Hexapeptide Derivatives of Glycopeptide Antibiotics: Tools for Mechanism of Action Studies

Hexapeptide (des-N-methylleucyl) derivatives of LY264826 wereprepared in order to examine further the role of N-substitutedhydrophobic side chains in defining the mechanisms of actionof semisynthetic glycopeptide antibiotics. The hexapeptide ofLY264826 binds to the cell wall intermediate analog L-Lys-D-Ala-D-Alawith a 100-fold lower affinity than LY264826 and inhibits Micrococcusluteus almost 200-fold more poorly than LY264826 does. Alkylationof the 4-epi-vancosamine moiety of the disaccharide significantlyenhanced the antibacterial activity of the hexapeptide. Alkylationdid not affect the binding affinity for D-alanyl-D-alanine residues;however, it did enhance dimerization 7,000-fold and enhancedbinding to bacterial membrane vesicles noticeably compared withthe levels of dimerization and binding for the unsubstitutedhexapeptide. The findings from this study complement those presentedin an earlier report (N. E. Allen, D. L. LeTourneau, and J.N. Hobbs, Jr., J. Antibiot. 50:677-684, 1997) and are consistentwith the conclusion that the enhanced antibacterial activitiesof semisynthetic glycopeptide antibiotics derive from the abilityof the hydrophobic side chain to markedly affect both dimerizationand binding to bacterial membranes.

INTRODUCTION

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Introduction

The heptapeptide moiety of glycopeptide antibiotics such asvancomycin forms a carboxylate-binding pocket, which binds toD-alanyl-D-alanine (D-Ala-D-Ala) peptidyl residues of disaccharidepentapeptide cell wall intermediates (4, 26, 31, 33). Bindingto D-Ala-D-Ala residues can lead to inhibition of transglycosylationand/or transpeptidation; these interactions in turn lead tobacterial growth inhibition (33). LY264826 (A82846B, chloroeremomycin,or chloroorienticin A) differs from vancomycin in having a 4-epi-vancosaminesugar substituted for vancosamine in the disaccharide attachedat residue 4 and an additional 4-epi-vancosamine attached atresidue 6 of the linear heptapeptide (Fig. 1) (29). The antibacterialactivity of LY264826 is approximately four- to eightfold greaterthan that of vancomycin (24, 27, 35). Several derivatives ofLY264826 with N-substituted alkyl hydrophobic side chains onthe 4-epi-vancosamine of the disaccharide sugar and exquisiteantibacterial activities against both vancomycin-susceptibleand -resistant bacteria have been described (12, 25, 27, 28,34).

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FIG. 1. Chemical structures of glycopeptide antibiotics included in this study. Me, methyl.

Despite differences in antibacterial activities, vancomycin,LY264826, and the hydrophobic side chain derivatives of LY264826share nearly identical binding affinities for D-Ala-D-Ala residueswhen they are measured in solution with a model peptide (2,3). VanA and VanB-type strains of vancomycin-resistant enterococci(VRE) contain D-alanyl-D-lactate (D-Ala-D-Lac) depsipeptidylresidues to which vancomycin binds very poorly (8, 32). Manyof the hydrophobic side chain derivatives of LY264826 have verygood activities against VRE (27, 28), yet these same agentshave nearly the same binding affinities as vancomycin and LY264826for D-Ala-D-Lac residues in solution (2, 3). Clearly, the antibacterialactivities of the side chain derivatives cannot be explainedon the basis of simple binding affinities for D-Ala-D-Ala andD-Ala-D-Lac residues measured in solution.

LY264826 and its derivatives (as well as some other membersof the glycopeptide class) have a strong tendency to self-associateand form homodimers (2, 3, 15, 18). The extent of dimerizationof LY264826 is approximately 2 orders of magnitude greater thanthat of vancomycin (2, 15, 22). The N-substituted derivativesof LY264826 are even more strongly dimerized, with the extentof dimerization apparently determined by the nature of the sidechain (3). The N-substituted derivatives also demonstrate agreater tendency to interact with bacterial membranes, a processthat could serve to anchor these agents at the in vivo targetsite (2, 3, 5, 6, 11, 37). Our studies (3) revealed a relativelyhigh degree of correlation between the extent of dimerizationof glycopeptide antibiotics and antibacterial activity and avery high degree of correlation between antibacterial activityand the concentration of a D-Ala-D-Ala-containing tripeptideligand needed in the growth medium to antagonize antibacterialactivity. The antagonism experiments are particularly pertinentto the mechanisms of action of these glycopeptides because theresults imply that growth inhibition is accompanied by strongerinteractions with D-Ala-D-Ala-containing cell wall intermediatesat the in vivo target site than with the D-Ala-D-Ala-containingtripeptide ligand in solution. The combined effect of dimerizationand membrane anchoring can lead to cooperative interactionsthat facilitate strong intramolecular effects at the targetsite that can fully account for the enhanced antibacterial activities.These kinds of interactions are not predicted by estimatingassociation constants in free solution. However, the contributionof intramolecular effects to the binding of glycopeptide antibioticsto D-Ala-D-Ala and D-Ala-D-Lac residues at the bacterial surfacehas been demonstrated by nuclear magnetic resonance and surfaceplasmon resonance techniques with a variety of model systems,namely, phosphatidylcholine vesicles (13, 37), lipid monolayers(10, 11), and a whole-cell antagonist binding assay (5).

In this report, we directly address the question of whetheraddition of an alkyl side chain can compensate for a major reductionin antibacterial activity due to a modification of the carboxylate-bindingpocket. The des-N-methylleucyl derivative of LY264826 (LY312607)lacks the N-terminal amino acid of the heptapeptide core (24)which is crucial for tight binding of D-Ala-D-Ala residues intothe carboxylate-binding pocket (38). The antibacterial activityand D-Ala-D-Ala residue binding affinity of LY312607 are 2 ordersof magnitude lower than those of LY264826. We report here thatN substitution of LY312607 results in significantly enhanceddimerization, membrane binding, and antibacterial activity,even though it has no effect on simple binding affinity forD-Ala-D-Ala-containing peptidyl residues in solution.

MATERIALS AND METHODS

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Materials and Methods

Chemicals. LY264826 was from Eli Lilly & Company. The semisyntheticderivative LY307599 (4-phenylbenzyl) was prepared as describedpreviously (12). Analytical and preparative high-pressure liquidchromatography (HPLC) analyses were conducted as described previously(12). Hexapeptides were prepared by standard Edman degradationtechniques (7, 24) as described below. N,N'-Diacetyl-L-Lys-D-Ala-D-Alawas purchased from Sigma Chemical Co.

Experimental procedures. (i) Preparation of LY312607. The triacetate salt of LY264826 (A82846B) (1.0 g, 0.56 mmol)was dissolved in 20 ml of H₂O-pyridine (1:1 [vol/vol]) and treatedwith phenyl isothiocyanate (0.10 ml, 0.84 mmol). The resultingmixture was stirred at room temperature for 1 h, at which timeHPLC analysis indicated complete consumption of the startingmaterial. The reaction mixture was concentrated in vacuo, andthe crude product was purified by preparative HPLC to give 0.74g (68% yield) of the desired thiourea intermediate. Fast atombombardment mass spectrometry (FAB-MS): calculated for C₈₀H₉₃Cl₂N₁₁O₂₆S,1,725.5; obtained, 1,728.3. A sample of the purified thioureaintermediate (133.4 mg, 0.0772 mmol) was suspended in 25 mlof CH₂Cl₂, cooled to 0°C, and then treated with trifluoroaceticacid (0.25 ml). After 1 h the reaction mixture was warmed toroom temperature and stirred for an additional 2 h. The solventwas removed in vacuo, and the crude product was purified bypreparative HPLC to give 51.7 mg (46% yield) of LY312607 asa white powder. FAB-MS: calculated for C₆₆H₇₅Cl₂N₉O₂₅, 1463.4;obtained, 1,466.7.

(ii) Preparation of LY314015. LY307599 (41 mg, 0.023 mmol) was dissolved in 4 ml of H₂O-pyridine(1:1 [vol/vol]) and treated with phenyl isothiocyanate (4.0µl, 0.033 mmol). The resulting mixture was stirred atroom temperature for 3 h, at which time HPLC analysis indicatedcomplete consumption of the starting material. The reactionmixture was concentrated in vacuo to give the crude thioureaintermediate as a white solid. The thiourea derivative was suspendedin 10 ml of CH₂Cl₂, cooled to 0°C, and then treated withtrifluoroacetic acid (0.25 ml). After 30 min the reaction mixturewas warmed to room temperature and stirred for an additional1 h. The solvent was removed in vacuo, and the crude productwas purified by preparative HPLC to give 14.0 mg (37% yield)of LY314015 as a white powder. FAB-MS: calculated for C₇₉H₈₅Cl₂N₉O₂₅,1,629.5; obtained, 1,632.5.

Ligand binding. Affinity capillary electrophoresis was used to measure the levelof binding of N,N'-diacetyl-L-Lys-D-Ala-D-Ala to glycopeptidesby published procedures (9, 17). Electrophoretic mobilitieswere measured with a P/ACE 5000 (Beckman Instruments) equippedwith a fused-silica capillary (57 cm [50 cm to the detector]by 75 µm [inner diameter]). Mesityl oxide was used asa neutral marker. Analytes were detected at 214 nm. The runbuffer was sodium phosphate; the ionic strength of the bufferwas varied from 20 to 75 mM and the pH was varied from 6.7 to7.1 to optimize the peak shape. The voltage was consequentlyvaried from 30 kV for buffer with a low ionic strength to 7.5kV for buffer with a high ionic strength; polarity was positiveto negative.

The difference between the electrophoretic mobility of a glycopeptidewith a given ligand concentration in the run buffer and theelectrophoretic mobility of the glycopeptide in the absenceof ligand in the run buffer ( ${Delta}$ µ_G,L) was calculated by theequation

(1)
where l_c is the total lengthof the capillary; l_d is the length of the capillary to the detector;V is voltage, t_G and t_EO are the migration times of the glycopeptideand the neutral marker, respectively, in run buffer with noligand added; and t_G,L t_EO,L are the migration times of theglycopeptide and the neutral marker, respectively, with ligandadded (17). Binding constants (K_b) were estimated from the relationship

(2)
where [L] is the ligand concentration, and ${Delta}$ µ_G,L^maxis the mobility of the glycopeptide when it is saturated withligand. K_b was determined as the negative slope from a plotof ${Delta}$ µ_G,L/[L] against ${Delta}$ µ_G,L (17).

Dimerization. The extent of dimerization of the glycopeptide antibiotics wasmeasured by capillary zone electrophoresis. The methodologyused to measure the mobility of glycopeptide antibiotics asa function of their concentration and the mathematics used toestimate dimerization constants (K_dim) have been described previously(21).The electrophoretic mobility (µ_obs) of a glycopeptidewas calculated by the equation

(3)
Theconcentrations of the glycopeptides as they passed the detectorwere determined by using the peak height, as explained previously(1). K_dim were calculated by the relationship

(4)
whereµ_d is the electrophoretic mobility of a hypothetical samplecontaining all dimer, µ_m is the electrophoretic mobilityof a hypothetical sample containing all monomer, and [G]_t isthe concentration of glycopeptide (monomer plus dimer) (21).Equation 4 was fitted to µ_obs and [G]_t by nonlinear regressionanalysis with a curve-fitting program (JMP; SAS Institute).

Membrane binding. Membrane vesicles were prepared by treatment of Bacillus megateriumwith N-acetylmuramidase, DNase, and RNase by previously publishedmethods (20). Binding of glycopeptides to membrane vesicleswas measured as described previously (2).

Antibacterial activity and antagonism assays. MICs were determined for Micrococcus luteus ATCC 9341 grownin TY broth (10 g of tryptone per liter, 5 g of yeast extractper liter, 5 g of NaCl per liter) in a microplate format. Eachwell contained 200 µl of TY broth, and the inoculum wasa 1:40,000 dilution of an overnight culture. The plates wereincubated overnight at 35°C and read at 650 nm in a ThermoMaxplate reader (Molecular Devices). Antagonism of antibacterialactivity by N,N'-diacetyl-L-Lys-D-Ala-D-Ala was determined byusing the same microplate format and procedure, except thatN,N'-diacetyl-L-Lys-D-Ala-D-Ala was added to the wells priorto inoculation. Each glycopeptide concentration was tested forinhibition in the presence of a range of N,N'-diacetyl-L-Lys-D-Ala-D-Alaconcentrations. The molar excess (ME) is the ratio of [N,N'-diacetyl-L-Lys-D-Ala-D-Ala]:[glycopeptide]in the well containing the lowest N,N'-diacetyl-L-Lys-D-Ala-D-Alaconcentration that completely suppressed growth inhibition.The values given in Table 1 represent the mean ME calculatedfor several inhibitory concentrations of glycopeptide. ME valuesvaried by 1 or 2 dilutions when they were tested with differentglycopeptide concentrations. Therefore, ME values were determinedover a fivefold range around the MIC and were averaged to maximizethe accuracy of the ME estimate.

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TABLE 1. Binding of cell wall ligand, dimerization, antibacterial activity, and antagonism by heptapeptide and hexapeptide derivatives of glycopeptide antibiotics

RESULTS AND DISCUSSION

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Results and Discussion

N-substituted derivatives of LY264826 have greater antibacterialactivities than the unsubstituted parent compound derived, atleast in part, from the augmentation of dimerization and membraneanchoring (3, 5, 37). Strongly dimerized glycopeptides havethe advantage that their interactions with D-Ala-D-Ala residues(and the D-Ala-D-Lac residues of vancomycin-resistant bacteria)can be influenced and driven by intramolecular effects (5, 6,10, 11, 23, 30, 37). These kinds of interactions are much strongerand more complex than the bimolecular interactions that typifythe actions of more weakly dimerized glycopeptides such as vancomycin.Findings from this laboratory (2, 3) and others (5, 11, 37)support the notion that the enhanced antibacterial activitiesof LY264826 and its N-substituted derivatives against both vancomycin-susceptibleand -resistant bacteria derive from the cooperative effectsof dimerization and membrane anchoring.

Hexapeptide derivatives of glycopeptide antibiotics lack theN-terminal amino acid of the carboxylate-binding pocket andtherefore lack one of the hydrogen bonds necessary for bindingto cell wall precursors. Loss of this amino acid is accompaniedby a 100-fold reduction in binding affinity and a comparableloss in antibacterial activity (38). We have examined the influenceof the addition of a hydrophobic side chain to the hexapeptideof LY264826 on its antibacterial properties. Table 1 presentsthe binding constants for N,N'-diacetyl-L-Lys-D-Ala-D-Ala, dimerizationconstants, MICs for M. luteus, and a measure of the ME of N,N'-diacetyl-L-Lys-D-Ala-D-Alarequired to completely antagonize growth inhibition for LY264826,LY312607 (the des-N-methylleucyl hexapeptide analog of LY264826),LY307599 (the N-phenylbenzyl side chain derivative of LY264826),and LY314015 (LY312607 substituted with an N-phenylbenzyl sidechain) (see Fig. 1 for the structures of these compounds).

The hexapeptide LY312607 is nearly 200 times less active thanLY264826 against M. luteus. The MICs of LY312607 for other gram-positivebacteria, including vancomycin-susceptible and -resistant bacteria,were higher but followed the same pattern (data not shown).The binding affinity for N,N'-diacetyl-L-Lys-D-Ala-D-Ala wasreduced 100-fold. Interestingly, the extent of dimerizationof LY312607 was approximately 100-fold less than that of LY264826,and less free N,N'-diacetyl-L-Lys-D-Ala-D-Ala ligand was requiredfor suppression of bacterial growth inhibition by LY312607 thanfor suppression of bacterial growth inhibition by LY264826.The intermolecular hydrogen bond formed between the amino groupof the 4-epi-vancosamine on residue 6 and the carbonyl on residue2 of the complementary half of the dimer is important for dimerization(18, 22); loss of the N-methylleucine may interfere with thisinteraction and compromise dimerization. The increased MIC ofLY312607 could be the result of a reduction in both the bindingaffinity and the extent of dimerization of this compound.

The hexapeptide LY314015 contains an N-phenylbenzyl side chainsubstitution. The presence of the hydrophobic side chain onthis molecule did not affect the binding affinity for N,N'-diacetyl-L-Lys-D-Ala-D-Alabut did enhance the extent of dimerization by 4 orders of magnitudeover that for the hexapeptide without a side chain. LY314015dimerized as well as LY307599 (a heptapeptide) (Fig. 1 and Table1), consistent with the side chain playing a significant rolein dimerization (3). The antibacterial activity of LY314015was also an order of magnitude greater than that of LY312607but was still more than an order of magnitude less than thatof LY307599. The fact that a 1,032-fold ME of N,N'-diacetyl-L-Lys-D-Ala-D-Alawas sufficient for suppression of growth inhibition by LY314015whereas a 16,500-fold ME was required for suppression of growthinhibition by LY307599 may be related to the reduced bindingaffinity that LY314015 has for D-Ala-D-Ala-containing peptidylresidues.

Hydrophobic side chains on glycopeptide antibiotics facilitateinteractions with membranous structures (2, 3, 5, 6, 11, 22).We and others have proposed that hydrophobic side chains actas anchors at the membrane outer interface and facilitate intramoleculareffects, leading to very tight interactions with D-Ala-D-Alaresidues of cell wall intermediates. The N-phenylbenzyl hexapeptidederivative (LY314015) showed greater binding than the hexapeptidewithout a side chain to B. megaterium membrane vesicles (Fig.2). Increased membrane anchoring and enhanced dimerization byLY314015 can reasonably account for the increased ME of N,N'-diacetyl-L-Lys-D-Ala-D-Alarequired to suppress in vitro antibacterial activity. The MICof LY314015 is higher than that of LY307599, and the ME of N,N'-diacetyl-L-Lys-D-Ala-D-Alaneeded to antagonize inhibition by LY314015 is lower than theamount needed to antagonize inhibition by LY307599. Nevertheless,the differences between the values measured for the unsubstitutedhexapeptide and the hexapeptide with a side chain are dramaticand signify that addition of the side chain to the hexapeptidewas able to compensate for the reduced binding affinity of themodified carboxylate-binding pocket to D-Ala-D-Ala residues.

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FIG. 2. Binding of hexapeptide derivatives to membrane vesicles.

N-substituted derivatives of vancomycin (with alkyl and arylsubstitutions on the vancosamine sugar) with enhanced activityagainst vancomycin-susceptible and -resistant bacteria havebeen reported (14, 16). N-Chlorophenylbenzyl-vancomycin wasfound to be 30 times more active than vancomycin against vancomycin-susceptibleenterococci and 100 times more active than vancomycin againstVRE. The same group (14, 19) reported that the hexapeptide (des-N-methylleucyl)derivative of N-chlorophenylbenzyl-vancomycin was nearly asactive as the heptapeptide parent compound against VRE. Theinvestigators proposed that the unexpected antibacterial activityof the hexapeptide of N-chlorophenylbenzyl-vancomycin againstboth vancomycin-susceptible and -resistant bacteria was dueto direct inhibition of the transglycosylase enzyme and arguedthat the weak binding of the hexapeptide derivative to dipeptidylresidues could not explain the observed inhibitory effects.The fact that both N-chlorophenylbenzyl-vancomycin and N-chlorophenylbenzyl-des-N-methylleucyl-vancomycininhibited peptidoglycan formation and induced lipid II accumulationin an in vitro particulate membrane-based assay with eithera pentapeptide or a tetrapeptide substrate was cited as furtherevidence that these agents were acting directly on transglycosylasewithout directly binding to D-Ala-D-Ala or D-Ala-D-Lac residues(14, 16).

We have shown that N substitution of LY264826 with a hydrophobicside chain dramatically enhances dimerization, membrane interactions,and antibacterial activity without affecting simple bindingaffinity for D-Ala-D-Ala or D-Ala-D-Lac residues (1-3). Oritavancin(LY333328) is the N-chlorophenylbenzyl derivative of LY264826undergoing clinical trials for the treatment of serious infectionscaused by gram-positive bacteria, including those involvingVRE (28, 36). We (2, 3) and others (5, 6, 11, 37) have proposedthat classic chelate phenomena and the attendant intramoleculareffects can explain the enhanced antibacterial activities ofthese derivatives in vivo. We have not directly measured theeffects that dimerization and membrane anchoring have on interactionswith cell wall intermediates at the in vivo target site. Nevertheless,it is difficult to reconcile the results from antagonism experiments(note the ME values in Table 1) with a mechanism that does notaccommodate a role for a direct interaction with dipeptidyland didepsipeptidyl residues. It should be noted that the antagonismstudies were conducted with D-Ala-D-Ala-containing bacteria.A role for dimerization and anchoring in the mechanism of actionagainst D-Ala-D-Lac-containing bacteria derives from previouswork of others (10, 11, 13, 37) with various model systems andby inference from our studies reported here.

This study further emphasizes the significant role played byhydrophobic side chains in the mechanisms of action of semisyntheticglycopeptide antibiotics. We have no evidence for a direct effectof any of the N-substituted derivatives of LY264826 on transglycosylationor transpeptidation. However, despite the high correlationsamong the extent of dimerization, the MIC, and the degree ofantagonism (3), our findings do not rule out alternative mechanismsincluding those that do not rely on interaction with D-Ala-D-Alaor D-Ala-D-Lac residues (14, 16, 19). We believe that the moststraightforward explanation for the extraordinary activitiesof these compounds is through an association with cell wallresidues made possible by dimerization and membrane anchoring.We previously reported (3) that an analog of vancomycin withan N-phenylbenzyl substitution (the vancomycin analog of LY307599)dimerized readily (2 orders of magnitude more readily than vancomycin,as measured by capillary electrophoresis) and showed significantbinding to membrane vesicles. Although we did not examine des-N-methylleucylderivatives of vancomycin in detail, we note that N-substitutedderivatives of both vancomycin and LY264826 have enhanced activitiesagainst vancomycin-susceptible and -resistant bacteria, showenhanced extents of dimerization and membrane anchoring, andhigher ME values. The findings argue in favor of related mechanismsof action for both the vancomycin- and LY264826-type glycopeptidesat the in vivo target site.

FOOTNOTES

* Corresponding author. Present address: 1481 E. 77th St., Indianapolis, IN 46240. Phone: (317) 259-4933. Fax: (317) 277-4522. E-mail: norrisallen{at}comcast.net.

REFERENCES

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