the authors tested their compound in animal models. A neutropenic thigh infection model revealed a >2-log decrease in CFU against ATCC strains. Next a lung infection model was tested with an MDR strain of K. pneumoniae and required 2 mg/kg of G0775 to be bacteriostatic and 20 mg/kg to be bactericidal, confirming its ability to overcome resistance mechanisms. Finally, in a mucin peritonitis model, mice were able to survive for 84 hours on 5 mg/kg delivered twice on day 0 of the infection compared to ciprofloxacin dosed at 80 mg/kg.

The novelty of this new class of arylomycins highlights the potential of repurposing and using structure guided design to generate new classes of antibiotics with extremely powerful binding kinetics. While the mechanism of entry is still not clearly understood the authors suggest it is likely not porin mediated since porin deletions did not alter MIC and porin mutations were not found in any G0775 resistant mutants. Although self-directed uptake was proposed as the mechanism of entry, the loss of G0775’s positive charge did not completely remove its activity, suggesting further research is required.

Overall this work has shown the utility in modifying existing antibiotics to generate novel functions and allowing a better understanding of compound entry to gram negative species. This research and others show the promise and potential of antimicrobial research and shed new light on the power of structure guided design and repurposing of existing antibiotics.

Original research article: “Optimized arylomycins are a new class of Gram-negative antibiotics”

Nature 12 September 2018, 561, 189-194

Dr. Dinesh Fernando

@DineshMFernando