Tackling the outer membrane: facilitating compound entry into Gram-negative bacterial pathogens

Abstract

Emerging resistance to all available antibiotics highlights the need to develop new antibiotics with novel mechanisms of action. Most of the currently used antibiotics target Gram-positive bacteria while Gram-negative bacteria easily bypass the action of most drug molecules because of their unique outer membrane. This additional layer acts as a potent barrier restricting the entry of compounds into the cell. In this scenario, several approaches have been elucidated to increase the accumulation of compounds into Gram-negative bacteria. This review includes a brief description of the physicochemical properties that can aid compounds to enter and accumulate in Gram-negative bacteria and covers different strategies to target or bypass the outer membrane-mediated barrier in Gram-negative bacterial pathogens.

Fig. 1. Entry routes of a wide range of antimicrobials via outer membrane of Gram-negative bacteria.

The figure represents the complex architecture of the cell envelope of Gram-negative bacteria which acts as a barrier for antimicrobial agents to enter the cell because of their specific physio-chemical composition. The figure illustrates the routes of entry followed by a wide range of antimicrobials to enter the outer membrane. The ways include membrane disruption by competing with divalent cations of membrane (as followed by agents such as PMBN, dendrimers, anti-microbial peptides), hydrophilic β-barrel proteins called porins (as followed by fluoroquinolones, β-lactams, tetracyclines, chloramphenicol), targeting outer membrane proteins (e.g.,darobactin, murepavadin, MRL-494). Further, the antibacterials such as macrolides utilizes membrane diffusion mechanism to enter the bacterial outer membrane. Adding on to the entry routes, antibacterials such as albomycin make use of transport proteins to cross outer membrane of bacterial cell. However, the accumulation of antimicrobials inside bacterial cells is compensated by the presence of outer membrane efflux pumps that export the entered antimicrobial agents and other foreign substances out of the cell. Figure created with BioRender.com.

Fig. 2. The figure illustrates the physicochemical parameters of compounds studied to overcome the outer membrane barrier of Gram-negative bacteria.

Of these rules, traditional drug space of orally bioavailable compounds (Lipinski rules) suggests that a druggable molecule should possess an av. mol. wt. of <500 Da, with <5 hydrogen bond donors, ≤10 hydrogen bond acceptors and calculated octanol-water partition coefficient (clogP ≤ 5). These physicochemical properties were further narrowed down by O’Shea and Moser’s, according to which, a Gram-negative targeting compound must possess an average mol. wt. of 414 Da with clogP ~ 0.1. Later, according to the eNTRy rules proposed by Hergenrother and colleagues, compounds with ionizable nitrogen, globularity ≤0.25 and rotational bond ≤5 are more likely to accumulate in Gram negatives. Glob globularity, RB number of rotatable bonds. Figure created with BioRender.com.

Fig. 3. Examples illustrating limitations of eNTRy rules of broadening the antibacterial spectrum of antibiotics.

A Erythromycin, a macrolide class antibiotic, was modified to 9(S)-erythromycyclamine to improve antibacterial spectrum by introducing primary amine to the structure. In contrast to the eNTRy rules, introduction of primary amine does not make any difference in the anti-bacterial spectrum of the compound. B Similarly, modification of Pleuromutilin to Pleuromutilin-amine by introduction of primary amine was not effective in improving its antibacterial activity. C Azithromycin, a macrolide class antibiotic, having RB=7 is studied to enter Gram-negative bacteria by self-promoted uptake, which is again in contrast to the eNTRy rules where RB of the compound to accumulate in Gram-negatives should ≤5.

Fig. 4. Structures of commonly studied antibiotic hybrids.

A Cefamandole-omadine conjugate, a cephalosporin and 2-mercaptopyridine-N-oxide or pyrithione (metal chelator and ATP synthesis inhibitor) conjugate and B ciprofloxacin‑tobramycin conjugate that effectively targets Gram-negative bacteria via dual mode of action.

Fig. 5. Structure of common siderophore‑antibiotic conjugates.

A Cephalosporin conjugate cefiderocol (Fetroja), an FDA approved siderophore cephalosporin conjugate for the treatment of hospital acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP). B Monosulfactam BAL30072, a monocyclic beta-lactam antibiotic with potent activity against drug-resistant Gram-negative pathogens.