Insect Peptides with Improved Protease-Resistance Protect Mice Against Bacterial Infection

Insect peptides with improved protease-resistance protect mice against bacterial infection

LASZLO OTVOS, JR.,1 KRISZTINA BOKONYI,1 ISTVAN VARGA,1 BALINT I. OTVOS,1,2 RALF HOFFMANN,1,3 HILDEGUND C.J. ERTL,1 JOHN D. WADE,4 AILSA M. McMANUS,5 DAVID J. CRAIK,5 and PHILIPPE BULET2 1 The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104 2 Unité Propre du CNRS No. 9022, “Réponse Immunitaire et Développement chez les Insectes,” Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France 3 Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany 4 Howard Florey Institute, Parkville 3052, Victoria, Australia 5 Centre for Drug Design and Development, University of Queensland, Brisbane 4072, Queensland, Australia ~Received October 13, 1999; Final Revision January 11, 2000; Accepted January 21, 2000!

Abstract At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads. Keywords: antibacterial peptides; in vivo anti-infection assay; metabolite; nuclear magnetic resonance spectroscopy; serum stability; stereospecific target protein

Antibacterial peptides and glycopeptides isolated from insects the apidaecins ~Casteels & Tempst, 1994!~18 amino acid resi- ~Gillespie et al., 1997; Bulet et al., 1999! are promising candidates dues!, the drosocins ~19 residues!, and the pyrrhocoricins ~20 res- for drug development due to their unusual mode of action. While idues!. The last two groups contain a disaccharide in midchain many antibacterial peptides from other origins kill bacteria by position ~Bulet et al., 1993; Cociancich et al., 1994!. The presence disrupting the cell membrane, some of the insect peptides bind to of the sugar increases the in vitro antibacterial activity of drosocin, an as yet unidentified stereospecific target molecule, presumably a but decreases the activity of pyrrhocoricin and lebocin, a some- protein or glycoprotein ~Bulet et al., 1996!. This unusual mecha- what more distant family member ~Hara & Yamakawa, 1995; Bulet nism of action will likely attract the attention of the pharmaceutical et al., 1996; Hoffmann et al., 1999!. Pyrrhocoricin is more active industry, which is anxiously awaiting novel antimicrobial com- against Gram-negative bacteria than drosocin in vitro, but the pep- pounds that can break drug tolerance in nosocomial infections. tide is almost completely inactive against Gram-positive strains. Significantly, no drug resistance other than increased proteolytic Drosocin is moderately active against Gram-positive bacteria. activity could be attributed to these molecules ~Boman, 1995!. The When glycosylated drosocin alone is injected into mice, the three most studied groups of the insect antibacterial peptides are glycopeptide shows no toxicity ~Hoffmann et al., 1999!. However, the glycopeptide cannot protect mice against Escherichia coli infection. This is probably due to the peptide’s rapid decomposi- Reprint requests to: Laszlo Otvos, Jr., The Wistar Institute, 3601 Spruce tion in mammalian sera ~Hoffmann et al., 1999!. While drosocin St., Philadelphia, Pennsylvania 19104; e-mail: [email protected]. needs up to 12 h to kill bacteria in vitro ~Cudic et al., 1999!,itis 742 In vivo active antibacterial peptides 743 completely degraded in diluted human and mouse sera within a 4-h peptidase cleavage; ~4! however, most antibacterial peptides need period ~Hoffmann et al., 1999!. Both aminopeptidase and carboxy- a positive charge at or near the native amino terminus ~Vunnam peptidase cleavage pathways are observed. Pyrrhocoricin appears et al., 1997!; ~5! unnatural, glycosylated, or d-amino acid residues to be more resistant to degradation in mouse serum than drosocin, are likely candidates to improve stability in serum ~Powell et al., but decomposes relatively quickly in some batches of human se- 1993!; and ~6! cyclic peptides cannot be cleaved by exopeptidases. rum ~Hoffmann et al., 1999!. The first metabolites from serum In this first round of studies, we did not consider backbone mod- stability assays were identified, and it was shown that the metab- ifications to conserve the conformation of the peptide. As backbone- olites of drosocin and pyrrhocoricin, lacking as few as five amino modified versions of a membrane-active antibacterial peptide were terminal or two carboxy terminal amino acids become inactive in reported to improve proteolytic stability and yield analogs with vitro ~Bulet et al., 1996; Hoffmann et al., 1999!. Although exper- slightly modified activity spectrum ~Oh et al., 1999!, backbone iments measuring the rate of peptide degradation are fairly straight- modifications will be considered in future steps. Table 1 lists the forward, there are several serum batch-related factors that may synthetic peptides and the in vitro antibacterial activities. produce highly variable results ~Powell et al., 1993!. The use of pooled human sera somewhat improves the reproducibility of the In vitro antibacterial activity stability assays, although even commercial sera with the same catalog number are notoriously different. Therefore, in the current The in vitro antibacterial activity of the peptides was measured study, we investigated the stability of the pyrrhocoricin-related against two Gram-positive strains, Micrococcus luteus and Bacil- peptides in two different batches of pooled human serum ~approx- lus megaterium, and three Gram-negative strains, E. coli D22, imately 80 donors in each batch!. Agrobacterium tumefaciens, and Salmonella typhimurium. Al- The hypothesis concerning the two independent active sites is though these bacterial strains are not necessarily clinically rele- further supported by a recent model of the bioactive secondary vant, we used these models to obtain a direct comparison with the structure of drosocin that identifies two reverse turns, one at each antimicrobial activity of other related peptides, most importantly terminal region, as binding sites to the target molecule ~McManus drosocin and its fragments ~Hoffmann et al., 1999!. The experi- et al., 1999!. The situation is further complicated by the fact that ments were conducted over a seven-month period. To assess the the degradation speed and pathway of a given peptide in diluted variability of the assay, peptide 18 was reassayed three months mouse serum is somewhat different from those observed in diluted after the original study. The differences in the IC50 values between human serum. The stability of the peptides is markedly increased 180a and 180b demonstrate this variability. Basically identical re- in insect hemolymph where they manifest their biological function sults were obtained against M. luteus, B. megaterium, E. coli D22, ~Hoffmann et al., 1999!. and S. typhimurium, indicating the high reproducibility of the as- We decided to design modified peptides based on pyrrhocoricin. say. The fourfold difference in the IC50 value against A. tumefa- Our goal was to retain the peptide’s high antibacterial potency in ciens is in line with the culture-to-culture variability of this strain. vitro and obtain good metabolic stability and lack of toxicity for From the peptides made for biochemistry investigations, we learned ensuing in vivo efficacy assays and drug development. We selected that the two putative binding sites cannot be separated ~peptides 2 pyrrhocoricin over drosocin because ~1! it is more potent against and 3!. In fact, an equimolar mixture of shorter peptides 2 and 3 some strains of Gram-negative bacteria, ~2! glycosylation is not remained inactive in all five bacterial strains studied. An analog needed for full biological activity, and ~3! the peptide is more made of only D-amino acids was similarly inactive ~peptide 4!, stable in mouse serum. Importantly, we wanted to obtain peptides indicating that pyrrhocoricin, like drosocin, binds stereospecifi- for which the degradation pathway in mouse serum ~the most cally to a target protein. The N-terminus of the native glycopeptide suitable test animal for in vivo efficacy assay! is similar to that in ~peptide 5! could not be blocked by acetylation without a major human serum ~the modified peptides are targeted for human ther- loss of antibacterial activity. Pyrrhocoricin analogs containing la- apy!. In addition, we wanted to determine the bioactive conforma- bels and an additional lysine at the N-terminus ~peptides 6 and 7! tion of native pyrrhocoricin and compare it with that of the published retain biological activity and can later be used to isolate and char- structure of native drosocin to establish the secondary structural acterize the bacterial target protein. requirements for antibacterial activity. Turning to the drug candidates, modification at either termini reduced the potency of unmodified pyrrhocoricin. From the only N- or C-terminally modified peptides, 1-amino-cyclohexyl- Results and discussion carboxylic acid at the amino-~peptide 11! and the acetylated 2,3- diamino-propionic acid at the carboxy-terminus ~peptide 12! Design of peptides appeared to retain most of pyrrhocoricin’s antibacterial activity. Pyrrhocoricin has the amino acid sequence: VDKGSYLPRPTP- For drug development, both termini have to be modified to block PRPIYNRN, with Thr11 glycosylated in the native peptide. In our or at least slow down exopeptidase cleavage that was observed on earlier studies, we synthesized the peptide with a free amino- the native and unglycosylated peptides. Peptides containing mod- terminus and an amidated carboxy-terminus. In the current study, ifications at both termini featured acetylation together with posi- an extensive series of fragments and analogs was made for bio- tively charged amino acid addition, incorporation of unnatural amino chemical characterization of the peptide and for drug design pur- acids, such as Chex or Dap~Ac!, glycosylation, imide formation, poses. The following findings were considered in the design of and D-amino acid substitution or cyclization ~peptides 13–23!. pyrrhocoricin analogs for biotechnological uses: ~1! the main sites Considering the requirement for submicromolar activity against at of the degradation of unmodified pyrrhocoricin are the N- and the least one strain, peptide 19, Chex-pyrrhocoricin-Dap~Ac!, and pep- C-termini, indicating mainly exopeptidase cleavage; ~2! an endo- tide 18, Ac-R-pyrrhocoricin-Dap~Ac!, were selected for further peptidase cleavage site was also identified between Ser6 and Tyr7; studies, which included cell toxicity and serum stability as well as ~3! N-terminal acetylation usually protects peptides from amino- metabolism assays. These peptides showed remarkable high activ- 744 L. Otvos et al.

Table 1. In vitro antibacterial activity of pyrrhocoricin-based peptides a

IC50 in mM against Micrococcus Bacillus E. coli Agrobacterium Salmonella Peptide no. Peptide composition luteus megaterium D22 tumefaciens typhimurium

1 Pyrrhocoricin 1–20 10 5 Ͻ0.075 0.075 0.6 Analogs for biochemistry studies 2 Pyrrhocoricin 1–9 — — — — — 3 Pyrrhocoricin 10–20 — — — — — 4 All d-pyrrhocoricin 1–20 40 — — — — 5 Ac-pyrrhocoricin GalNAc ~T11! ——10— — 6 Biotin-K-pyrrhocoricin 1–20 5 — Ͻ0.15 1.25 1.25 7 Fluorescein-K-pyrrhocoricin 1–20 80 — 5 20 40 N-terminal modifications 8 Ac-K-pyrrhocoricin 1–20 40 — 0.6 5 40 9 Ac-K-VDK-pyrrhocoricin 1–20 80 — 0.3 10 5 10 Ac-R-pyrrhocoricin 1–20 5 — 0.6 2.5 2.5 11 Chex-pyrrhocoricin 2–20 5 10 0.3 1.25 2.5 C-terminal modification 12 Pyrrhocoricin 1–19-Dap~Ac! 520Ͻ0.30 Ͻ0.30 Ͻ0.30 Dual modifications 13 Ac-K-pyrrhocoricin 1–19-D20 — — 2.5 40 — 14 Ac-K-pyrrhocoricin 1–20 imide ~N20! 5 20 0.3 1.25 2.5 15 Ac-K-pyrrhocoricin 1–20 GlcNAc ~N20! 40 — 0.6 5 2.5 16 Ac-K-pyrrhocoricin 1–20 Ac3GlcNAc ~N20! 40 40 0.6 2.5 20 17 Ac-K-pyrrhocoricin 1–19-Dap~Ac! 10 40 0.6 1.25 1.25 18a Ac-R-pyrrhocoricin 1–19-Dap~Ac! 10 80 0.3 0.6 2.5 18b Ac-R-pyrrhocoricin 1–19-Dap~Ac! 10 40 0.3 2.5 2.5 19 Chex-pyrrhocoricin 2–19-Dap~Ac! 10 40 0.6 5 2.5 20 d-val-pyrrhocoricin 2–19-D-asn 10 — 0.6 2.5 5 External and internal modification 21 Chex-pyrrhocoricin 2–19-~A5F6!-Dap ~Ac! 54020— — Cyclic derivatives 22 Cyclo-@K-pyrrhocoricin 1–19-D20# ———— — 23 Cyclo-@K-pyrrhocoricin 1–20-14-7# 0.6 2.5 0.6 2.5 5

a Abbreviations: V, valine; D, aspartic acid; K, lysine; G, glycine; S, serine; Y, tyrosine; L, leucine; P, proline; R, arginine; T, threonine; I, isoleucine; N, asparagine; A, alanine, F, phenylalanine; Ac, acetyl; Chex, 1-amino-cyclohexane-carboxylic acid; Dap~Ac!, b-acetyl-2,3-diamino propionic acid; GlcNAc, 2-acetamido-2-deoxy-glucose; GalNAc, 2-acetamido-2-deoxy-galactose; Gal, galactose; D-val, D-valine; D-asn, D-asparagine; fluorescein, 5~6!carboxy-fluorescein. The inhibitory concentration ~IC50! is expressed as the concentration tested at which 50% growth inhibition is observed. Growth inhibition assays were performed at 30 8C for 24 h in Luria–Bertani rich nutrient medium. No activity up to 80 mM concentration is indicated by —. Peptide 22 was tested up to 20 mM concentration. All amino acids were of the L-configuration, except for peptides 4 and 20, in which the indicated amino acids were of the D-configuration. All carbohydrates were of the D-configuration.

ity against Gram-negative bacteria. Significantly, the best analog Cytotoxicity of pyrrhocoricin and its analogs for killing both Gram-negative and Gram-positive bacteria was cyclic peptide cyclo-@K-pyrrhocoricin 1-20–14-7#~peptide 23!, To test whether native and modified pyrrhocoricin peptides were which demonstrated a broad activity spectrum at low micromolar toxic to mammalian cells, pyrrhocoricin, Chex-pyrrhocoricin- concentrations. In this peptide, a lysine residue was added to the Dap~Ac!, Ac-R-pyrrhocoricin-Dap~Ac!, melittin ~positive con- amino terminus of the peptide, which is cyclized with an eight- trol!, and peptide 31D, a T-cell epitope ~negative control! were residue spacer between the original N- and C-termini. The spacer mixed with sheep erythrocytes. At a final concentration of 40 mM, corresponded to the middle domain of pyrrhocoricin and was in- only melittin lysed the red blood cells ~Table 2!. When the exper- corporated backward to remain in register with the original copy of iments were repeated with pyrrhocoricin and the Chex-pyrrhocoricin- this fragment. This arrangement retained the native orientation of Dap~Ac! peptide up to 256 mM concentration, the peptides remained the bioactive domains ~the original termini!. The synthesis of this completely nontoxic to sheep erythrocytes. In another toxicity as- peptide in quantities large enough for toxicity and in vivo efficacy say, COS cell survival was studied upon addition of pyrrhocoricin. studies, as well as detailed conformational analysis, is currently in Cell counts and rate of proliferation remained unchanged after progress in our laboratory. addition of peptide up to 50 mg0mL concentration. In vivo active antibacterial peptides 745

Table 2. Hemolytic activity of pyrrhocoricin and some of its analogs a

Absorbance at 405 nm

Peptide Peptide no. 40 mM 256 mM

Pyrrhocoricin 1 0.108 6 0.015 0.135 6 0.005 Chex-pyrrhocoricin 2–19-Dap~Ac! 19 0.110 6 0.049 0.143 6 0.006 Ac-R-pyrrhocoricin 1–19-Dap~Ac! 18 0.126 6 0.019 Melittinb ~positive control! 2.990 6 0.06 31Dc ~negative control! 0.160 6 0.021

a The UV absorbance of 40 or 256 mM test and control peptides was measured after 1 h incubation with sheep erythrocytes in triplicate. b Melittin: GIGAVLKVLTTGLPALISWIKRKRQQ ~purchased from Bachem Biosciences, King of Prussia, Pennsylvania!. In our hands, the lowest concentration in which melittin lysed sheep erythrocytes was 5 mM, in agreement with the findings of Wade and coworkers ~Wade et al., 1990!. c 31D: AVYTRIMMNGGRLKR, a T-helper cell epitopic peptide ~Ertl et al., 1989!.

Peptide stability in mammalian serum but batch-to-batch variations result in highly variable decomposition curves. The mouse serum was identical to the one that was In vivo stability of peptides in blood is currently modeled well by used to characterize the metabolic behavior of drosocin ~Hoffmann in vitro stability in serum or plasma ~neglecting renal and hepatic et al., 1999!. When evaluating the first metabolites after 45 min clearance!~Powell et al., 1993!. Serum stability studies represent digestion ~Table 3!, it was evident that the C-terminal asparagine one of the most important secondary screening assays in peptide is cleaved off the unmodified pyrrhocoricin ~peptide 1!. In con- drug development, largely because they eliminate peptides that trast, the modified C-terminal residue stayed on the peptides con- have short half-lives and are, therefore, unlikely to be therapeuti- taining a carboxy-terminal Dap~Ac! residue that replaced Asn20 cally effective ~Powell et al., 1993!. Because diluted serum in- ~peptides 18 and 19!. The Ac-R-N-terminal peptide ~peptide 18! creases peptide recovery as well as retards the reaction kinetics to produced more internal cleavage products close to the amino ter- a manageable rate, the experiments were performed using 25% minus than the Chex-N-terminal peptide ~peptide 19!, most nota- aqueous sera. It has been demonstrated earlier that the degradation bly a fragment in which the VDK tripeptide fragment was missing. rates are linearly proportional to serum concentration ~Powell et al., While for unmodified pyrrhocoricin ~peptide 1! the degradation 1992!. We used mouse serum and two different batches of pooled products were different in the two human sera, for the Chex- human sera. In our experience, reproducible results can be ob- pyrrhocoricin-Dap~Ac! peptide ~peptide 19! they were very simi- tained by using a given serum preparation over a long time period, lar. They were also closer to those observed after digestion with the

Table 3. Degradation products of pyrrhocoricin peptides after a 45 min digestion with 25% mammalian sera a

Degradation product

Peptide no. Peptide composition Serum -C1 -C2 -C3 -C6 -N3 -N5 -N3,C2 -N5,C2

1 Pyrrhocoricin Mouse 220000 0 0 Human 1 343102 0 0 Human 2 220000 0 0 19 Chex-pyrrhocoricin 2–19-Dap~Ac! Mouse 020002 0 0 Human 1 042002 0 1 Human 2 040002 0 1 18 Ac-R-pyrrhocoricin 1–19-Dap~Ac! Mouse 020000 0 0 Human 1 053023 1 2 Human 2 050021 0 0

aThe missing residues are indicated ~e.g., -C2 means that the C-terminal two residues were cleaved off!. For Ac-R-pyrrhocoricin- Dap~Ac!~peptide 18!, the N-terminal residue numbers correspond to unmodified pyrrhocoricin. The relative amounts of the degradation products are estimated based on the MALDI-MS peak heights. 0 ϭ not detected; 1 ϭ very weak ~below 5,000!,2ϭweak ~5–10,000!,3ϭmedium ~10–15,000!,4ϭstrong ~15,000–uncleaved molecular ion at 25–30,000!,5ϭvery strong ~above uncleaved molecular ion!. All identified degradation products are listed. The mouse and the human 1 serum are approximately one year old, kept frozen. The human 2 serum is a month old ~identical SIGMA catalog number as human 1!. 746 L. Otvos et al. mouse serum. Mice being the species for ensuing in vivo efficacy stead of the bacteria. Neither peptide alone showed any toxicity at studies, mouse serum appeared to be a good model for the study of the 50 mg0kg dose. To assess the success of the infection, five the in vivo efficacy of peptide 19. mice were infected with E. coli, but received DS5 instead of the Figure 1 shows the kinetics of the degradation for peptides 1 and test peptides. Of these control mice, two died on the third day and 19. The degradation curves for unmodified pyrrhocoricin ~pep- the other three became clinically ill, which included decreased tide 1! and the Chex-pyrrhocoricin-Dap~Ac! peptide ~peptide 19! activity and head tilt. When the efficacy of the peptides was stud- were similar. Unmodified pyrrhocoricin was somewhat more sta- ied, the Chex-pyrrhocoricin-Dap~Ac! derivative ~peptide 19! pro- ble than peptide 19 in mouse serum and in the weaker human tected all 15 mice, regardless of the dose. For unmodified serum, but less stable in the more active human serum. Although pyrrhocoricin ~peptide 1!, the mice that were injected with 10 and the lack of exopeptidase cleavage sites reduced the degradation 25 mg0kg survived without any clinical signs of disease. However, rate of peptide 19 compared to peptide 1, this loss of protease from the 50 mg0kg group, one mouse died and the other four activity was compensated for by increased endopeptidase activity showed clinical signs of disease. This mirrors the drosocin studies C-terminal to Ser5 and Asn18. In support of this, metabolism in which the peptide was not toxic alone, but became toxic ~or studies of the broad spectrum cyclo-@K-pyrrhocoricin 1-20–14-7# augmented the infection! when the mice were infected and became derivative ~peptide 23! indicated a single endopeptidase cleavage compromised ~Hoffmann et al., 1999!. Increased mortality is an site between Asn18 and Arg19 in both mouse and human sera. In often detected pharmacological phenomenon when high doses of accordance with the increased number and amount of internal deg- drugs are administered to challenged animals. Such an effect was radation products near the amino termini, the Ac-R-pyrrhocoricin- not found for the Chex-pyrrhocoricin-Dap~Ac! peptide. All mice Dap~Ac! peptide ~peptide 18! degraded considerably faster than showed some enlargement of the abdominal region during the either peptide 1 or peptide 19 ~data not shown!. Remarkably, after study; therefore, at termination, the mice were necropsied. Stom- 8 h of digestion, in mouse serum, where the in vivo experiments achs and intestines were observed to be enlarged due to the pres- were planned, both peptides 1 and 19 had 20% of the initial amounts ence of food or stool. There were no other findings. These data intact. Because we currently use the peptides well above the effi- indicate that the increase of the proteolytic stability of the pyrrho- cacious concentrations, this 20% remaining uncleaved peptide coricin peptides compared to drosocin resulted in peptides capable amount can successfully protect the experimental animals from of protecting mice against bacterial infection. The results also war- live bacterial challenge. rant further pharmaceutical development with peptides based on the Chex-pyrrhocoricin-Dap~Ac! lead. This analog showed good potency, complete lack of toxicity, and similarity in the degrada- In vivo antibacterial activity tion pathway between human and mouse sera. The 10–50 mg0kg Peptides 1 and 19 were submitted to in vivo toxicity and antibac- dosage range was applied to obtain direct comparison with the terial efficacy assays ~Fig. 2!. Mice of the CD-1 strain were intra- drosocin study ~Hoffmann et al., 1999!. Our next experiments will venously infected with E. coli strain ATCC 25922. Peptides were include the identification of the lowest active dose, optimal mode intravenously injected 1 h after infection at 10, 25, and 50 mg0kg of delivery, and determination of in vivo activity spectrum of doses, followed by a second injection after 5 h. The survival rate peptide 19 and peptide 23. was compared with that of control mice who were submitted to the same peptide treatment, but received 5% dextrose ~DS5! in- Conformation of native pyrrhocoricin Because native pyrrhocoricin could not be shortened nor could the amino acid composition be changed without a loss of in vitro antibacterial activity, we hypothesized that the peptide has to as- sume a certain secondary structure to bind stereospecifically to the target protein. Accordingly, we determined the bioactive conformation of native glycosylated pyrrhocoricin and its nonglycosylated analog ~peptide 1! by two-dimensional nuclear magnetic resonance ~NMR! spectroscopy. Here, the structures are described in comparison with the recently published conformation of drosocin ~McManus et al., 1999!. It appeared that the structure of pyrrhocoricin, like drosocin, was largely random coil, and there was little change in the backbone conformation upon glycosylation. For pyrrhocoricin, however, there was a subpopulation with organized structure at both the N- and C-termini. Figure 3A and 3B summa- rize the short- and medium-range nuclear Overhauser effects ~NOEs! and show that there were a series of dNN~i,iϩ1! NOEs present at both the N- and C-termini of the pyrrhocoricins. Additional d ϩ Fig. 1. Stability of pyrrhocoricin ~peptide 1!~open symbols! and Chex- aN~i,i 2! pyrrhocoricin-Dap~Ac!~peptide 19!~closed symbols! in 25% aqueous NOEs were found in the spectra of the glycosylated derivative. mammalian sera. The peptides were incubated in serum on the same day in Taken together, these data indicated the presence of reverse turns triplicate, and the resulting samples were chromatographed over a two- at the pharmacologically important terminal regions. The increase week period. The incubation temperature was 37 8C. Squares correspond to in the turn potential at the termini compared to drosocin ~McManus digestion in mouse serum, and triangles correspond to digestion in human sera. The experimental data were collected in triplicate and were fitted et al., 1999! may explain the increased in vitro antibacterial activ- to an exponential equation by the SlideWrite program for graphical ity of pyrrhocoricin ~Hoffmann et al., 1999!. A comparison of the representation. aH NMR shifts with random coil values ~Merutka et al., 1995! In vivo active antibacterial peptides 747

Fig. 2. In vivo antibacterial activity of pyrrhocoricin ~peptide 1!~open symbols! and Chex-pyrrhocoricin-Dap~Ac!~peptide 19!~closed symbols!. Three mice per group were used for toxicity ~broken lines!, and five mice per group were used for efficacy studies ~solid lines!. Five additional mice were infected with E. coli ATCC 25922 for negative controls and received 5% dextrose ~DS5! instead of test peptides ~dots and dashes with crosses!.

is shown in Figure 3C. These results suggested that the middle ism ~CD! spectroscopy ~data not shown!. Nonglycosylated domain of pyrrhocoricin, like drosocin, had an extended structure. pyrrhocoricin ~peptide 1! exhibited a linear unordered r reverse Both the unordered-turn conformational equilibrium and the pres- turn conformational transition upon going from water to trifluoro- ence of extended regions were further verified by circular dichro- ethanol as solvents. The CD spectrum of the native glycopeptide

Fig. 3. Summary of NOE connectivities ~A and B! and deviations of the aH chemical shifts from their “random coil” values ~C! for nonglycosylated pyrrhocoricin ~peptide 1!, and its native counterpart, containing a Gal-GalNAc disaccharide moiety on Thr11. The random coil values ~Merutka et al., 1995! are corrected for sequence-specific shift of 0.29 ppm for residues preceding Pro. For the NOE connectivities, the intensities are indicated by the thickness of the line. 748 L. Otvos et al. recorded in water was more similar to those of unordered peptides Biospectrometry Workstation by standard methods. Mass spectra ~type U spectra!. However, the type C spectrum, and therefore the verified the anticipated composition of the peptides. final turn structure, was stabilized at a lower trifluoroethanol concentration ~50%! for the glycopeptide compared to the nonglyco- In vitro antibacterial and hemolytic assays sylated analog, fully supporting the NMR findings. The broadening of the negative band between 210 and 220 nm in the aqueous spec- Antibacterial assays were performed in sterile 96-well plates ~Nunc tra identified the presence of extended structures for both peptides. F96 microtiter plates! with a final volume of 100 mL as described In summary, according to the NMR data, the bioactive confor- earlier ~Bulet et al., 1996!. Briefly, 90 mL of a suspension of a mation of native pyrrhocoricin involves two reverse turns at the midlogarithmic phase bacterial culture at an initial 600 nm ultra- termini bridged by an extended peptide segment in the middle violet ~UV! absorbance of 0.001 in Luria-Bertani–rich nutrient domain. This hypothetical structure is supported by the antibacte- medium was added to 10 mL of serially diluted peptides in steril- rial activity of the cyclic peptide analogs. Cyclization stabilizes ized water. The final peptide concentrations ranged between 0.15 reverse turns ~Woody, 1985! and was expected to improve potency, and 80 mM. Plates were incubated at 30 8C for 24 h with gentle but the cyclic pyrrhocoricin derivative, without expanding the cy- shaking, and growth inhibition was measured by recording the cle ~peptide 22!, lost activity compared to the analog linear peptide increase of the UV absorbance at 600 nm on a SLT Labinstruments ~peptide 13!, probably due to distortion of the extended domain in 400 ATC microplate reader. The higher temperature compared to the middle of the peptide. When the ring size was increased by traditional antibacterial assays ~25 8C! was applied to increase the repeating an internal octapeptide fragment ~peptide 23!, the result- sensitivity of the assay. Hemolytic activity was assayed with sheep ing cyclic analog became highly active against both Gram-negative erythrocytes suspended in Alsever’s solution ~BioWhittaker, Walk- and Gram-positive bacterial strains. This hypothesis will be tested ersville, Maryland!, and diluted with Dulbecco’s phosphate-buffered experimentally when higher amounts of the cyclo-@K-pyrrhocoricin saline, pH 7.2. Thirty microliters of 1% suspension of the red blood 1-20–14-7# peptide are available for detailed CD and NMR stud- cells was incubated with agitation at 39 8C with 30 mLof40– ies. High resolution conformational analysis of the drug lead Chex- 256 mM peptides dissolved in phosphate-buffered saline for 1 h pyrrhocoricin-Dap~Ac! peptide ~peptide 19! is also currently in and centrifuged at 1,000 g for 5 min. Fifty microliters of the progress in our laboratories. This structure will be reported later, supernatant was collected, and the release of hemoglobin was de- together with the complete pharmacological profile of this peptide tected by measuring the UV absorbance at 405 nm. analog. In vivo toxicity and efficacy studies Materials and methods In vivo toxicity and antibacterial activity of pyrrhocoricin and the Chex-pyrrhocoricin-Dap~Ac! derivative were evaluated at Chrysa- Peptide synthesis lis Preclinical Services Corporation ~Olyphant, Pennsylvania!. The Peptides were assembled by a Milligen 9050 continuous-flow conditions were designed to duplicate those during the similar automated synthesizer on a Fmoc-PAL-polyethylene-glycol- assay of drosocin ~Hoffmann et al., 1999!. According to this pro- polystyrene copolymer resin with an initial load of 0.17 mmol0g tocol, male mice of CD-1 strain ~Harlan Sprague Dawley, Inc., ~PerSeptive Biosystems, Warrington, United Kingdom!. Standard Stamford, Connecticut! were intravenously infected in the tail with Fmoc-chemistry was used throughout ~Fields & Noble, 1990! with 1,000,000 colony forming units ~0.2 mL! of E. coli strain ATCC 4 mol excess of the acylating amino acids and HATU ~1-hydroxy- 25922. To obtain better infection, mice were also fed with E. coli. 7-azabenzotriazole uranium salt! activation, recommended for the With this infection strategy, one of five control mice died on day synthesis of complex peptides ~Angell et al., 1994!. The side-chain 1, and two of five mice died on day 2. All the control mice re- protecting groups were trityl for Asn, tert-butyl ether for Tyr, Ser, maining alive became clinically sick by day 2. The pyrrhocoricin and Thr, tert-butyl ester for Asp, 2,2,5,7,8-pentamethyl-chroman- peptides were intravenously injected 1 h after infection at doses of 6-sulfonyl for Arg, and tert-butyloxy-carbonyl for Lys. Fmoc-1- 10, 25, and 50 mg0kg, followed by a booster injection of the amino-cyclohexane-carboxylic acid was purchased from Neosystem peptides after5hofinfection. Mice were observed at 1 and 5 h, 1 Laboratoire ~Strasbourg, France!. Fmoc-diamino-propionic acid from and 2 days postinfection for clinical signs or mortality, and were Bachem Biosciences ~King of Prussia, Pennsylvania! was acety- compared with control mice who received 5% dextrose ~DS5! lated with equimolar amount of pentafluorophenyl acetate prior to instead of peptides ~negative controls!, or were submitted to the peptide synthesis and was used for peptide assembly without any same peptide treatment, but received 50 mg0kg of DS5 instead of further purification. The glycopeptides were synthesized from the bacteria ~toxicity!. Clinical signs included decreased activity commercially available glycoamino acid building blocks, in- and head tilt. cluding Fmoc-Thr@Gal~Ac4!-GalNAc~Ac2!#-OH and Fmoc- Asn@GlcNAc~Ac !#-OH ~Novabiochem, San Diego, California!. 3 NMR spectroscopy Peptides were cleaved from the solid support by trifluoroacetic acid in the presence of m-cresol ~2.5%!, ethane–dithiol ~2.5%!, 1H NMR spectra were recorded on a Bruker DMX 750 MHz thioanisole ~5%!, and water ~5%! as scavengers for 2–3 h. Deacet- spectrometer at temperatures in the range 283–298 K. The sample ylation of the sugar hydroxyl groups was accomplished bya2min consisted of 600 mg of native glycosylated or nonglycosylated treatment with 0.1 M NaOH ~Otvos et al., 1994!. After cleavage, pyrrhocoricin in 125 mL of 50% TFE-d3:50% H2O solution in a peptides were purified by reversed-phase high-performance liquid 2.5 mm NMR tube. Two-dimensional total correlation spectros- chromatography. The final products were characterized by matrix- copy ~TOCSY! and NOE spectroscopy ~NOESY! spectra were assisted laser desorption0ionization mass spectrometry at the Wis- recorded in the phase-sensitive mode using time-proportional phase tar Institute Protein Microchemistry Laboratory on a Voyager incrementation for quadrature detection in the f1-dimension ~Marion In vivo active antibacterial peptides 749

& Wüthrich, 1983!. TOCSY spectra were recorded using an Cudic M, Bulet P, Hoffmann R, Craik DJ, Otvos L Jr. 1999. Chemical synthesis, MLEV-17 mixing scheme ~Bax & Davis, 1985! with mixing times antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects. Eur J Biochem 266:549–558. of 80 ms, 16 scans, and 512 increments. NOESY spectra were Ertl HCJ, Dietzschold B, Gore M, Otvos L Jr, Larson JK, Wunner WH, Ko- recorded with mixing times of 100 and 300 ms, 64 scans, and 512 prowski H. 1989. Induction of rabies virus-specific T helper cells by syn- increments. All two-dimensional spectra were collected over 4,096 thetic peptides that carry dominant T-helper cell epitopes of the viral data points in the f2 dimension, with a spectral width of 9,800 Hz ribonucleoprotein. J Virol 63:2885–2892. Fields GB, Noble RL. 1990. Solid-phase peptide synthesis using 9-fluorenyl- in both dimensions. The water proton signal was suppressed using methoxycarbonyl amino acids. Int J Pept Protein Res 35:161–214. the WATERGATE method ~Piotto et al., 1992!, consisting of two Gillespie JP, Kanost MR, Trenczek T. 1997. Biological mediators of insect sine-shaped gradient pulses on either side of a binomial 3-9-19 immunity. Annu Rev Entomol 42:611–643. Hara S, Yamakawa M. 1995. A novel antibacterial peptide family isolated from pulse of 10 kHz field strength. Spectra were referenced to DSS. the silkworm, Bombyx mori. Biochem J 310:651–656. The data were processed on a Silicon Graphics ~SGI 4D030! com- Hoffmann R, Bulet P, Urge L, Otvos L Jr. 1999. Range of activity and metabolic puter using the UXNMR software package. The f1-dimension was stability of synthetic antibacterial glycopeptides from insects. Biochim Bio- zero-filled to 4,096 real data points, with f1- and f2-dimensions phys Acta 1426:459–467. Marion D, Wüthrich K. 1983. Application of phase-sensitive two-dimensional being multiplied by a squared sine function and Gaussian function, correlated spectroscopy ~COSY! for measurements of 1H-1H spin-spin cou- respectively, prior to Fourier transformation. pling constants in proteins. Biochem Biophys Res Commun 113:967–974. McManus A, Otvos L Jr, Hoffmann R, Craik DJ. 1999. Conformational studies by NMR of the anti-microbial peptide, drosocin and its non-glycosylated Acknowledgments derivative: Effects of glycosylation on solution conformation. Biochemistry 38:705–714. The authors wish to thank Dr. Magdalena Blaszczyk-Thurin for critical Merutka G, Dyson HJ, Wright PE. 1995. “Random coil” 1H chemical shifts reading of the manuscript. This work was supported by NIH Grant GM obtained as a function of temperature and trifluoroethanol concentration for 45011 ~to L.O.!. D.J.C. is an Australian Research Council Senior Fellow. the peptide series GGXGG. J Biomol NMR 5:14–24. Oh JE, Hong SY, Lee K-H. 1999. 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