The challenge of converting Gram-positive-only compounds into broad-spectrum antibiotics

Abstract

Multidrug resistant Gram-negative bacterial infections are on the rise, and there is a lack of new classes of drugs to treat these pathogens. This drug shortage is largely due to the challenge of finding antibiotics that can permeate and persist inside Gram-negative species. Efforts to understand the molecular properties that enable certain compounds to accumulate in Gram-negative bacteria based on retrospective studies of known antibiotics have not been generally actionable in the development of new antibiotics. A recent assessment of the ability of >180 diverse small molecules to accumulate in Escherichia coli led to predictive guidelines for compound accumulation in E. coli. These "eNTRy rules" state that compounds are most likely to accumulate if they contain a nonsterically encumbered ionizable Nitrogen (primary amines are the best), have low Three-dimensionality (globularity ≤ 0.25), and are relatively Rigid (rotatable bonds ≤ 5). In this review, we look back through 50+ years of antibacterial research and 1000s of derivatives and assess this historical data set through the lens of these predictive guidelines. The results are consistent with the eNTRy rules, suggesting that the eNTRy rules may provide an actionable and general roadmap for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.

Keywords: Gram-negative bacteria; antibacterials; molecular properties; multidrug resistance.

Figure 1

Components of the Gram-negative cellular envelope. (A) The cellular envelope of Gram-negative bacteria (right) contains two lipid membranes, while Gram-positive bacteria (left) have only one lipid membrane. The outer leaflet of the outer-membrane of Gram-negative bacteria is composed of lipopolysaccharides (LPS). Most small molecules are unable to passively diffuse at an appreciable rate through the LPS layer. Instead, molecules enter through channel proteins called porins (yellow). Almost all compounds, however, are subject to multidrug efflux pumps. (B) The structure of lipid A. The core oligosaccharide is linked to the 6′ carbon. (C) Top view of OmpF, the prototypical porin. A negatively-charged loop extends into the center of the cavity across from a positively-charged wall, creating a narrow constriction site and preventing the passage of many compounds. The crystal structure was generated from PDB 1opf using the Molecular Operating Environment software.

Figure 2

Antibiotic structures and spectrum of activity. Broad-spectrum antibiotics (effective against Gram-positive and Gram-negative pathogens), and Gram-positive-only antibiotics. Although information on the precise mode of entry into Gram-negative bacteria is not definitive, data suggests that antibiotics can enter Gram-negative bacteria through porins, the self-promoted uptake, or through a combination of both. *Colistin is Gram-negative only.

Figure 3

Workflow for the accumulation assay. After a culture of bacteria is grown to mid-log phase, bacteria are harvested and washed with sterile phosphate-buffered saline (PBS). The pellet is then resuspended in a much smaller volume of PBS, and aliquoted into Eppendorf tubes. The cultures are then incubated with compound of interest for 10 minutes. To separate the bacteria from extracellular compound, cultures are layered on cold silicone oil and centrifuged. The oil forms a layer between the bacterial pellet and the supernatant. After disposal of both the supernatant and the oil, the pellets are resuspended in water and the bacteria are lysed by freeze-thawing. The lysate is centrifuged to remove cellular debris, and the supernatant is analyzed by LC-MS/MS.

Figure 4

Validation of compound accumulation assay in E. coli. (A) Compounds that are active against E. coli have significantly higher accumulation than low-activity antibiotics and ampicillin. Statistical significance was determined by using a two-sample Welch's t-test (one-tailed test, assuming unequal variance) relative to the negative controls. P values relative to the average of the low-accumulating controls; (B) Accumulation comparison of ciprofloxacin, tetracycline, and chloramphenicol for E. coli ΔompR vs parental strain E. coli BW25113. (C) Co-treatment with colistin enhances the accumulation of low-accumulating antibiotics in E. coli. For B and C statistical significance was determined by using a two-sample Student's t-test (two-tailed test, assuming equal variance). *P < 0.05, *P < 0.01, *P < 0.001 All experiments were performed in biological triplicate. Error bars represent s.e.m. Data for this figure is taken from Richter et al. with permission.

Figure 5

eNTRy Rules. (A) Compounds are most likely to accumulate in Gram-negative bacteria if they contain a non-sterically encumbered ionizable Nitrogen (we observed primary amines are the best), have low Three-dimensionality (Globularity ≤ 0.25), and are relatively Rigid (rotatable bonds ≤ 5). Additionally, some degree of hydrophobicity is required. (B) Flexibility (as measured by the number of rotatable bonds) plotted against globularity for the primary amines tested in Richter et al. with sufficient hydrophobicity. (C) Flexibility versus globularity for antibiotics. Antibiotics included are Gram-positive-only drugs containing an ionizable nitrogen (triangles), and Gram-negative actives that are believed to enter cells through porins (circles). Data for this figure is taken from Richter et al. with permission.

Figure 6

Appending a sterically-unencumbered amine on relatively flat and rigid antibiotics can enhance their activity against Gram-negative bacteria. (A) Seven examples^{, , -,} of conversions of Gram-positive-only compounds into broad-spectrum antibiotics via addition of a primary amine, all are consistent with the eNTRy rules. (B) Two examples of conversions to broad-spectrum antibiotics that are not consistent with the eNTRy rules, both converted compounds are diamines and may enter Gram-negative bacteria through the self-promoted uptake pathway. RB = rotatable bonds; Glob = globularity; Broad-spectrum = activity against both Gram-positive and Gram-negative bacteria; PA = P. aeruginosa; MIC = minimum inhibitory concentration.

Figure 7

A sterically-unencumbered amine on large and flexible Gram-positive-only antibiotics does not enhance their activity against Gram-negative bacteria. (A) Existing Gram-positive-only antibiotics that have a primary amine but do not meet the shape requirements for broad-spectrum activity. (B) Addition of a primary amine to compounds that do not meet the shape requirement does not broaden the spectrum of activity.^{-, ,} RB = rotatable bonds; Glob = globularity.