An efflux-susceptible antibiotic-adjuvant with systemic efficacy against mouse infections.

Scarcity of effective treatments against sepsis is daunting, especially under the contemporary standpoints on antibiotics resistance, entailing the development of alternative treatment strategies. Here, we describe the design and antibiotic adjuvant properties of a new lipopeptide-like pentamer, decanoyl-bis.diaminobutyrate-aminododecanoyl-diaminobutyrate-amide (C10BBc12B), whose sub-maximal tolerated doses combinations with inefficient antibiotics demonstrated systemic efficacies in murine models of peritonitis-sepsis and urinary-tract infections. Attempts to shed light into the mechanism of action using membrane-active fluorescent probes, suggest outer-membrane interactions to dominate the pentamer's adjuvant properties, which were not associated with typical inner-membrane damages or with delayed bacterial growth. Yet, checkerboard titrations with low micromolar concentrations of C10BBc12B exhibited unprecedented capacities in potentiation of hydrophobic antibiotics towards Gram-negative ESKAPE pathogens, with an apparent low propensity for prompting resistance to the antibiotics. Assessment of the pentamer's potentiating activities upon efflux inhibition incites submission of a hitherto unreported, probable action mechanism implicating the pentamer's de-facto capacity to hijack bacterial efflux pumps for boosting its adjuvant activity through repetitive steps including outer-membrane adhesion, translocation and subsequent expulsion.

Mechanistic Studies of Antibiotic Adjuvants Reducing Kidney's Bacterial Loads upon Systemic Monotherapy.

We describe the design and attributes of a linear pentapeptide-like derivative (C14(ω5)OOc10O) screened for its ability to elicit bactericidal competences of plasma constituents against Gram-negative bacteria (GNB). In simpler culture media, the lipopeptide revealed high aptitudes to sensitize resilient GNB to hydrophobic and/or efflux-substrate antibiotics, whereas in their absence, C14(ω5)OOc10O only briefly delayed bacterial proliferation. Instead, at low micromolar concentrations, the lipopeptide has rapidly lowered bacterial proton and ATP levels, although significantly less than upon treatment with its bactericidal analog. Mechanistic studies support a two-step scenario providing a plausible explanation for the lipopeptide's biological outcomes against GNB: initially, C14(ω5)OOc10O permeabilizes the outer membrane similarly to polymyxin B, albeit in a manner not necessitating as much LPS-binding affinity. Subsequently, C14(ω5)OOc10O would interact with the inner membrane gently yet intensively enough to restrain membrane-protein functions such as drug efflux and/or ATP generation, while averting the harsher inner membrane perturbations that mediate the fatal outcome associated with bactericidal peers. Preliminary in vivo studies where skin wound infections were introduced in mice, revealed a significant efficacy in affecting bacterial viability upon topical treatment with creams containing C14(ω5)OOc10O, whereas synergistic combination therapies were able to secure the pathogen's eradication. Further, capitalizing on the finding that C14(ω5)OOc10O plasma-potentiating concentrations were attainable in mice blood at sub-maximal tolerated doses, we used a urinary tract infection model to acquire evidence for the lipopeptide's systemic capacity to reduce the kidney's bacterial loads. Collectively, the data establish the role of C14(ω5)OOc10O as a compelling antibacterial potentiator and suggest its drug-like potential.

Eliciting improved antibacterial efficacy of host proteins in the presence of antibiotics.

We recently reported the aptitude of a membrane-active lipopeptide (C10OOc12O) to sensitize gram-negative bacilli (GNB) to host antibacterial proteins. Here we explored the potential of harnessing such capacity in the presence of antibiotics. For this purpose, we compared Escherichia coli sensitization to antibiotics in broth and plasma; assessed inner and outer membrane damages using scanning electron microscopy, dyes, and mutant strains; and assessed the ability to affect disease course using the mouse peritonitis-sepsis model for mono- and combination therapies. We found that by altering permeability of both outer and inner membranes, subinhibitory concentrations of C10OOc12O can transiently sensitize GNB to diverse cytoplasm-targeting antibiotics in simple media. Sensitization was maintained in plasma, where C10OOc12O instigated greater bactericidal activities, including in the presence of a bacteriostatic antibiotic (erythromycin). Single-dose administrations of rifampin and C10OOc12O to E. coli-infected mice resulted in 55% vs. 0, and 36% viability, respectively, for combined and individual treatments. Combining C10OOc12O and erythromycin has similarly improved mice protection from developing fatal sepsis. Consequently, the data confirmed that C10OOc12O renders GNB sensitive to both endogenous and exogenous antibacterials, and suggested that the tripartite concomitant presence increases therapeutic efficacy synergistically. This approach might expand the available treatment options to comprise antimicrobials with low permeability and/or efflux issues.-Jammal, J., Zaknoon, F., Mor, A. Eliciting improved antibacterial efficacy of host proteins in the presence of antibiotics.

A broad-spectrum bactericidal lipopeptide with anti-biofilm properties.

Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C14KKc12K) and investigated its properties in various media. Mechanistic studies suggest that C14KKc12K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C14KKc12K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C14KKc12K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC50 at 3 and 6 µM, respectively for GPB and GNB. C14KKc12K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.

Sensitization of Gram-Negative Bacilli to Host Antibacterial Proteins.

To address the need for novel alternatives to antibiotics, we attempted to sensitize gram-negative bacilli to innate antibacterial protagonists. We report a lipopeptide-like sequence (C10OOc12O) that inflicted outer membrane damage at a low micromolar range, whereas measurable bacterial growth inhibition in broth medium required >10-fold higher concentrations. In serum, however, C10OOc12O induced antibacterial activity in a manner suppressible by anticomplement antibodies or heat treatment and acted synergistically with exogenous lysozyme in broth and serum media. Upon subcutaneous administration, C10OOc12O exhibited high circulating levels that correlated with significant therapeutic efficacies, using either the mouse peritonitis-sepsis model or the thigh infection model. These findings are consistent with the view that, by damaging the outer membrane, C10OOc12O was able to enhance gram-negative bacilli susceptibility to antibacterial components of the immune humoral arm. Such lipopeptides may therefore be useful in fighting gram-negative bacilli threats through sensitization to endogenous and/or exogenous antibacterial proteins such as lysozyme and complements.

Sensitization of Gram-negative bacteria to rifampin and OAK combinations.

While individually inefficient against Gram-negative bacteria, in-vitro combinations of rifampin and OAK were mutually synergistic since sub-minimal inhibitory concentrations of one compound have potentiated the other by 2-4 orders of magnitude. Synergy persisted in-vivo as single-dose systemic treatment of Klebsiella infected mice resulted in 10-20% versus 60% survival, respectively accomplished by individual and combined compounds. This outcome was achieved without drug formulation, rather, pharmacokinetic considerations have inspired the therapeutic regimen.

Improved bacterial detection using immobilized acyl-lysyl oligomers.

The global need to improve bacterial detection in liquid media has motivated multidisciplinary research efforts toward developing new approaches that overcome the shortcomings of traditional techniques. We recently proposed the use of oligomers of acylated lysyls (OAKs) in their resin-linked form (ROAKs) for the efficient, robust, and inexpensive filtration of bacteria. Here, to investigate the potential for the use of ROAKs in downstream applications, we first examined the capacity of ROAKs to capture bacteria as a function of environmental conditions and structure-activity relationships (SARs). We next assessed their ability to release the captured bacteria and then combined both abilities to improve real-time PCR outcomes. ROAKs were able to deplete liquid samples of bacterial content after incubation or continuous flow, illustrating the efficient capture of different bacterial species under a wide range of ionic strength and pH conditions. We also show circumstances for the significant release of captured bacteria, live or dead, for further analysis. Finally, the SAR study revealed a shorter ROAK derivative exhibiting a capture capacity similar to that of the parent construct but the increased recovery of ROAK-bound bacteria, enabling improvement of the detection sensitivity by 20-fold. Collectively, the data support the potential usefulness of a simple, robust, and efficient approach for rapid capture/analysis of bacteria from tap water and, possibly, from more complex media.

Sensitization of gram-negative bacteria by targeting the membrane potential.

Toward generating new tools for fighting multidrug-resistant (MDR) bacteria, we assessed the ability of a membrane-active peptide to sensitize gram-negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr-AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh-infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram-negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide-like sequence, N(α)(ω7)dodecenoyl-lysyl-[lysyl-aminododecanoyl-lysyl]-amide (designated C12(ω7)K-ß12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton-motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram-negative bacteria to include efflux substrates.

Antibacterial properties of an oligo-acyl-lysyl hexamer targeting Gram-negative species.

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 µM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

Antiplasmodial properties of acyl-lysyl oligomers in culture and animal models of malaria.

Our previous analysis of antiplasmodial properties exhibited by dodecanoyl-based oligo-acyl-lysyls (OAKs) has outlined basic attributes implicated in potent inhibition of parasite growth and underlined the critical role of excess hydrophobicity in hemotoxicity. To dissociate hemolysis from antiplasmodial effect, we screened >50 OAKs for in vitro growth inhibition of Plasmodium falciparum strains, thus revealing the minimal requirements for antiplasmodial potency in terms of sequence and composition, as confirmed by efficacy studies in vivo. The most active sequence, dodecanoyllysyl-bis(aminooctanoyllysyl)-amide (C(12)K-2α(8)), inhibited parasite growth at submicromolar concentrations (50% inhibitory concentration [IC(50)], 0.3 ± 0.1 µM) and was devoid of hemolytic activity (<0.4% hemolysis at 150 µM). Unlike the case of dodecanoyl-based analogs, which equally affect ring and trophozoite stages of the parasite developmental cycle, the ability of various octanoyl-based OAKs to distinctively affect these stages (rings were 4- to 5-fold more sensitive) suggests a distinct antiplasmodial mechanism, nonmembranolytic to host red blood cells (RBCs). Upon intraperitoneal administration to mice, C(12)K-2α(8) demonstrated sustainable high concentrations in blood (e.g., 0.1 mM at 25 mg/kg of body weight). In Plasmodium vinckei-infected mice, C(12)K-2α(8) significantly affected parasite growth (50% effective dose [ED(50)], 22 mg/kg) but also caused mortality in 2/3 mice at high doses (50 mg/kg/day × 4).

Experimental conditions that enhance potency of an antibacterial oligo-acyl-lysyl.

Oligo-acyl-lysyls (OAKs) are synthetic mimics of host defense peptides known to exert antibacterial activity both in cultures and in animal models of disease. Here, we investigated how environmental conditions (temperature, pH, and ionic strength) affect the antibacterial properties of an octamer derivative, C(12)K-7alpha(8). Data obtained with representative bacteria, including the Gram-negative bacterium Escherichia coli and the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, showed that OAK's potency was proportionally affected by pH changes and subsided essentially throughout a wide range of salt concentrations and temperature values, whereas antistaphyloccocal activity was relatively more vulnerable. It was rather the mode of action that was most susceptible to the environmental changes. Thus, OAK's bactericidal effect was limited to a growth-inhibitory effect under acidic pH, low temperatures, or high salt concentrations, whereas basic pH or high temperatures have enhanced the bactericidal kinetics. Properties of binding to model phospholipid membranes provided evidence that correlated the differential modes of action with variable binding affinities. Interestingly, combination of the optimal incubation conditions resulted in a remarkable increase in potency, as expressed by a 16- to 32-fold reduction in the MIC value and by much faster bactericidal rates (>99% death induced within minutes versus hours) compared with the standard incubation conditions. Collectively, the data suggest that OAKs might be useful in developing design strategies for robust antimicrobial peptides that are able to affect a pathogen's viability under a large spectrum of incubation conditions.

A miniature mimic of host defense peptides with systemic antibacterial efficacy.

Oligomers of acylated lysines (OAKs) are synthetic mimics of host defense peptides (HDPs) with promising antimicrobial properties. Here we challenged the OAK concept for its ability to generate both systemically efficient and economically viable lead compounds for fighting multidrug-resistant bacteria. We describe the design and characterization of a miniature OAK composed of only 3 lysyls and 2 acyls (designated C(12(omega7))K-beta(12)) that preferentially targets gram-positive species by a bacteriostatic mode of action. To gain insight into the mechanism of action, we examined the interaction of OAK with various potential targets, including phospholipid bilayers, using surface plasmon resonance, and Langmuir monolayers, using insertion assays, epifluorescence microscopy, and grazing incidence X-ray diffraction, in a complementary manner. Collectively, the data support the notion that C(12(omega7))K-beta(12) damages the plasma-membrane architecture similarly to HDPs, that is, following a near-classic 2-step interaction including high-affinity electrostatic adhesion and a subsequent shallow insertion that was limited to the phospholipid head group region. Notably, preliminary acute toxicity and efficacy studies performed with mouse models of infection have consolidated the potential of OAK for treating bacterial infections, including systemic treatments of methicillin-resistant Staphylococcus aureus. Such simple yet robust chemicals might be useful for various antibacterial applications while circumventing potential adverse effects associated with cytolytic compounds.

Antibacterial properties and mode of action of a short acyl-lysyl oligomer.

We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC(12)-2 beta(12), which was recently suggested to represent the shortest OAK sequence that retains nonhemolytic antibacterial properties. A growth inhibition assay against a panel of 48 bacterial strains confirmed that NC(12)-2 beta(12) exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC(12)-2 beta(12) demonstrated a bacteriostatic mode of action, unlike previously described OAKs that were bactericidal and essentially active against gram-negative bacteria only. The results of various experiments with binding to model phospholipid membranes correlated well with those of the cytotoxicity experiments and provided a plausible explanation for the observed activity profile. Thus, surface plasmon resonance experiments performed with model bilayers revealed high binding affinity to a membrane composition that mimicked the plasma membrane of staphylococci (global affinity constant [K(app)], 3.7 x 10(6) M(-1)) and significantly lower affinities to mimics of Escherichia coli or red blood cell cytoplasmic membranes. Additional insertion isotherms and epifluorescence microscopy experiments performed with model Langmuir monolayers mimicking the outer leaflet of plasma membranes demonstrated the preferential insertion of NC(12)-2 beta(12) into highly anionic membranes. Finally, we provide mechanistic studies in support of the view that the bacteriostatic effect resulted from a relatively slow process of plasma membrane permeabilization involving discrete leakage of small solutes, such as intracellular ATP. Collectively, the data point to short OAKs as a potential source for new antibacterial compounds that can selectively affect the growth of gram-positive bacteria while circumventing potential adverse effects linked to lytic compounds.

Design and characterization of a broad -spectrum bactericidal acyl-lysyl oligomer.

Previously characterized chemical mimics of host defense peptides belonging to the oligo-acyl-lysyl (OAK) family have so far failed to demonstrate broad-spectrum antibacterial potency combined with selectivity toward host cells. Here, we investigated OAK sequences and characterized a promising representative, designated C(12)K-3beta(10), with broad-spectrum activity (MIC(90) = 6.2 microM) and low hemotoxicity (LC(50) > 100 microM). Whereas C(12)K-3beta(10) exerted an essentially bactericidal effect, E. coli bacteria were killed faster than S. aureus (minutes versus hours). Mechanistic studies addressing this difference revealed that unlike E. coli, S. aureus bacteria undergo a transient rapid bactericidal stage that over time converts to a bacteriostatic effect. This behavior was dictated by interactions with cell wall-specific components. Preliminary efficacy studies in mice using the thigh infection model demonstrated the OAK's ability to significantly affect bacterial viability upon single-dose systemic treatment (2 mg/kg).

Structure-activity relationships of antibacterial acyl-lysine oligomers.

We describe structure-activity relationships that emerged from biophysical data obtained with a library of antimicrobial peptide mimetics composed of 103 oligoacyllysines (OAKs) designed to pin down the importance of hydrophobicity (H) and charge (Q). Based on results obtained with OAKs displaying minimal inhibitory concentration < or = 3 microM, the data indicate that potent inhibitory activity of the gram-negative Escherichia coli and the gram-positive Staphylococcus aureus required a relatively narrow yet distinct window of HQ values where the acyl length played multiple and critical roles, both in molecular organization and in selective activity. Thus, incorporation of long-but not short-acyl chains within a peptide backbone is shown to lead to rigid supramolecular organization responsible for poor antibacterial activity and enhanced hemolytic activity. However, sequence manipulations, including introduction of a tandem lysine motif into the oligomer backbone, enabled disassembly of aggregated OAKs and subsequently revealed tiny, nonhemolytic, yet potent antibacterial derivatives.

Effects of acyl versus aminoacyl conjugation on the properties of antimicrobial peptides.

To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptide's global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.