INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

This paper describes a series of N-substituted glycine trimers (herein generically referred to as peptoids) with antibacterial activity. Peptoids (7) have been used for a variety of applications. Recently, lipid-modified peptoid molecules, referred to as liptoids, have been used to deliver DNA to mammalian cells (9) and peptoid-peptide conjugates have been demonstrated to bind to SH3 domains with a higher affinity than peptides alone (19). Combinatorial chemistry peptoid libraries are part of the collection of small molecule libraries that the Chiron Corporation synthesizes and screens for pharmaceutical activity. In another report we describe the peptoid library that led us to individual antibacterial peptoid molecules (18). In this paper we characterize the biological properties of some of these new antibacterial molecules.

The structure of peptoids distinguishes them from the seemingly similar peptides and other molecules known to have antibacterial properties. Peptoids have been shown to be resistant to proteolysis (14), and it is likely that the pharmacokinetics and pharmacodynamics of these molecules will be different than those of previously described molecules. It is possible to robotically make a great variety of peptoids, and this diversity offers the potential for designing desirable properties into compounds from this class. The aim of this study was to characterize the biological properties of a few select antimicrobial peptoids in order to evaluate them as future drug development candidates.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. The strains obtained from the American Type Culture Collection (ATCC, Rockville, Md.) included Staphylococcus aureus strains (ATCC 25923, 29213, 13709) Streptococcus pneumoniae (ATCC 49619), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC 35667), Escherichia coli (ATCC 25922, ATCC 43827 ML-35), and Pseudomonas aeruginosa (ATCC 27853). All other strains listed in the tables, figures, or text are from the strain collection of the Center for Research in Anti-Infectives and Biotechnology, Creighton University.

Media for in vitro assays. Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England), Mueller-Hinton cation-adjusted broth (MHCA) (Becton Dickinson, Cockeysville, Md.), and brain heart infusion (BHI) broth (Difco, Detroit, Mich.) were used for in vitro assays.

Peptoid synthesis. The choice of the substituents (amine building blocks) for the CHIR29498 and analogue molecules was described previously (18), and the chemistry for peptoid synthesis is described in detail in Figliozzi et al. (7). The structures for the peptoids described within this study are shown in Fig. 1.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Structure of DHAA and the peptoids. CHIR26240 is a dimer peptoid, and the other peptoids shown are trimers. CHIR32133 is one of the series of six positional isomers shown in Table 1. The bold A, B, and C near the substituents of CHIR32133 are used to define substituent positions on the peptoid scaffold. By using this molecule to define the positions, the substituents in the positional isomers are thus described as e.g., BCA. The structures for the six positional isomers of CHIR32133 described in Table 1 are 32133 (shown above), ABC; 33090, ACB; 33091, BAC; 33092, BCA; 33670, CAB; and 33671, CBA.

In vitro susceptibility testing. Custom microbroth dilution trays (MIC2000, Dynatech Laboratories, Inc., Chantilly, Va.) were prepared with MHB. Chiron compounds (concentrated stocks of 20 mM in 100% dimethyl sulfoxide [DMSO]) were diluted into MHB and tested in a doubling dilution series (0.6 to 40 µM) (16). The final DMSO concentration was below 1% in these assays, and appropriate DMSO controls showed no effect on growth (data not shown). The comparison agents vancomycin and gentamicin were tested in the range of 1 to 128 µg/ml. Bacterial strains were incubated overnight at 37°C in MHB and inoculated into the custom trays (final inoculum, 10⁴ CFU). Trays were incubated at 37°C for 18 to 24 h before results were read.

Time-kill studies in the presence and absence of chloramphenicol. S. aureus SA4 was grown overnight in MHB to a culture density of approximately 10⁹ CFU/ml. Cells were then diluted 1:100 in sterile physiologic saline solution, and a 1-ml aliquot of this suspension was then added to 99 ml of prewarmed MHB or MHB containing 8 µg of chloramphenicol/ml to achieve initial culture densities of approximately 5 × 10⁵ CFU/ml. Cultures were incubated at 37°C with agitation, and peptoid antimicrobials were added 5 min after inoculation. Sampling aliquots were removed from all cultures immediately after peptoid addition (t = 0 h) and then again at 30 min and 1, 2, 4, 6, and 24 h after peptoid addition. Aliquots containing chloramphenicol were treated for 5 min at 37°C with chloramphenicol acetyltransferase in the presence of acetyl coenzyme A to inactivate the drug and prevent carryover. All other aliquots were treated in parallel by addition of an equal amount of sterile MHB. Treated samples were then serially diluted in sterile saline and plated in duplicate by adding 1-ml aliquots to molten MHA. Solidified plates were then incubated at 37°C for a minimum of 18 h before viable counts were determined. Data from duplicate plate counts were averaged, and the resulting values were plotted on a log scale against time. The lower limit of detection in this assay was approximately 1 CFU/ml.

Leakage assays. E. coli ML-35 (lacI lacZ+ lacY) was utilized for an inner membrane leakage assay (21). Logarithmic-phase bacteria (6 × 10⁶ CFU) were incubated in 0.6 ml of a 10 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl and 1.5 mM o-nitrophenyl--D-galactopyranoside (ONPG) at 37°C. At time zero, defined amounts of test compounds were added, and the conversion of substrate to product was measured by spectrophotometric readings of absorbance at 405 nm over time. Sonicated ML-35 cells were used as a positive control for total lysis and to demonstrate that the test compounds did not stimulate or inhibit the -galactosidase activity. Gramicidin S (GS; Sigma) was included in the assay as a positive control for drug-induced leakage (11).

Flow cytometry. The flow cytometric determination of bacterial fluorescence was carried out according to Mason et al. (12, 13). Propidium iodide (PI; Molecular Probes, Inc.) was diluted from a 1-mg/ml stock solution to 100 µg/ml in MHCA medium. S. aureus ATCC 25923 was grown in MHCA broth and diluted in MHCA to a final concentration of 10⁸ CFU/ml. One milliliter of culture was incubated with the appropriate drug for 30 min or 2 h. The cultures were then centrifuged for 10 min at 10,000 × g, the supernatant was aspirated, and the bacterial pellet was then resuspended in 1 ml of fresh MHCA broth. Two hundred microliters of the bacterial suspension was incubated in the dark for 5 min at room temperature with 0.8 ml of MHCA plus the PI working solutions.

Hemolysis assay. Blood from female BALB/c mice or male humans was drawn with heparin as the anticoagulant. The blood was processed and assayed (20) immediately. The erythrocytes (RBC) were spun at 1,000 × g, washed three times with 10 volumes of phosphate-buffered saline (PBS), and then resuspended in PBS. Washed RBC were treated with diluted test compound or control fluid. Triton X-100 (0.1%) was used for positive-control hemolysis values, and DMSO was used as the negative control since all tested compounds were dissolved in DMSO. The final DMSO concentration for each test sample was 1%. The samples were incubated in Costar 96-well round-bottomed microtiter plates at 37°C for 60 min. Plates were centrifuged at 1,000 × g for 5 min, and 100 µl of supernatant was transferred to deep-well 96-well plates and diluted with 900 µl of PBS. A 200-µl aliquot of the diluted supernatant was transferred to a Corning 96-well flat-bottomed plate and read in a spectrophotometer at 560 nm to evaluate heme release. Human blood was tested due to our interest in peptoids as future therapeutic molecules, and mouse blood was tested to determine whether hemolysis would be a problem in the animal model.

Murine infection assay. Master stocks of S. aureus (Smith, ATCC 13709) were prepared by growth to log phase in BHI broth and then frozen in 0.5-ml aliquots at 80°C in 10% glycerol. On the morning of the experiment, 0.25 ml of thawed bacterial stock was inoculated into 20 ml of BHI and grown at 37°C for approximately 7 h until a density of ~5 × 10⁹ CFU/ml was achieved as measured by absorption at 600 nm in relation to a growth curve plotted under identical conditions. Plate counts were made from stock dilutions to confirm the exact CFU per milliliter used in the experiment, and values ranged from 0.75 × 10⁸ to 2.5 × 10⁸ CFU/mouse.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Activity profiles. MIC data for dehydroabietylamine (DHAA) and the peptoid compounds are shown in Table 1. MICs for these compounds are presented as micromolar values to allow for direct comparison of the candidate drugs based on the number of molecules delivered rather than the weight of drug delivered. Peptoid trimers consist of a peptoid backbone (poly-glycine) with three substituted nitrogens (Fig. 1). Likewise, dimer peptoids are defined as a peptoid backbone with two substituted nitrogens.

Killing kinetics. Although CHIR29498 and CHIR29496 differ at the C terminus (Fig. 1), both peptoids demonstrated very similar growth inhibition profiles against the strains tested (Table 1), and both demonstrated rapid killing in assays in which treated S. aureus were plated minutes after treatment (data not shown). CHIR29496 was further analyzed by a comparison of the killing kinetics in the presence and absence of chloramphenicol (Fig. 2). Peptoid killing activity was not eliminated by protein synthesis inhibition. Incubation of SA4 cells with 8 µg of chloramphenicol/ml was used to produce bacteriostasis through reversible blockage of protein synthesis. Since viable counts from the initial (t = 0 h) aliquots from peptoid-containing cultures were substantially below those of the growth control culture, all graph traces were plotted using the t = 0 h growth control count as the initial point. This can be justified in that all cultures were inoculated with equal amounts of a common culture within a few seconds of each other. The lower counts in the peptoid-containing cultures are therefore the result of 5 min of peptoid activity on the culture (the time required for chloramphenicol acetyltransferase inactivation of chloramphenicol prior to plating in drug-free agar).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Effect of protein synthesis inhibition on antibacterial activity. The data are numbers of viable cells (CFU/milliliter) in aliquots removed at various times after drug exposure compared to those for a drug-free control culture. This experiment utilizes the S. aureus strain SA4 obtained from the Center for Research in Anti-Infectives and Biotechnology, Creighton University, strain collection. It is a methicillin-susceptible S. aureus clinical isolate obtained in Florida in 1971.

Leakage assays. To see if exposure to peptoid antimicrobials caused leakage of cellular contents, an inner membrane leakage assay was performed (21). E. coli ML-35 (ATCC43827) constitutively produces cytoplasmic -galactosidase and is both lactose and permease deficient. Extracellular hydrolysis of the substrate ONPG is therefore only possible if -galactosidase is released from the cells through membrane leakage or cellular lysis. Figure 3 shows steadily increasing levels of extracellular enzyme activity (measured by absorbance at 405 nm) when this strain was exposed to the trimer peptoid CHIR29498 at or above MIC levels. Given the rapidly bactericidal activities we observed with these compounds (Fig. 2), the slow, steadily increasing levels of extracellular activity suggest leakage from intact cells rather than release through complete lysis. Further, though levels of released enzyme were higher in the presence of higher concentrations of drug, the rates of release remained similar. The kinetics shown in Fig. 3 suggest that while both drug levels produced similar final rates of enzyme efflux, more time was required to achieve this level of permeabilization at the lower drug concentration. The dimer peptoid CHIR26240 produced an initial small release of -galactosidase which leveled off within 10 min of drug addition. DHAA produced no increase in extracellular enzyme activity at the levels tested for both the dimer and trimer peptoids. The steadily increasing levels of released enzyme seem to be correlated with bactericidal activity as neither CHIR26240 nor DHAA was active against E. coli at the levels tested but CHIR29498 was. GS, which is known to cause leakage (11), was included for reference (Fig. 3).

View larger version (23K): [in this window] [in a new window]   FIG. 3.   CHIR29498 and leakage of -galactosidase from E. coli ML-35. (A) Profile of -galactosidase activity in the supernatant of CHIR29498- or GS-treated cells compared to that for DMSO-treated cells. (B) -Galactosidase activities from the supernatants of CHIR26240-, DHAA-, and DMSO-treated cells.

Hemolysis assay. We utilized a simple assay in which washed mouse or human RBC were exposed to CHIR29498 or 0.1% Triton X-100 and released heme was measured (20). It was apparent (Fig. 4) that CHIR29498 caused heme leakage from washed RBC at some value greater than its corresponding MIC for staphylococci (Table 1). The hemolytic activity of CHIR29498 was fairly modest compared to that of other cell-membrane-active molecules such as magainin 2 (1) or the positive control GS.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Hemolysis measurements of washed human RBC treated with CHIR29498, CHIR26240, DHAA, ampicillin or DMSO. Values are represented as the percentages of total lysis compared to lysis by 0.1% Triton X-100.

Murine infection assay. In the absence of detailed knowledge of the pharmacokinetics of CHIR29498, we sought in vivo verification of the efficacy of this compound in a model of S. aureus infection and treatment. In this model, CD-1 mice were infected via i.p. injection with S. aureus (Smith, ATCC 13709) and then either immediately (Table 3), or after a defined delay (Table 4), dosed with a single i.p. injection of the drug to be tested. In preliminary experiments, threefold inoculum increments between 3 × 10⁶ and 3 × 10⁸ CFU/mouse were tested, and the bacterial dose causing 100% lethality after i.p. infection was determined to be approximately 10⁸ CFU/mouse. Test compounds were then evaluated based on their ability to protect mice from this otherwise lethal inoculum. The infection load was heavy to allow for unambiguous interpretation of the number of animals rescued from the infection by the treatment.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The peptoids described in this paper are easily synthesized molecules which display rapid bactericidal activities against a panel of gram-positive and gram-negative bacteria, including both drug-sensitive and multiply-resistant isolates. The peptoid compounds tested in these studies were generally slightly more active against gram-positive than gram-negative isolates. The bactericidal activities of the peptoids were found to be protein synthesis independent, and rearrangement within a peptoid molecule did not seem to greatly affect potency as positional isomers of a tested molecule retained most of the parent molecule's activity.

Antibacterial peptoids have an activity profile that is similar to that of the extensively described antibacterial peptides (cecropins, defensins, attacins, diptericins, magainins, protegrins [4, 10, 15, 17, 24]). The smallest natural antibacterial peptide to date is indolicidin (6), which is 13 amino acids in length. Screening of synthetic peptide libraries for antibacterial activity identified activity in 6-mer synthetic peptides which were most potent when unnatural amino acids were included in the synthesis (2, 3). By contrast, we have identified antibacterial peptoids, such as CHIR26240, which are smaller than these synthetic antibacterial peptides. Studies of antibacterial peptides have shown that the D and the L forms were equally potent, suggesting a target that does not require a lock-and-key interaction (22). Likewise, positional isomers of an antibacterial peptoid tested in this study retained most of the activity of the parent molecule. It is commonly accepted that antibacterial peptides cause membrane leakage, perhaps by interfering with the charge of the phosphate on phospholipids of the membranes. In some cases the amphipathic nature of the peptoids may lead directly to pore formation in membranes. In reality, the mechanism of the membrane disruption for membrane-disrupting compounds has yet to be fully elucidated. However, a profile for such membrane-active compounds can be compiled from the peptide and peptoid literature: rapid bactericidal activity independent of protein synthesis, rapidly induced leakage (as measured by leakage assays), and active molecules with equally active isomers.

CHIR29498 was not found to be aggressively hemolytic in vitro at concentrations that were effectively antimicrobial. It did, however, cause hemolysis at higher concentrations. We do not know if hemolysis is responsible for the high-dose (60 mg/kg) animal toxicity observed in this study (described in Materials and Methods).

In vivo antimicrobial efficacy of the peptoid compounds was demonstrated in a simple S. aureus mouse infection model. This model is used when little is known about the pharmacokinetic properties of a lead molecule. To make the i.p.-i.p. model more stringent, we delayed the time between infection and drug treatment. CHIR29498 protected infected animals even when treatment was delayed by up to 110 min; however, the drop in efficacy upon time delay (Table 4) compared to that of immediate dosing (Table 3) indicates that CHIR29498 may require optimization for improved absorption or stability within the body. Although this is a very simple model, it was capable of discriminating among CHIR29498, CHIR26240, and DHAA, all of which were active in vitro.

Finally, although we did not know that DHAA was antibacterial when it was first selected for the combinatorial libraries from which these peptoids were derived, adding properties to an already active moiety may be yet another useful application of peptoid chemistry. We have identified active peptoids that do not contain independently active substituents and thus would like to emphasize that it is possible to create antimicrobial peptoids from building blocks which do not have independent activity.