Polymyxins with truncated fatty acyl chains are weakly or non-bactericidal due to the absence of the lipid tail which is crucial for eliciting the lethal action of polymyxins (Vaara, 1992). Nonetheless, polymyxin decapeptides (deacylated polymyxin (DAPB) and colistin), PMB and colistin nonapeptides, and PMB octapeptide and heptapeptide demonstrated the ability to permeabilize the OM (Kimura et al., 1992; Vaara, 1992). Among these analogs, PMBN (Figure 2) was the most effective OM permeabilizer and retains the ability to bind to the LPS (Tsubery et al., 2000; Moison et al., 2017). It synergizes in vitro with antibiotics such as rifampicin, novobiocin, and erythromycin against E. coli, K. pneumoniae, and P. aeruginosa (Vaara, 2019). It also demonstrates in vivo efficacy in combination with novobiocin and erythromycin, and protected mice infected with K. pneumoniae and P. aeruginosa (Ofek et al., 1994). While PMBN was less toxic than polymyxins in several animal studies, it was found that it was still as nephrotoxic as PMB, and was therefore no longer considered for clinical use (Vaara, 1992, 2010). PMBN is still contemporarily used as a tool for the development of OM permeabilizers, evident in several studies (Vaara, 2019). It has been widely investigated since its discovery, and its well-characterized properties have established PMBN as the benchmark for evaluating novel OM permeabilizers (Vaara and Vaara, 2010; Vaara, 2019; Wesseling and Martin, 2022).

The nephrotoxicity of PMBN prompted the development of polymyxins with three positive charges. NAB7061 possessed variations in the linear peptide segment with an octanoyl as the fatty acid tail, the C-terminal Dab replaced with an aminobutyryl residue, and absence of the N-terminal Dab (Vaara et al., 2008). NAB7061 was notably synergistic with rifampin, clarithromycin, azithromycin, erythromycin, and mupirocin against E. coli, K. pneumoniae, and Enterobacter cloacae (Vaara et al., 2008; Vaara et al., 2010). Rifampicin and clarithromycin potentiation was also observed in A. baumannii (Vaara et al., 2008). The in vivo potency of the combination therapy of NAB7061 and erythromycin was demonstrated in an experimental E. coli murine peritonitis model (Vingsbo Lundberg et al., 2010).

Further optimization led to the development of SPR741, with the N-terminal lipid tail substituted with an acetyl group and one of the Dab residues replaced with serine (Vaara et al., 2010; Figure 2). SPR741 synergized with multiple antibiotics such as rifampicin (Corbett et al., 2017; Zurawski et al., 2017; Vaara, 2019), clarithromycin (Corbett et al., 2017; Vaara, 2019), erythromycin (Corbett et al., 2017), azithromycin (Corbett et al., 2017; Stainton et al., 2018), ceftazidime (Eckburg et al., 2019), and piperacillin-tazobactam (Eckburg et al., 2019) against a variety of Gram-negative bacteria. The phase 1 studies of SPR741 demonstrated its safety and pharmacokinetics in combination with ceftazidime, aztreonam, and piperacillin-tazobactam (Eckburg et al., 2019). However, further development of SPR471 was discontinued in favor of the standalone antibacterial agent SPR206 (Figure 2), a potent, non-nephrotoxic PMB derivative which may offer improved safety and efficacy profiles (Prasad et al., 2022). It demonstrated both in vitro activity and in vivo efficacy against MDR Gram-negative bacteria (Brown et al., 2019; Bruss et al., 2021, 2023). In comparison to SPR741, SPR206 bears an aminobutyrate N-terminus with a 3-chlorophenyl moiety at the β-position (Brown et al., 2019).

Dilipid polymyxins were synthesized with the hypothesis that additional hydrophobic character in the lipid component would enhance its ability to insert into membranes (Domalaon et al., 2018a). The Dab amine side chain was acylated with various hydrophobic components: butyl, octyl, and dodecyl lipids, as well as adamantyl and biphenyl groups. Among these series of compounds, the butyric acid derivative (Figure 2) potentiated β-lactams, tetracyclines, fluoroquinolones, fosfomycin, trimethoprim, chloramphenicol, novobiocin, vancomycin, clindamycin, linezolid, and rifampicin against P. aeruginosa PAO1 in a manner comparable to PMBN (Domalaon et al., 2018a). While the dilipid polymyxins did not necessarily improve the activity against Gram-negative bacteria, it conferred anti-Gram-positive activity, implying that incorporation of another lipid potentially strengthens its interactions with the lipoteichoic acid in Gram-positive bacteria via hydrophobic effects. The increased hydrophobicity could also result in non-specific lysis of eukaryotic cells. A preliminary assessment showed that the dilipid butyric acid derivative was non-hemolytic at 512 μg/mL similar to colistin and PMBN (Domalaon et al., 2018a).

Substitution of the Dab amines to guanidines was postulated to favor interactions with the OM (Kim et al., 2021). In comparison to amines, guanidinium groups have a higher pK_a and remain protonated across a wide pH range, including physiological pH (Blondeau et al., 2007; Kim et al., 2021). The delocalization of the positive charge and planar Y-shape geometry allow them to bind with high affinity to oxoanions (Blondeau et al., 2007), such as the phosphates on the core sugars and lipid A of the LPS (DeLucia et al., 2011). In a recent study, cationic peptide-based adjuvants with guanidine-containing arginine residues showed greater antibiotic potentiation compared to those with lysine and Dab (Ramirez et al., 2020). The resulting guanidinylated colistin (GCol) and PMB (GPMB) (Figure 2) synergized with a panel of antibiotics against reference and MDR strains of Gram-negative bacteria (Ramirez et al., 2022). The activity spectrum of rifampicin and erythromycin were expanded to Gram-negative bacteria, with minimum inhibitory concentrations (MICs) below the interpretative susceptibility breakpoint against MDR clinical isolates (Ramirez et al., 2022). In particular, rifampicin potentiation by the guanidinylated polymyxins was higher than PMBN in colistin-resistant P. aeruginosa, E. coli, K. pneumoniae, and E. cloacae (Ramirez et al., 2022). The guanidinylated polymyxins also enhanced the activity of ceftazidime and aztreonam, and their respective combinations with the BLI avibactam against MDR and β-lactamase harboring P. aeruginosa, respectively (Ramirez et al., 2022). Furthermore, the triple combinations of guanidinylated colistin with ceftazidime/avibactam or aztreonam/avibactam were shown to be bactericidal (Ramirez et al., 2022). Despite the lack of standalone activity, fluorescent assays measuring the uptake of the membrane impermeable probe, N-phenyl-1-naphthylamine (NPN), demonstrated that the guanidinylated polymyxins permeabilized the OM in a time- and concentration-dependent manner similar to PMBN (Ramirez et al., 2022). Unfortunately, changing the amines to guanidines did not improve the cytotoxicity relative to the parent PMB (Ramirez et al., 2022).

Polymyxins are also utilized as pharmacophores in the design of antibiotic hybrids, wherein two or more bioactive scaffolds are covalently linked while preserving their distinct mechanisms of action, resulting in a synergistic effect (Domalaon et al., 2018b). For instance, vancomyxins (Figure 3) are created by covalently linking vancomycin to polymyxin E nonapeptide (PMEN) (Van Groesen et al., 2021). The LPS binding of the hybrids are maintained, resulting in the improved activity of the conjugated vancomycin against Gram-negative bacteria relative to the dual combination of vancomycin and PMEN (Van Groesen et al., 2021). Against Gram-positive bacteria, the potency of vancomycin is retained, with some enhancement against vancomycin-resistant strains (Van Groesen et al., 2021). Compared to clinically used polymyxins, the hybrids also showed reduced nephrotoxicity (Van Groesen et al., 2021). PMEN was conjugated to a β-hairpin peptide macrocycle inspired by murepavadin, a peptidomimetic antibiotic which inhibits the LPS transport protein LptD in P. aeruginosa (Luther et al., 2019). The potent activity of the resulting chimeric peptidomimetic antibiotics (CPA) (Figure 3) against MDR pathogens arises from their ability to target both the LPS and BamA (Luther et al., 2019). BamA is an OMP and a key component of the β-barrel assembly machinery (BAM), which is responsible for the folding and integration of β-barrel proteins in the OM. Aside from antibiotics, PMBN was covalently attached to the FDA-approved photosensitizer chlorin e6 (Ce6) via different linkers (Wu et al., 2024). The ability of PMBN to permeabilize the OM allowed diffusion of Ce6 into the bacterial membranes, resulting in photobactericidal activity (Wu et al., 2024). The optimized derivative possessing an amino-3,6-dioxaoctanoic linker (P2pCe6) (Figure 3) showed selective imaging of Gram-negative bacteria, improved biocompatibility, higher reactive oxygen species (ROS) production, and strong photoinactivation of Gram-negative bacteria (Wu et al., 2024). P2pCe6 was also effective in treating P. aeruginosa infections and accelerating wound healing in Galleria mellonella and mouse models, respectively (Wu et al., 2024).

The use of polymers, conjugates, liposomes, gels, fibers, and membranes to improve the delivery of polymyxins has been widely studied (Dubashynskaya and Skorik, 2020). These drug carriers are designed to control the release of polymyxins, ensuring targeted delivery, increased bioavailability, improved chemical and physical stability, and enhanced accumulation in membranes and biofilms (Dubashynskaya and Skorik, 2020). As a result, these systems increase the local concentration of polymyxins, allowing for reduced dosages and lower toxicity (Dubashynskaya and Skorik, 2020). A self-forming peptide-based hydrogel was utilized for the sustained delivery of PMB. The hydrogels were formed by mixing PMB with a solution of varying peptide amphiphiles with tunable mechanical properties (Shi et al., 2021). The system released the drug over 5 days, resulting in prolonged antibacterial activity against Gram-negative bacteria (Shi et al., 2021). The localized release of PMB reduced P. aeruginosa-related mortality in a G. mellonella burn wound model (Shi et al., 2021). Moreover, the inclusion of fusidic acid in the polymyxin hydrogel enhanced the activity against S. aureus and A. baumannii, highlighting the potential application of this delivery system in combination therapy (Shi et al., 2021).

PMB has also been strategically anchored to the carrier, facilitating the co-delivery of companion antibiotics (Wu et al., 2022). For instance, linezolid was encapsulated inside mesoporous silica particles, and the outer surface was coated with PMB (Otri et al., 2024). The PMB coating disrupted bacterial membranes, enhancing the penetration and efficacy of linezolid (Otri et al., 2024). The system dramatically improved activity against E. coli, as well as Gram-positive bacteria (Otri et al., 2024). Another study involved the development of anionic liposomes loaded with fosfomycin, with the cationic PMB adsorbed onto the surface, enabling selective bacterial targeting against A. baumannii (Zhu et al., 2023). In vitro and in vivo tests showed that these PMB–fosfomycin liposomes achieved stronger antibacterial and anti-inflammatory effects than free drug mixtures, while also reducing the nephrotoxicity of PMB (Zhu et al., 2023). PMB-based platforms utilizing covalent organic nanoparticles, nanocrystals, and liposomes (Le Guern et al., 2017; Jiang et al., 2021; Cui et al., 2024) have also been applied in photodynamic therapy, wherein they act synergistically with a photosensitizer.

Tobramycin conjugates have also been documented to synergize with several antibiotic classes, notably β-lactams and/or BL/BLI combinations (Idowu et al., 2019a, 2020; Idowu et al., 2020; Berry et al., 2021). These hybrid molecules consist of a secondary domain linked to tobramycin via an alkyl tether and lack ribosomal binding activity with the C-5 position used as a point of attachment. The structure–activity relationship (SAR) studies of these tobramycin conjugates have been reported, demonstrating the significance of the tether length and hydrophobicity, physicochemical properties of the secondary moiety, and number of cationic groups (Idowu et al., 2020). The various secondary motifs that have been conjugated to tobramycin include antibiotics [rifampicin (Idowu et al., 2019c), PMB₃ (Domalaon et al., 2017) and fluoroquinolones (Gorityala et al., 2016a, 2016b; Dhiman et al., 2024)], antiparasitic agents [niclosamide (Berry et al., 2021)], EPIs (Yang et al., 2017) (NMP, paroxetine, and dibasic peptide analog of MC-04,124), chelating agents [cyclam (Idowu et al., 2019a), deferiprone (Gandhi et al., 2023)], and peptides (Lyu et al., 2017, 2019). Homodimeric analogs wherein tobramycin was conjugated to another tobramycin unit was also developed (Idowu et al., 2019b; Idowu et al., 2020). Despite the vast structural complexities between the secondary domains, they are mainly lipophilic or polybasic in nature and influence the amphiphilicity of the tobramycin conjugates with the accompanying aliphatic linker. Reducing the overall cationicity to mitigate toxicity was also accomplished by replacing the aminoglycoside scaffold with nebramine (Ammeter et al., 2019; Yang et al., 2019), the hydrolysis product of tobramycin which lacks the kanosamine sugar.

The most notable among these conjugates are the tobramycin-ciprofloxacin and -cyclam hybrids and the homodimeric tobramycin (Figure 5). The tobramycin hybrid conjugated to the piperazine of ciprofloxacin via a C12 chain (Figure 5) synergized with fluoroquinolones against MDR P. aeruginosa (Gorityala et al., 2016a), various β-lactams and BL/BLI combinations against β-lactamase-harboring P. aeruginosa (Idowu et al., 2020), as well with mitomycin C, an anticancer drug, against MDR Gram-negative bacteria (Domalaon et al., 2019). Further derivatives of this conjugate employed variation of the fluoroquinolone or aminoglycoside moiety (Gorityala et al., 2016b; Yang et al., 2019), conversion of the tobramycin amino groups to guanidines (Idowu et al., 2020), and modification of the linkers and point of attachment (Dhiman et al., 2024). The tobramycin- and nebramine-ciprofloxacin derivatives demonstrated comparable potentiation of rifampicin, fluoroquinolones, and minocycline against P. aeruginosa (Yang et al., 2019). Similarly, no significant differences in the potentiation of β-lactams and BL/BLIs against P. aeruginosa harboring β-lactamases were observed between the guanidinylated and non-guanidinylated tobramycin-ciprofloxacin hybrids (Idowu et al., 2020). In varying the flexibility and hydrophobic threshold of the tether, the 12-long carbon chain linker demonstrated enhanced tetracycline potentiation in comparison to the less hydrophobic polyethylene glycol and shorter C6 chain linkers, as well as the more rigid biphenyl tether (Dhiman et al., 2024). The tobramycin-cyclam hybrid with an eight-long carbon chain linker (Figure 5) enhanced in vitro activity and in vivo efficacy of β-lactam monotherapy and BL/BLI dual combinations against β-lactamase-harboring P. aeruginosa (Idowu et al., 2019a). Meanwhile, the homodimeric tobramycin, which comprises of a 1,4-dibutyl-1,2,3-triazole core joining the two tobramycin molecules, in combination with ceftolozane, was more active than ceftolozane/tazobactam against MDR or extensively drug-resistant (XDR) P. aeruginosa (Idowu et al., 2020). The tobramycin homodimer (Figure 5) was also found to be more potent than PMBN in potentiating novobiocin against MDR/XDR Gram-negative bacteria (Idowu et al., 2019b). Both tobramycin-cyclam and homodimeric tobramycin were comparable with their nebramine counterparts in potentiating β-lactams and BL/BLIs. The optimal linker length for these hybrids and homodimers were determined to be 8 or 12-carbon atoms long (Idowu et al., 2020).

A recent development to the tobramycin- and nebramine-based hybrids and homodimers, is the addition of a third domain (Dhiman et al., 2023b; Dhiman et al., 2023a; Figure 5). These structures consist of a central 1,3,5-triazine framework, appended with various combinations of tobramycin, ciprofloxacin, NMP, and cyclam to yield tobramycin-based chimeras (Dhiman et al., 2023b). The chimeric compound which contained 2 units of tobramycin and ciprofloxacin (Figure 5) synergized with fluoroquinolones and BL/BLI combinations against fluoroquinolone-resistant and β-lactamase-harboring P. aeruginosa, respectively (Dhiman et al., 2023b). In the chimeric series, compounds with two tobramycin moieties demonstrated higher antibiotic potentiation (Dhiman et al., 2023a). These results led to the incorporation of 3 units of tobramycin or nebramine with varying hydrocarbon chains to generate trimeric molecules (Dhiman et al., 2023a). The resulting tobramycin and nebramine trimers (Figure 5) were also synergistic with β-lactams and BL/BLI combinations against MDR P. aeruginosa (Dhiman et al., 2023a).

Amphiphilic tobramycins were revisited for their capacity as OM permeabilizers, as prior derivatives were assessed solely for their immunomodulatory effects (Guchhait et al., 2015). Following established protocols (Guchhait et al., 2015; Idowu et al., 2019c) and SAR studies (Idowu et al., 2020), the amenable C-5 position was chosen for the installment of several lipophilic moieties to investigate their influence on OM permeabilization, given their critical role in facilitating the insertion within the lipid-rich region of the LPS. The derivatives were further optimized by converting the amines to guanidine functions (Figure 6), based on the same rationale for the development of guanidinylated polymyxins (Ramirez et al., 2020, 2022). The optimized guanidinylated tobramycin biphenyl derivative synergized with β-lactams, restoring the susceptibility of MDR and β-lactamase harboring P. aeruginosa to ceftazidime and aztreonam in both dual and triple combinations with avibactam, respectively (Ramirez et al., 2023). In particular, the triple combination of ceftazidime/avibactam plus guanidinylated tobramycin biphenyl resulted in a bactericidal effect (Ramirez et al., 2023). An indication of the OM permeabilizing capability of guanidinylated tobramycin biphenyl was substantiated by the time- and adjuvant concentration-dependent uptake of NPN, as well as the potentiation of rifampicin (Ramirez et al., 2023). More importantly, the derivative was non-cytotoxic against HEK293 cells up to 106 times the active concentration (Ramirez et al., 2023). The hit molecule also emphasized that the guanidinium and biphenyl groups were key structural features (Ramirez et al., 2023). Guanidinylation and/or alkylation resulted in a loss of antibacterial activity (Ramirez et al., 2023). These findings were consistent with previous studies, indicating the importance of the amino groups and C-5 hydroxyl in binding with the ribosomal RNA (Ramirez et al., 2023). However, the requirement of both components for synergism with β-lactam antibiotics was in contrast with previously synthesized tobramycin conjugates, wherein guanidinylation was not necessary and only showed comparable antibiotic potentiation. While the guanidinylated tobramycin derivatives were overall less cytotoxic compared to the polymyxins, guanidinylation still contributed to a slight increase in cytotoxicity (Ramirez et al., 2023).

The alkylation at the singular C-5 position in the aminoglycoside scaffold was further extended to include all other hydroxyl groups. Previously, Fridman et al. developed a synthetic strategy which allowed etherification of all alcohols in the tobramycin and nebramine scaffold (Berkov-Zrihen et al., 2015). Several of these cationic amphiphiles demonstrated promising low hemolytic activity and the ability to interact with LPS or disrupt the bacterial membrane (Berkov-Zrihen et al., 2015). Thus, these previous findings guided the design of benzyl and isopentyl tobramycin and nebramine ethers as OM permeabilizers. In addition, tobramycin phenyl carbamates were developed to ascertain the optimal point of attachment, since earlier studies only evaluated the standalone activity of these derivatives (Bera et al., 2010). Further structural optimizations included synthesis of substituted benzyl analogs to determine the necessary hydrophobic threshold required for OM permeabilization. The previously developed tobramycin benzyl ether (Figure 6) sensitized reference and resistant isolates of Gram-negative bacteria to rifampicin, and the restored susceptibility of drug-resistant Escherichia coli to minocycline (Ramirez et al., 2024). The OM perturbation of the compound was verified by its ability to mediate uptake of NPN, synergize with other OM impermeable antibiotics such as novobiocin and vancomycin, and lose synergistic interactions in the presence of competing Mg²⁺ and Na⁺ cations (Ramirez et al., 2024). While rifampicin potentiation did not translate to Gram-positive bacteria, tobramycin benzyl ether exhibited potent standalone activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains (Ramirez et al., 2024). However, the derivative showed non-specific binding to serum proteins, evidenced by the reduced rifampicin potentiation with the addition of fetal bovine serum (FBS) (Ramirez et al., 2024).