Structure-activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues

Journal of Antimicrobial Chemotherapy (2003) 51, 821–831DOI: 10.1093/jac/dkg170Advance Access publication 13 March 2003

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© 2003 The British Society for Antimicrobial Chemotherapy

Structure–activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues

Neil J. Marshall1, Ryszard Andruszkiewicz2, Sona Gupta1, Sßawomir Milewski2 and John W. Payne1*

1School of Biological Sciences, University of Wales Bangor, Gwynedd LL57 2UW, UK; 2Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdaásk, Gdaásk, 80-952, Poland

Received 3 December 2002; returned 18 January 2003; revised 21 January 2003; accepted 21 January 2003

Synthetic glutamine analogues such as N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid(FMDP) inhibit purified glucosamine-6-phosphate synthase, an intracellular enzyme that isessential for microbial cell wall synthesis, but they are inactive against intact organismsbecause they cannot enter the cell. However, when the analogues are linked to a peptide they canbe actively transported, and FMDP peptidomimetics show broad-spectrum antimicrobial activ-ity. To characterize this process in more detail, the antibacterial activities of various syntheticpeptidomimetics containing glutamine analogues have been determined against isogenicstrains of Escherichia coli in which one or more of its three peptide transporters Dpp, Opp andTpp have been mutated. In addition, their affinities for DppA and OppA, the binding-protein com-ponents of the transporters, have been measured. In general, antibacterial activities against thevarious transport mutants correlated with binding to DppA and OppA. Xaa-FMDP compoundshave greater activities than FMDP-Xaa analogues. To explore structure–activity relationshipsfor the peptidomimetics, molecular modelling was used to determine the conformational formsthey adopt in solution. The relative bioactivities of the peptidomimetics correlated with thepercentage of conformers that had backbone torsions matching those previously defined forthe molecular recognition templates of the peptide transporters. However, the large size of theN-terminal residue in the FMDP-Xaa analogues appears to interfere with transport and thus tolimit antibacterial activity. Overall, the results provide the structural rationale for the identifi-cation in silico of analogues with optimal bioactivities, which decreases the need for extensivechemical syntheses and testing.

Keywords: cell wall biosynthesis, peptide transport, molecular modelling, molecular recognition

Introduction

When designing novel antimicrobial agents, enzymes involvedin the biosynthesis of microbial cell walls are generally goodtargets. In this regard, glucosamine-6-phosphate synthase[L-glutamine: D-fructose-6-phosphate amidotransferase (hex-ose isomerising)], which is responsible for the synthesis ofD-glucosamine-6-phosphate, is particularly attractive becauseit is involved in the first step in the formation of the coreamino-sugar N-acetyl glucosamine that is an essential part ofthe unique peptidoglycan and chitin components of the cellwalls of bacteria and fungi, respectively. Although the analo-gous enzyme is present in man, its inhibition is not a problem

because of the slow turnover of its relevant polysaccharideproducts. Thus, compounds that are able to inhibit thisenzyme can potentially be broad-spectrum antimicrobialagents, having both antibacterial and antifungal activities.Several well-studied glutamine analogues, e.g. 6-diazo-5-oxo-L-norleucine, azaserine and anticapsin, do inhibit the enzymebut lack specificity and inhibit other enzymes that use gluta-mine as a substrate.1,2 A rationally designed glutamine ana-logue, N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoicacid (FMDP), was shown to be both a strong and selectiveinhibitor of the enzyme in vitro, inhibiting the enzyme byforming a covalent linkage with the Cys residue located in theglutamine-binding domain.3–5 Unfortunately, FMDP proved

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*Corresponding author. Tel: +44-1248-382349; Fax: +44-1248-370731; E-mail: [email protected]

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ineffective against whole cells because it could not gain accessto the intracellular location of the enzyme.3,6 Subsequently,FMDP was converted into an active antimicrobial agent byincorporating it as part of a peptide, which allowed its activeaccumulation by peptide transporters in the form of apeptidomimetic prodrug susceptible to intracellular peptidaseaction.3,6,7

FMDP-peptides have been shown to inhibit variousbacterial species, including Escherichia coli (ATCC 9637),Shigella sonnei 433, Staphylococcus aureus 209P, Bacilluspumilus 1697, Bacillus subtilis3,6,8,9 and also fungi, e.g. Can-dida albicans.6,9 However, the inhibition assays allowed onlya rough ranking of relative antibacterial activities, e.g. forE. coli MICs of 100 or >200 mg/L,3 or the inhibitory effects ofa single amount of each inhibitor using disc diffusion assays.8

The rate-limiting step for antimicrobial activity is generallyuptake, although for B. subtilis uptake rates were generallyfaster than the intracellular hydrolysis rates that liberated freeFMDP.8 Furthermore, certain of these compounds havedemonstrated chemotherapeutic activity in a mouse model ofgeneralized candidiasis,10,11 showing them to be potentiallyvaluable, broad-spectrum antimicrobial compounds.

Molecular modelling of small peptides (two to fiveresidues) derived from protein hydrolysis has allowed identi-fication of the conformational forms they adopt in solution,and revealed how various peptide transporters have evolvedcomplementary specificities to recognize different conforma-tional types as ligands.12,13 From evaluation of the structuraland electronic features needed for this ligand recognition,individual molecular recognition templates (MRTs) for themicrobial peptide transporters have been defined.14–17 Thefollowing features are involved in defining MRTs: (i) chargedN-terminal α-amino and C-terminal α-carboxylate groups,permitting charge and hydrogen-bond donor and acceptorproperties; (ii) backbone torsion angles psi, phi and omega;(iii) chirality at α-carbons; (iv) N–C distance between ter-minal amino and carboxylate groups; (v) chi space torsionsand size of side chains; (vi) H-bond donor and acceptor prop-erties of peptide bond atoms; and (vii) charge fields aroundN-terminal α-amino group and C-terminal α-carboxylate. Todefine an MRT, each of these seven features must be of acertain type, e.g. positively-charged N-terminal α-aminogroup or L-chirality, or value, e.g. psi torsion angle of 150°.Knowing these MRTs, compounds can be modelled to assesstheir match to these parameters and thus their potential asputative transport ligands.

In the present study, structure–activity relationships havebeen explored for FMDP peptides using E. coli. The substratespecificities of the generic peptide transporters Opp, Dpp andTpp have well-characterized features that allow them to bedistinguished from each other, but they also share certain sub-strate recognition features, so that some peptides can be takenup by more than one transporter.14,17 Dpp and Opp are typical

ABC transporters energized by ATP; each comprises fourmembrane proteins plus a periplasmic peptide binding pro-tein DppA or OppA, respectively.17 In contrast, Tpp lacks abinding protein and comprises a single membrane protein thatis energized by a proton-motive force.17 To be transported byDpp or Opp, ligands must be recognized and bound by theirrespective binding proteins. A strong correlation existsbetween the affinity of ligands for these binding proteins anduptake rates through the respective transporters.12,14,16,18 Thus,compounds can be evaluated as potential transport ligands bymeasuring their abilities to compete for binding of radio-actively labelled ligands to DppA and OppA, which is notonly more convenient than directly assaying transport but inrequiring less sample is particularly convenient when littlematerial is available, as with many of the peptidomimeticshere. For antibacterial compounds, such binding studiescan complement inhibition assays. Here, structure–activityresults have been obtained using three approaches: first, anti-bacterial activities against isogenic strains of E. coli thatpossess different complements of the peptide transporters;secondly, peptide binding to purified peptide binding pro-teins; and thirdly, molecular modelling to derive structuralinformation on the conformations adopted by the peptido-mimetics in solution. The results should aid the design of anti-microbial peptidomimetic prodrugs of glutamine analogues.

Materials and methods

Peptide compounds

Peptidomimetics containing FMDP, N3-(fumaramoyl)-L-2,3-diaminopropanoic acid (FCDP) or N4-(4-methoxy-fumaramoyl)-L-2,4-diaminobutanoic acid (FMDB) were syn-thesized as described previously.3,8 The iodinated peptideligands, Gly[125I]Tyr and [125I2]TyrGlyGly were prepared asdescribed previously.18 The synthetic inhibitor, alafosfalin[Ala-L-1-aminoethylphosphonic acid; AlaAla(P)] was a giftfrom Roche Products, Welwyn Garden City, UK.14,18

Determination of the antibacterial activities of peptidomimetics

Peptidomimetics were tested using the following strains, theisolation and characterization of which were described previ-ously:18 a parental strain, E. coli K-12 Morse 2034 (trp, leu)(CGSC 5071), which is a wild-type with respect to Opp, Dppand Tpp; and three isogenic mutants: (i) strain PA0183(∆opp), derived from strain Morse 2034, in which the deletionextends from tonB to tdk; (ii) strain PA0333 (∆opp,dpp)derived from PA0183; and (iii) strain PA0410 (∆opp,tpp) alsoderived from PA0183. Inhibition was determined using thefollowing disc diffusion assay, adapted from recommendedprocedures.19 Bacteria were grown in minimal ‘A’ medium18

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supplemented with 0.5 mM L-Leu, 0.2 mM L-Trp, 0.05 mMFeCl3 and 0.4 % (w/v) D-glucose at 37°C with rotary shaking.Soft agar overlays (0.6 % w/v agar), similarly supplementedwith Leu, Trp and glucose, were inoculated with exponential-phase bacteria to give ∼1 × 107 cells/mL, to allow semi-con-fluent growth, and used to overlay a plate (1.5 % w/v agar) ofthe same composition. Typically, six filter paper discs wereplaced on the surface, 10 µL amounts of different concentra-tions of the peptidomimetics were added, giving a range of30–300 nmol, and the plates were incubated at 37°C. After 17h incubation, the appearance of any inhibition zones wasnoted and their diameters measured. Antibacterial activitywas expressed as the amount of inhibitor (nmol) required toproduce an inhibition zone of 25 mm and was determinedfrom semi-logarithmic plots of log10 concentrations of inhibi-tor versus inhibition zone diameters. These isogenic strainsall have the same rates of growth in the above liquid mediumand on these agar plates, so zone sizes are not susceptible todifferent growth rates18 (data not shown).

Assays for peptide binding to DppA and OppA

DppA and OppA were isolated from strain Morse 2034 by anosmotic shock procedure, purified to homogeneity by sequen-tial use of ion-exchange chromatography, freed from anyendogenous ligands using reverse-phase chromatography,lyophilized and stored at –20°C, as described.14,18 Competi-tive filter-binding assays were carried out using Gly[125I]Tyras the radioactive ligand for DppA, and [125I2]TyrGlyGly forOppA, as described previously.18 All competitor peptido-mimetics were tested at a molar ratio of 10:1 relative to theligands, and some were tested at other molar concentrations.As control competitors, AlaAla was used with DppA andAlaAlaAla with OppA. Controls were performed in whicheither the binding protein or a competing peptide was omitted.

Molecular modelling of peptides and peptidomimetics

Conformational analysis of zwitterionic peptides and peptido-mimetics was carried out using SYBYL software (Tripos Inc.,St Louis, MO, USA), in a similar manner to that described indetail previously.12–16,20,21 Briefly, starting structures wereassigned appropriate atom types and charges, and were energy-minimized before being subjected to random searches (2–5ksearch cycles) with an absolute energy cut-off of 40 kcal/mol,using a distance-based dielectric constant of 80 to simulate awater environment. Unique conformers were distinguishedby setting a root mean square threshold of 0.2 Å, together withchirality checking. The computed collection of conformersfor each compound was first ordered according to energy,before the percentage contribution of each conformer wasdetermined using a Boltzmann distribution. For each dipep-tide conformer, backbone torsion angles, psi (ψ) (Tor2),omega (ω) (Tor3) and phi (φ) (Tor4) were measured, together

with relevant side-chain torsions, chi (χ) and the distance(N–C) between the nitrogen of the N-terminal α-amino groupand α-carbon of the C-terminal α-carboxylate group. Fortripeptide conformers, analogous measurements were made,together with those for (ψi) Tor6, (ω2) Tor7 and (φi+1) Tor8(Figure 1). For each dipeptide compound, ψ and φ torsionalspace for the peptide unit was divided up into 36 10° sectorsand the percentage contributions of conformers havingparticular ψ–φ combinations were aggregated. Molecularmodelling of peptides has previously indicated that con-formers adopt a limited set of torsional combinations, whichhave been classified by dividing ψ and φ conformationalspace into 12 30° sectors designated A1 to A12 (0° to ±180°)and B1 to B12 (0° to ±180°), respectively.12,14–16 In this way,particular conformational forms can be defined by referenceto their location within the conformational grid, e.g. A7B9.Analogous procedures were adopted for tripeptides.16 Acomplete profile of the conformers of any compound can bedisplayed using 3D pseudo-Ramachandran plots (3DPR) thatrelate cumulative percentage of conformer to backbone ψ andφ torsions.12,14–16,20

The maximum lengths and volumes of the side chains ofFCDP, FMDP, FMDB and of natural amino acids were com-puted by placing a volume mesh around the residues modelledin fully extended conformations. Lengths were measuredbetween the Cα atom and the outermost heavy atom of achain. Volumes were determined for the side chain only, i.e.excluding the α-amino and α-carboxylate, and were related tothe volume of a water molecule (17.7 Å3).

Figure 1. Schematic of tripeptide mimetic LysNva-FMDP showing theamino acid residues i–1, i and i+1, with their associated charges. Back-bone torsion angles: Tor2 is ψi–1, Tor3 is ω, Tor4 is φi, Tor6 is ψi, Tor7is ω2 and Tor8 is φi+1. Side chain torsion angle χ1 is shown for Lys. N–Cdistance is measured from the N-terminal α-amino nitrogen to theC-terminal α-carboxylate carbon.

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Results

Antibacterial activities of peptidomimetics

The antibacterial activities of the peptidomimetics weretested against the parent strain M2034 and peptide-transportmutants of E. coli (Table 1). Because only limited amounts ofthe synthetic peptidomimetics were available, all compoundswere initially tested against the parent strain and those that didnot give a measurable level of inhibition, e.g. Gly-FMDP(Table 1), were not tested with the mutants. In addition, testswith the mutants were performed on the basis of their sub-strate specificities, e.g. Opp favours peptides with three tofive residues and transports dipeptides poorly, whereas Dppand Tpp transport di- and tripeptides only but with differentspecificities.17,18,22 Alafosfalin, a well-characterized, synthetic,antimicrobial peptide prodrug (smugglin),7,22 was used(0.5 nmol) as a control to check reproducibility of assay pro-cedures: for example, an inhibition zone of 18.6 ± 0.9 mm wasfound for all assays with strain M2034.

From Table 1, possible trends can be inferred on theinhibitory activities of the various analogues towards strainM2034. First, Xaa-FMDP dipeptides are markedly moreactive than FMDP-Xaa dipeptides. This can be seen specific-ally when comparing Leu-FMDP and Met-FMDP withFMDP-Leu and FMDP-Met. Secondly, Xaa-FMDP dipep-tides appear in general to have greater activity than analogousoligopeptides. Thirdly, the antibacterial activities of analoguesof FMDP are higher than those of FMDB (compare Nva-FMDP with Nva-FMDB) and FCDP analogues (compareFMDP-Ala with FCDP-Ala). These observations agree withthe relative inhibitory activities of the free glutamine ana-logues against the purified target enzyme: their IC50 valuesbeing FMDP 15 µM, FMDB 56 µM and FCDP 100 µM.1

However, from these inhibition data with M2034, it is unclearwhether variations in activities arise primarily from differ-ences in uptake or in intracellular cleavage, making resultswith transport mutants helpful.

Active Xaa-FMDP analogues can generally be transportedby both Dpp and Tpp, i.e. strains PA0410 and PA0333 wereinhibited, with activity against the former usually beinggreater (Table 1). Their activities against strains M2034 andPA0183 indicated that uptake by Opp also contributed to theiroverall antibacterial activity (Table 1). For the three oligo-peptides, inhibition of wild-type M2034 and no antibacterialactivity against PA0183 (Opp–,Dpp+,Tpp+) indicate that theiruptake occurs exclusively by Opp (Table 1). Thus, althoughboth Dpp and Tpp can transport ‘folded’ conformers of manynatural tripeptides, these particular tripeptide mimetics areeffectively recognized only by Opp, which recognizes ‘elon-gated’ conformers.14,17,22

Binding of peptidomimetics to DppA and OppA

Several trends can be observed from the relative abilities ofthe dipeptide analogues to compete for binding withGly[125I]Tyr to DppA (Table 2). Xaa-FMDP dipeptides aregenerally better competitors than FMDP-Xaa dipeptides; thiswas exemplified specifically when comparing Leu-FMDPand Met-FMDP with FMDP-Leu and FMDP-Met. Most Xaa-FMDP dipeptides showed broadly similar levels of competi-tive ability, although no activity was detectable for Gly-FMDP and FMDP-FMDP. These results are all in broadagreement with the relative inhibitory activities of thesecompounds (Table 1), indicating that transport rates appear tobe the main determinant of antibacterial activities. AcNva-FMDP also showed no competitive activity (Table 2), inaccordance with the requirement for a protonated N-terminalα-amino group for ligand binding to DppA and transport byDpp.17,18,22 Consequently, lack of binding and presumedtransport is sufficient explanation for its failure to inhibit(Table 1). In addition to the results for a ligand:competitorratio of 1:10 (Table 2), Nva-FMDP and Nva-FMDB were alsoassayed at 1:1 and 1:5 ratios and found to inhibit 73% and

Table 1. Antibacterial activities of glutamine analogue peptidomimetics against strains of E. coli having different peptide transporters

Activities are expressed as the amounts in nmol producing inhibition zones of25 mm, obtained by extrapolation from plots of inhibition zone diametersversus amounts of peptidomimetics.aBacterial strains have the following phenotypes for peptide transporters:wild-type strain M2034 (Opp+,Dpp+,Tpp+); PA0183 (Opp–,Dpp+,Tpp+);PA0410 (Opp–,Dpp+,Tpp–); PA0333 (Opp–,Dpp–,Tpp+).bBecause these activities were so low only the wild-type strain was tested.cBecause no inhibition was detected with this Opp– strain the other strainscontaining the same opp deletion were not tested.Nva, norvalyl (2-amino pentanoic acid); Sar, sarcosyl (N-methyl glycyl); ND,not determined because of limiting material; NI, no inhibition was detected atany amount up to the maximum 300 nmol tested.

Peptidomimetics M2034a PA0183a PA0410a PA0333a

Leu-FMDP 80 200 450 180Lys-FMDP 180 250 240 NIMet-FMDP 90 ND 750 500Nva-FMDP 60 ND 60 500Phe-FMDP 80 250 600 1000Tyr-FMDP 200 550 900 >1000Val-FMDP 60 80 100 550Nva-FMDB 150 190 NI NIFMDP-Ala 700 NI NI NIGly-FMDP >3000b

AcNva-FMDP >5000b

FMDP-Leu >1000b

FMDP-Met >3500b

FMDP-FMDP >5000b

FCDP-Ala >5000b

FCDP-Met >5000b

Nva-FMDP-Nva 230 NIc

SarNva-FMDP 290 NIc

LysNva-FMDP 150 NIc

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93%, and 89% and 100%, respectively. Similarly, FCDP-Alaand FCDP-Met are better competitors than FMDP-Ala andFMDP-Met (Table 2), compatible with them being well trans-ported and implying that the very poor antibacterial activity ofthe FCDP-containing dipeptides (Table 1) relates to the lowerenzyme inhibition of the FCDP warhead compared withFMDP.1

As with Dpp, substrates for Opp can be ranked by theirabilities to bind to OppA,23 and these can be measured in ananalogous competitive assay using [125I2]TyrGlyGly asligand. The three tripeptide analogues showed comparablecompetitive binding to OppA (Table 2), which accords withtheir similar antibacterial activities and supports their trans-port by Opp (Table 1). Lys-FMDP showed a lower level ofbinding, as is normal for dipeptides, but in accordance withuptake by Opp contributing to the inhibition seen with thesecompounds in M2034 (Table 1).

Molecular modelling of peptides and peptidomimetics

Molecular modelling of various classes of natural and syn-thetic peptides has allowed identification of the bioactiveconformational forms recognized by different peptide trans-porters and peptidases, and provided an understanding ofstructure–activity relationships for substrates of these pro-teins.12–14,16,20 Backbone torsions are a very important featureof MRTs for peptide transporters.12,14–17 For the ω peptide

bond only the trans form is recognizable.24,25 A variety ofvalues can be adopted by ψ and φ torsions. Dipeptides mostlyadopt Tor2 of A4 (–50° to –85°), A7 (+140° to –175°) andA10 (+50° to +85°), and Tor4 of B2 (+40° to +85°), B9 (–50°to –95°) and B12 (–130° to +175°) (Figure 1), and thesetorsions help to determine the ligand specificities of peptidetransporters.12,14–17 Thus, in the present case, conformationalanalysis of the peptidomimetics can provide an estimate of theextent to which the conformer repertoire of each matches theMRTs for substrate(s) of each peptide transporter. This evalu-ation of the peptidomimetics as putative ligands of Dpp, Tppand Opp can be related to the ligand binding data, and, in turn,to the antibacterial results, so as to explore the basis forstructure–inhibition relationships. Random search conforma-tional analysis for each peptidomimetic and related Xaa-Glndipeptides showed they occurred as several hundred distinctconformers (results not shown), comparable to the finding formost other natural peptides.15,16 The various conformers forany one compound, e.g. 768 for Val-FMDP, are distinguish-able either by having different combinations of backbone tor-sions (ψ, ω and φ), or having comparable backbone torsionsbut different orientations of side chains (χ torsions). Using arandom search procedure, the minimum energy conformationidentified may or may not be the actual global minimum con-former and it may or may not be a bioactive form. However,this is not of great importance here, for what is criticallyimportant when considering molecular recognition of flexiblemolecules is to consider the whole population of conforma-tional forms and to be able to estimate with good precision thepercentage of conformers that match MRTs.13,21 Thus, there isa direct correlation between the proportion of conformers thatmatch a particular MRT and the bioactivity of the compound,e.g. affinity to the ligand, transport rate, etc.12–14,20,24

For these compounds, the minimum energy form typicallyaccounts for ∼8–30% of the total conformer pool, and thereare broad similarities between the energies of Xaa-Glnpeptides and their respective peptidomimetics. Because allthe peptidomimetics comprise L-amino acids with chargedN- and C-terminal groups, when the minimum energy con-formers have backbone torsions that match those for peptidetransporter MRTs they can be considered as putative ligands.For each peptidomimetic, data for the percentage of its con-formers having backbone torsions and lengths matching theMRTs for Dpp, Tpp and Opp are shown in Table 3. Three-dimensional pseudo-Ramachandran plots showing the com-plete conformer distribution for representative inhibitors areshown in Figure 2. From these results, several general trendscan be seen regarding structure–activity relationships for theinhibitors. For the Xaa-FMDP compounds, there is broadsimilarity in their conformer profiles with those of the relatedXaa-Gln dipeptides, and they have comparable proportions ofconformers matching the different MRTs, e.g. Tyr-FMDP(65%, 18%) and Tyr-Gln (64%, 17%); Val-FMDP (55%,

Table 2. Competitive binding of peptidomimetics to DppA and OppA

The percentage inhibition of binding to DppA was determined usingGly[125I]Tyr as ligand, and for OppA [125I2]TyrGlyGly was used as ligand. Themolar ratio of peptidomimetic to ligand was 10:1 in all cases.

Peptidomimetics

Percentage inhibition of binding to DppA Peptidomimetics

Percentage inhibition of binding to OppA

Gly-FMDP 0 SarNva-FMDP 83Leu-FMDP 80 LysNva-FMDP 89Met-FMDP 88 Nva-FMDP-Nva 91Nva-FMDP 99 Lys-FMDP 24Phe-FMDP 94Tyr-FMDP 73Val-FMDP 75AcNva-FMDP 0Nva-FMDB 93FMDP-Ala 14FMDP-Leu 36FMDP-Met 5FMDP-FMDP 0FCDP-Ala 49FCDP-Met 47

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19%) and Val-Gln (54%, 23%); Leu-FMDP (15%, 55%) andLeu-Gln (21%, 50%) for Dpp and Tpp, respectively. Thesepercentage values for the Xaa-FMDP peptidomimetics aregenerally in broad agreement with their relative antibacterialactivities (Table 1). The antibacterial activity of thesecompounds appears mainly to arise from uptake by Dpp, inaccordance with them having a higher proportion of con-formers that match the MRT for Dpp and, also, as a resultof the greater inherent transport capacity of Dpp.17,22 ForAcNva-FMDP, its inactivity can be attributed to substitutionof its N-terminal α-amino group, which must possess a posi-tive charge to achieve ligand binding to Dpp, Tpp and Opp.For both DppA and OppA, this involves interaction of thepositive charge on the α-amino group with the side-chaincarboxylate of an aspartic acid residue.12,17,22 The antibac-terial activity of Gly-FMDP is extremely poor, which is in

accordance with the general finding that any dipeptidecontaining Gly shows low transport activity by Dpp, Tpp andOpp.17 This arises from several causes. First, a high pro-portion of their conformers have cis ω bonds, which are notrecognized.14,24,25 Secondly, the small side chain allowsgreater flexibility of their ψ and φ torsions so they can adopt awider array of conformational forms, very few of whichmatch well the backbone torsions favoured for recognition bythe transporters. The need to define weightings for the relativecontributions of such conformers with torsions outside theoptimum range (Table 3) still needs to be adequatelyaddressed.15,24 Significantly, the most effective inhibitor ofmutant PA0333 (Tpp+) is Leu-FMDP (Table 1) and thisaccords well with the finding that (but for Lys-FMDP) it hasthe highest proportion of conformers (55%) that match pre-cisely the backbone torsions of the MRT for Tpp (Table 3,Figure 2). Thus, Lys-FMDP alone appears anomalous in therelationship between its antibacterial activity and conformerdistribution based on backbone torsions, and an explanationfor this is considered below.

The FMDP-Xaa analogues have conformer profilesbroadly similar to those of Xaa-FMDP and they also possesshigh proportions of conformers matching the backbone tor-sions of the MRTs for Dpp and Tpp (Figure 2, Table 3), and yetthey have significantly lower antibacterial activities (Table 1)and comparably poorer affinities for DppA (Table 2).Therefore, a further structural/electronic feature(s) must beresponsible for this difference in their bioactivities. Toexplore the possibility that this might arise from the size ofFMDP, the dimensions of the side chains for the analogueswere computed and compared with those for examples of thelargest side chains of protein amino acids. Results for lengths,molecular volumes and number of water equivalents were,respectively: FMDB (12.3 Å; 145 Å3; 9); FMDP (9.8 Å; 130Å3; 8); FCDP (8.6 Å; 117 Å3; 7); Gln (5.1 Å; 86 Å3; 5); Lys(6.4 Å; 107 Å3; 6) and Trp (5.4 Å; 128 Å3; 8). Thus, theglutamine analogues can adopt more extended conformationsthan any protein amino acid and some have larger volumes.

Results with Lys-FMDP provide a further test of the pre-sent approach of using conformational analysis to probestructure–activity relationships. Lys-FMDP inhibited theparent strain and mutants PA0183 (Dpp+,Tpp+) and PA0410(Dpp+) but showed no activity against mutant PA0333 (Tpp+)(Table 1), indicating that its uptake is predominantly by Dpp,a little by Opp, but not at all by Tpp. These results appear atodds with the finding that the majority of its conformers havebackbone torsions that match the MRT for Tpp (Table 3,Figure 2). However, particular properties of a side chain canalso markedly influence the molecular recognition of pep-tides, as illustrated above regarding size, and described earlierfor certain side chains that are charged and have particularχ torsions.12–14 Examination of the molecular structures ofA4B9, A4B12 conformational forms of Lys-FMDP, which

Table 3. Percentage of conformers for glutamine dipeptides and peptidomimetics with backbone torsions matching those for the MRTs of Dpp, Tpp and Opp

–, not determined.aPercentages for peptides containing Gly or Sar residues include conformerswith Tor2 values outside the torsion angles adopted by other amino acids;this approach is adopted because of the extended backbone torsional spaceaccessible to these residues although the conformers are not equally goodligands.13,15,16

Compounds Dpp Tpp Opp

Gly-FMDP 26a 53a –Val-FMDP 55 19 –Leu-FMDP 15 55 –Met-FMDP 41 14 –Phe-FMDP 57 13 –Tyr-FMDP 65 18 –Lys-FMDP 17 59 –Nva-FMDP 42 40 –AcNva-FMDP 11 46 –Nva-FMDB 50 16 –FMDP-Ala 71 11 –FMDP-Leu 37 16 –FMDP-Met 50 11 –FMDP-FMDP 27 9 –FCDP-Ala 67 14 –FCDP-Met 49 25 –Nva-FMDP-Nva – – 70SarNva-FMDP – – 60a

LysNva-FMDP – – 89AlaGln 39 30 –PheGln 70 11 –LysGln 44 28 –LeuGln 21 50 –SerGln 34 38 –ThrGln 45 31 –ValGln 54 23 –TyrGln 64 17 – by guest on N

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have the lowest energies and are most abundant, indicates thatboth side chains are contiguous and fully extended in whichtrans χ torsions predominate, allowing stabilizing interactionsthat are unique to these conformational forms (Figure 3).Thus, Lys-FMDP is quite atypical, and in its A4(B9,B12)conformers the side chains appear as a ‘fused unit’. In con-trast, natural dipeptides having charged or polar N-terminalresidues, e.g. LysGln has mainly A7 conformers in which theside chains are separated.13,15

The results showing inhibition of the parent strain butnot of mutant PA0183 (Opp–,Dpp+,Tpp+) by the tripeptideanalogues indicate that Opp is exclusively responsible fortheir transport (Table 1). In support of this, each showed goodbinding to OppA (Table 2). The steric problems of the longside chain of FMDP described above would be exacerbatedin ‘folded’ conformations of the tripeptides that are putativeligands of Dpp and Tpp, precluding them from being recog-nized by these transporters.12,17

Discussion

All microorganisms have peptide transporters to absorb theproducts of protein breakdown. Several transporters usuallycoexist; these have overlapping and complementary specific-ities that have evolved to optimize uptake of the nutritionallyimportant components of the peptide pool. However, compe-tition between microorganisms has also led to them producinga wide variety of natural antimicrobial peptidomimeticsdesigned to exploit peptide transporters for their delivery.These peptidomimetics usually comprise an impermeablecompound, which is inhibitory to an intracellular target,linked into a peptide so as to smuggle the antimicrobial agentinto the competing microorganisms in the form of a pro-drug.7,17,22,23,26 Amongst the many examples of such naturalantibacterial and antifungal agents, e.g. bacilysin, valclavam,lindenbein, bialaphos, polyoxins and nikkomycins, somehave been studied for their therapeutic potential.7,23,26 This

Figure 2. Three-dimensional pseudo-Ramachandran plots for the dipeptides (a) LysGln and (c) TyrGln, and the dipeptide mimetics (b) Lys-FMDPand (d) Tyr-FMDP. The aggregated percentages of conformers with particular combinations of backbone torsions ψ (Tor2) and φ (Tor4) are plottedagainst those torsions. The axes are labelled with the torsional ranges that are best recognized by Dpp: A7 (+140° to –175°) with B9 (–50° to –95°)and B12 (–130° to +175°); and by Tpp: A4 (–50° to –85°) and A10 (+50° to +85°) with B9 (–50° to –95°) and B12 (–130° to +175°). Conformers withB2 torsions (+40° to +85°) are not recognized by either transporter.

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approach has also been explored for targeting a variety ofsynthetic antimicrobial agents.7,26,27 This peptide prodrugconcept has wider potential benefits, since peptide trans-porters in the gut and kidney with similar specificities canendow such therapeutic agents with oral activity. Recentstudies have sought to define the structural basis for ligandrecognition by peptide transporters, so as to allow arational approach to the design and synthesis of peptido-mimetics with either antimicrobial or other therapeutic activ-ities.12–14,17,24,28,29

Examples of the glutamine analogue peptidomimeticsstudied here have been shown previously to have broad-spectrum antimicrobial activities and chemotherapeuticactivity in a mouse model of candidiasis. In addition, exten-sive data have been obtained previously on their transport andcleavage by microorganisms, but these have not provided

an understanding of the structural basis for their individualactivities.8–11 In the present study, the relative antibacterialactivities of a series of these peptidomimetics against isogenicstrains of E. coli differing only in their complement of peptidetransporters, has shown the extent to which the different trans-porters recognize and absorb the various compounds. Eachstrain has an identical complement of peptidases, so intra-cellular cleavage of peptidomimetics is comparable in each.Given the limited availability of compounds, the disc diffu-sion assay was chosen as a suitable test to obtain quantitativeantibacterial data that was dependent upon the uptake of thevarious compounds through distinguishable peptide trans-porters. In this assay, a query can always be raised regardingpossible variations in diffusion rates of compounds andwhether hypothetically this may influence zone sizes, butonly in very rare cases would it be possible to measure diffu-

Figure 3. Ball-and-stick representations of specific low energy conformers derived from random searches: (a) Lys-FMDP (A4B12); (b) TyrGln(A7B12); (c) Leu-FMDP (A4B9); and (d) FMDP-FMDP (A7B2). The structures were created in CPK colours, but atoms retain recognizableshading when displayed in black and white. To aid identification, for Lys-FMDP the structure is orientated with the protonated N-terminal α-aminogroup of Lys at the bottom left, the side chain protonated ε-amino group of Lys at the top left, the C-terminal α-carboxylate of FMDP at the bottomright, and with the carbonyl oxygen of the peptide bond directed behind and the nitrogen of the peptide bond directed forwards. The peptide bond isorientated analogously for the other structures.

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sion with any precision in these assays so as to check thisspeculation. In general, diffusion may be influenced by mass,shape, pKa, hydrophobicity, etc., but for compounds asclosely related to each other as these it seems improbable thatany such variations could have any significant effect on diffu-sion rates. Certainly, no correlation exists between the sizes ofthe compounds and their zone sizes. Thus, as is usual with thisassay, it appears sensible to accept the inhibition zones asdirectly reflecting antimicrobial activities. This approach issupported by the correlations between the antibacterial assaysand the results obtained from measurements of the relativeaffinities of the compounds for the peptide-binding proteinsDppA and OppA, which are the components responsible forligand specificities in the Dpp and Opp transporters. Ingeneral, there was good agreement between ligand bindingand antibacterial activity; and this same conclusion hasbeen reached previously for other antimicrobial peptido-mimetics.17,18

Explanation of the structural basis for these activities wasprovided by comparison with results of molecular modellingof the various compounds. These peptidomimetics are thefirst example of a series of antimicrobial compounds forwhich such an analysis has been attempted. They are flexiblemolecules that exist as a variety of conformers in solution, andto understand how this determines their molecular recog-nition, transport, antibacterial activities, etc. it is necessary toanalyse the complete repertoire of conformers for eachcompound. Previous analysis of natural peptide substrates hasidentified the important structural and electronic features, e.g.charged termini, particular backbone torsions, L-chirality,etc., which must be possessed by a compound for it to be well-recognized by peptide transporters in microorganisms andother species. Thus, Dpp recognizes dipeptide conformerswith torsions of A7 paired with B9 and B12, whereas Tpp rec-ognizes a different set of dipeptide conformers with torsionsof A4 and A10 paired with B9 and B12; Dpp and Tpp alsorecognize ‘folded’ tripeptide conformers with the above tor-sional values and appropriate N–C distances. Opp recognizes‘elongated’ conformers of tri- and higher oligopeptides (andto a lesser extent dipeptides) with A7 combined with B9 andB12 and N–C distances greater than ∼5.5 Å.12,14–16 These con-clusions have been corroborated by studies of bound peptideligands in crystal structures of DppA and OppA.16,30–33 Thesestructural parameters have been incorporated into definitionsof MRTs for each microbial transporter, which also appear torelate to mammalian and plant transporters.12,13,16,24 Thus, inprinciple, comparison of the extent to which the conformersof the peptidomimetics match the MRTs allows estimation oftheir transport rates and antibacterial activities.

For most of the Xaa-FMDP compounds and the tripeptido-mimetics, structure–activity relationships were clear, withpreferences for Dpp, Tpp or Opp, and thus antibacterialactivities against the different strains, being related to the pro-

portions of conformers that adopted the particular backbonetorsions recognized by each transporter. However, a particu-lar strength of this study is how it has provided insight into theinfluence of side chain properties, e.g. overall size and χ tor-sions, on molecular recognition. Thus, the lower bioactivitiesof FMDP-Xaa compounds compared with the correspondingXaa-FMDP analogues, which are not explicable in terms oftheir backbone torsions, can be related to the dimensions ofthe side chain of FMDP. These values can be related to resultsof X-ray crystal studies on DppA and OppA, which show thatthey have water-filled pockets to accommodate the variety ofside chains found in natural peptides. These have evolved sothat different numbers of water molecules are displaceddepending on the size of the side chain.30–33 DppA possessestwo pockets: thus, for dipeptide ligands, the N- and C-residueside chains are readily accommodated.31 For DppA (and Tpp)to bind tripeptides, a tripeptide conformer must be ‘folded’ sothat its ψi–1 and φi torsions and the locations of its N- andC-terminal charged groups mimic those in dipeptide ligandconformers. This requires that the C-terminal region (secondpeptide bond and the central and C-terminal side chains) isrecognized as a ‘unit’ that fits into the C-terminal pocket.12,14–16

To achieve this, the C-terminal pocket needs to be larger thanthe N-terminal pocket and this is exactly what has beenfound.31 Consequently, the greater size of FMDP comparedwith the largest protein amino acids makes it likely that it isnot easily accommodated in the N-terminal water-filledpocket but it can be in the C-terminal one. This observationprovides a plausible explanation for the much poorer affinitiesof the FMDP-Xaa compounds compared with the Xaa-FMDPanalogues for DppA (Table 2) (and implicitly for Tpp) and fortheir lower antibacterial activities (Table 1). FMDP-FMDPshowed no binding to DppA and lacked measurable anti-bacterial activity. This can now also be rationalized on thebasis of its large side chains and also the fact that about 60% ofits conformers have unrecognizable B2 torsions (data notshown). With regard to the dimensions of the other glutamineanalogues, the even larger size of FMDB would be expectedto limit severely its transport as part of a peptidomimetic,which would further diminish its poor enzyme inhibition. Thesmall size of FCDP supports the finding that DppA bindsFCDP-Ala and FCDP-Met better than FMDP-Ala andFMDP-Met (Table 2), which endorses the suggestion that thepoor antibacterial activities of FCDP peptidomimetics areprobably a consequence of the intrinsically lower inhibitionof the target enzyme by this analogue. Nature endorses thisinfluence of side chain size on bioactivity. Thus, most naturalantimicrobial dipeptidomimetics have a protein amino acid astheir N-terminus, commonly Ala, and the larger antimicrobialcompound forms the C-terminus. This is invariably the casewhen the inhibitory moiety is large, as in valclavam, poly-oxins and nikkomycins.17,22,23,26,27 Interestingly, the naturalantibiotic lindenbein, FCDP-Ala, has the warhead at the

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N-terminus. However, as shown above, being the smallest ofthese glutamine analogues it can be accommodated in theN-terminal pocket, allowing it to be well bound by DppA(Table 2), although FCDP has poor inhibitory activity againstthe target enzyme (Table 1). Molecular modelling of Ala-FCDP, an alternative theoretical lindenbein analogue, showsthat compared with the natural form it possesses a much lowerproportion of conformers that match the MRT for Dpp(results not shown). It appears that nature chose the form bestsuited to deliver the antimicrobial moiety.

Lys-FMDP provides a further example of the ability of sidechains to affect molecular recognition. Its conformers aremostly A4(B9,B12), which are putative ligands specificallyfor Tpp, but it was completely inactive with this transporter.The explanation for this became clear from inspection of themodelled conformers in which their side chains interact withone another. This may improve stability but would seem tomake the ‘fused side chains’ too large to allow binding byTpp. With this insight, it would be relatively easy to evaluateconformer databases of related compounds to identify any inwhich the side chains are comparably close and so may consti-tute an unrecognizable ‘fused unit’.

In conclusion, conformational analysis of these peptido-mimetics has provided detailed insights into their structure–activity relationships. This approach would speed up identifi-cation of compounds with optimal bioactivities whilstdramatically decreasing the need for extensive chemicalsyntheses and testing. In addition, molecular modelling couldbe performed on, for example, FMDP-containing pseudopep-tides, having chemical features such as peptide-bond isosteresthat could enhance overall pharmacokinetics.34 In a broadercontext, the study extends the use of the concept of MRTs, andprovides important structural information that will aid thedesign not only of antimicrobial compounds but also of alltherapeutic agents targeted for delivery by peptide trans-porters.

Acknowledgements

We are grateful to Barry M. Grail, Gillian M. Payne andCraig J. Winstanley for their contributions to these studies.Financial support for this research was provided by the BritishSociety for Antimicrobial Chemotherapy (Grant GA313),and the Biotechnology and Biological Sciences ResearchCouncil (Grant P12847).

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Documents

Structure-activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues