A new web-based tool allowing rapid in silico prediction of the ability of candidate antibiotics to accumulate in gram-negative bacteria should enable subsequent prioritization of new compounds for synthesis and further evaluation, U.S. researchers reported Nov. 18, 2019, in Nature Microbiology

Known as eNTRyway, it is one of a growing number of web systems designed to facilitate antibacterial research, which should assist in the development of antibiotics with activity against gram-negative pathogens, such as Escherichia coli. 

Using the eNTRyway tool, “many compounds can be evaluated computationally[,] and then the most promising ones, as evaluated by the app, can be prioritized for synthesis and experimental testing,” said study leader Paul Hergenrother, a professor of chemistry in the Carl R. Woese Institute for Genomic Biology at the University of Illinois. 

This is a timely development. Over the past 50 years, just seven new antibiotic classes have been approved by the U.S. FDA, none of which are effective against the most problematic gram-negative ESKAPE pathogens responsible for about 75% of infections by antibiotic-resistant bacteria. 

Moreover, the number of patients dying from ESKAPE infections (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), has increased almost fourfold in just eight years. Four of the six ESKAPE pathogens – K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp. – are gram-negative. 

Despite advances in genomics and high-throughput screening, antibiotic classes to treat problematic gram-negatives that are structurally distinct or have new targets have remained elusive.  

The resultant low hit rate reported in extensive antimicrobial screening programs is mainly due to poor drug accumulation in gram-negatives, given their membrane impermeability and effective efflux pumps.  

Nevertheless, numerous new natural product and synthetic compounds have been discovered with promising activity against gram-positive bacteria, many of which act via novel mechanisms.  

Importantly, most such compounds would kill gram-negatives if they could accumulate inside those pathogens, but no method exists for effectively converting gram-positive-only compounds into broad-spectrum antibiotics.  

One potential approach involves adjusting antibiotic physiochemical properties to favor accumulation within bacteria. If that could be optimized chemically in gram-negatives, the early stage developmental attrition of antibiotics might be mitigated. 

However, that would require a comprehensive understanding of the physicochemical parameters allowing compound accumulation in gram-negative bacteria.  

In the new Nature Microbiology study, Hergenrother and his research team studied the ability of different compounds to accumulate in E. coli.  

They demonstrated that compounds having an ionizable nitrogen moiety, low three-dimensionality and low numbers of rotatable bonds, known as the eNTRy rules, had a significantly higher probability of bacterial accumulation.  

“Our estimate is that approximately 85 percent of compounds that meet these eNTRy criteria will accumulate in E. coli,” Hergenrother told BioWorld

To facilitate implementation of those eNTRy rules, the team developed the convenient, open-access, web-based eNTRyway chemoinformatics tool to predict a candidate compound’s accumulation in E. coli from its structure.  

Together with structure-activity relationships and X-ray crystallographic data, eNTRyway was used to re-design the gram-positive-only antibiotic drug (API-1251, Affinium Pharmaceuticals) into versions that accumulated in E. coli and had antibacterial activity against gram-negative pathogens. 

Currently in phase II trials for methicillin-resistant S. aureus (MRSA), Debio-1452 “possesses two of the three eNTRy criteria, making it a good candidate for re-design,” noted Hergenrother. 

The newly developed lead compound, Debio-1452-NH3, was shown to operate as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of FabI, an enzyme drug target in the bacterial fatty acid biosynthetic pathway. 

That is a significant finding, said Hergenrother, as “resistance mutants to this compound arise in the FabI gene, the mode-of-action of this compound is not changing, it is simply accumulating much better.” 

The researchers then validated that potent inhibition using in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations.  

They also demonstrated that Debio-1452-NH3 was well-tolerated in vivo and reduced the bacterial burden in mice, saving them from lethal infections by A. baumannii, K. pneumoniae and E. coli isolates.  

“Tolerability was assessed in mouse infection models with three different gram-negative ESKAPE pathogens, but further testing will give more insights into the probability of translational success,” said Hergenrother. 

Regarding efficacy, “the compound dramatically elevated the ability of mice to survive infection and reduced the bacterial burden in the lung,” he told BioWorld

Collectively, those findings show that eNTRyway facilitates the discovery and development of high-accumulating compounds in E. coli and serves as a general plan for converting gram-positive-only compounds into broad-spectrum antibiotics.  

“We hope this blueprint can be used by others to facilitate the identification of promising leads as gram-negative antibiotics. In the future, we will be applying this method/strategy to redesign other gram-positive-only antibiotics and moving the most promising compounds toward clinical development.” 

No Comments