Title: Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin
Abstract: θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins. θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins. Under normal circumstances Bacillus anthracis causes human infections only in individuals exposed to infected farm animals or their spore-contaminated products. The virulence of B. anthracis primarily derives from the hardiness of its spores, an anti-phagocytic capsule that surrounds its vegetative cells (1Green B.D. Battisti L. Koehler T.M. Thorne C.B. Ivins B.E. Infect. Immun. 1985; 49: 291-297Crossref PubMed Google Scholar), and two secreted binary toxins: lethal toxin (LeTx) 3The abbreviations used are: LeTx, lethal toxin; EdTx, edema toxin; EF, edema factor; HNP-1, human neutrophil peptide-1 (an α-defensin); LF, lethal factor; LGA, Lamarckian genetic algorithm; MAPKK-2, mitogen-activated protein kinase kinase-2; MEC, minimum effective concentration; PA, protective antigen; RC100IAA, reduced and alkylated RC-1; RC-1XX, various retrocyclin analogues; RC-1, retrocyclin-1; RTD, rhesus θ-defensin; SPR, surface plasmon resonance; RU, resonance unit(s); BSA, bovine serum albumin; r.m.s.d., root mean square deviation. 3The abbreviations used are: LeTx, lethal toxin; EdTx, edema toxin; EF, edema factor; HNP-1, human neutrophil peptide-1 (an α-defensin); LF, lethal factor; LGA, Lamarckian genetic algorithm; MAPKK-2, mitogen-activated protein kinase kinase-2; MEC, minimum effective concentration; PA, protective antigen; RC100IAA, reduced and alkylated RC-1; RC-1XX, various retrocyclin analogues; RC-1, retrocyclin-1; RTD, rhesus θ-defensin; SPR, surface plasmon resonance; RU, resonance unit(s); BSA, bovine serum albumin; r.m.s.d., root mean square deviation. and edema toxin (EdTx). Both toxins contain protective antigen (PA, 83 kDa). LeTx also contains lethal factor (LF, 90 kDa), and EdTx contains edema factor (EF, 89 kDa). The genes for all three toxin components, PA, LF, and EF, reside on the pXO1 plasmid (2Okinaka R.T. Cloud K. Hampton O. Hoffmaster A.R. Hill K.K. Keim P. Koehler T.M. Lamke G. Kumano S. Mahillon J. Manter D. Martinez Y. Ricke D. Svensson R. Jackson P.J. J. Bacteriol. 1999; 181: 6509-6515Crossref PubMed Google Scholar), and those responsible for capsule synthesis exist on the pXO2 plasmid (3Collier R.J. Young J.A. Annu. Rev. Cell Dev. Biol. 2003; 19: 45-70Crossref PubMed Scopus (470) Google Scholar). Both of these plasmids are required for in vivo virulence (3Collier R.J. Young J.A. Annu. Rev. Cell Dev. Biol. 2003; 19: 45-70Crossref PubMed Scopus (470) Google Scholar).EF is an adenylate cyclase (4Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3162-3166Crossref PubMed Scopus (756) Google Scholar) and LF is a zinc-dependent metalloprotease that selectively attacks certain MAPK kinases (5Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Crossref PubMed Scopus (885) Google Scholar, 6Vitale G. Pellizzari R. Recchi C. Napolitani G. Mock M. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 706-711Crossref PubMed Scopus (361) Google Scholar). PA is required to allow both of the other toxin components to enter host cells (7Gordon V.M. Klimpel K.R. Arora N. Henderson M.A. Leppla S.H. Infect. Immun. 1995; 63: 82-87Crossref PubMed Google Scholar). When PA binds a cellular receptor (8Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A. Nature. 2001; 414: 225-229Crossref PubMed Scopus (747) Google Scholar), it is cleaved into PA63 (63 kDa) and PA20 (20 kDa). The PA20 diffuses away, and the residual receptor-bound PA63 molecules self-associate into ring-shaped heptamers (9Milne J.C. Furlong D. Hanna P.C. Wall J.S. Collier R.J. J. Biol. Chem. 1994; 269: 20607-20612Abstract Full Text PDF PubMed Google Scholar) that bind EF or LF with high affinity (10Cunningham K. Lacy D.B. Mogridge J. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7049-7053Crossref PubMed Scopus (149) Google Scholar, 11Mogridge J. Cunningham K. Lacy D.B. Mourez M. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7045-7048Crossref PubMed Scopus (165) Google Scholar, 12Mogridge J. Cunningham K. Collier R.J. Biochemistry. 2002; 41: 1079-1082Crossref PubMed Scopus (183) Google Scholar). Oligomerization of PA63 leads to endocytosis, which transports the complexes to an acidic compartment (13Beauregard K.E. Collier R.J. Swanson J.A. Cell Microbiol. 2000; 2: 251-258Crossref PubMed Scopus (92) Google Scholar, 14Friedlander A.M. J. Biol. Chem. 1986; 261: 7123-7126Abstract Full Text PDF PubMed Google Scholar, 15Gordon V.M. Leppla S.H. Hewlett E.L. Infect. Immun. 1988; 56: 1066-1069Crossref PubMed Google Scholar). Here, the heptameric pre-pore changes into an integral-membrane pore (16Blaustein R.O. Koehler T.M. Collier R.J. Finkelstein A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2209-2213Crossref PubMed Scopus (239) Google Scholar, 17Milne J.C. Collier R.J. Mol. Microbiol. 1993; 10: 647-653Crossref PubMed Scopus (132) Google Scholar) that translocates EF or LF into the cytosol (18Wesche J. Elliott J.L. Falnes P.O. Olsnes S. Collier R.J. Biochemistry. 1998; 37: 15737-15746Crossref PubMed Scopus (171) Google Scholar). Immunization against PA is protective (19Friedlander A.M. Welkos S.L. Ivins B.E. Curr. Top. Microbiol. Immunol. 2002; 271: 33-60PubMed Google Scholar).Defensins are small, β-sheet peptides that collectively possess broad antibacterial, antifungal, and antiviral properties (20Ganz T. C. R. Biol. 2004; 327: 539-549Crossref PubMed Scopus (144) Google Scholar, 21Lehrer R.I. Nat. Rev. Microbiol. 2004; 2: 727-738Crossref PubMed Scopus (448) Google Scholar, 22Selsted M.E. Ouellette A.J. Nat. Immunol. 2005; 6: 551-557Crossref PubMed Scopus (934) Google Scholar, 23Lehrer R.I. Lichtenstein A.K. Ganz T. Annu. Rev. Immunol. 1993; 11: 105-128Crossref PubMed Scopus (895) Google Scholar). They are believed to be especially important as "first responders" to microbial and viral incursions and to play critical roles in defending the mucosal surfaces that line the respiratory, gastrointestinal, and genitourinary tracts. Humans express 6 different α-defensins and 30 or more β-defensins (24Scheetz T. Bartlett J.A. Walters J.D. Schutte B.C. Casavant T.L. McCray Jr., P.B. Immunol. Rev. 2002; 190: 137-145Crossref PubMed Scopus (31) Google Scholar, 25Schutte B.C. Mitros J.P. Bartlett J.A. Walters J.D. Jia H.P. Welsh M.J. Casavant T.L. McCray Jr., P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2129-2133Crossref PubMed Scopus (435) Google Scholar). Human α-defensins (HNPs) are potent noncompetitive inhibitors of the metalloprotease activity of anthrax LF. They can protect murine macrophages from B. anthracis LeTx in vitro and provide protection to mice when co-injected with a lethal dose of LeTx (26Kim C. Gajendran N. Mittrucker H.W. Weiwad M. Song Y.H. Hurwitz R. Wilmanns M. Fischer G. Kaufmann S.H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4830-4835Crossref PubMed Scopus (128) Google Scholar).θ-Defensins are cyclic octadecapeptides that are encoded by mutated α-defensin genes (27Nguyen T.X. Cole A.M. Lehrer R.I. Peptides. 2003; 24: 1647-1654Crossref PubMed Scopus (159) Google Scholar). They have been purified, as peptides, only from the leukocytes and bone marrow of rhesus macaques (28Selsted M.E. Curr. Protein Pept. Sci. 2004; 5: 365-371Crossref PubMed Scopus (99) Google Scholar, 29Tang Y.Q. Yuan J. Osapay G. Osapay K. Tran D. Miller C.J. Ouellette A.J. Selsted M.E. Science. 1999; 286: 498-502Crossref PubMed Scopus (614) Google Scholar, 30Leonova L. Kokryakov V.N. Aleshina G. Hong T. Nguyen T. Zhao C. Waring A.J. Lehrer R.I. J. Leukoc. Biol. 2001; 70: 461-464PubMed Google Scholar), whose three θ-defensins are named rhesus θ-defensins (RTDs) 1-3. Humans have multiple θ-defensin genes, including some that are transcribed. However, human genes and their transcripts contain a premature stop codon that aborts successful translation (27Nguyen T.X. Cole A.M. Lehrer R.I. Peptides. 2003; 24: 1647-1654Crossref PubMed Scopus (159) Google Scholar, 31Cole A.M. Hong T. Boo L.M. Nguyen T. Zhao C. Bristol G. Zack J.A. Waring A.J. Yang O.O. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1813-1818Crossref PubMed Scopus (264) Google Scholar). Retrocyclins 1-3 are synthetic θ-defensin peptides, whose structures are based on human multiple θ-defensin pseudogenes. Conceptually, they represent peptides last produced by apes whose eventual progeny included gorillas, chimps, and humans. The purpose of this study was to examine the effects of θ-defensins on vegetative cells and spores of B. anthracis and on the enzymatic and cytotoxic properties of anthrax LF.EXPERIMENTAL PROCEDURESRetrocyclin and Other PeptidesRetrocyclins (31Cole A.M. Hong T. Boo L.M. Nguyen T. Zhao C. Bristol G. Zack J.A. Waring A.J. Yang O.O. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1813-1818Crossref PubMed Scopus (264) Google Scholar) and HNP-1, -2, and -3 (32Wu Z. Powell R. Lu W. J. Am. Chem. Soc. 2003; 125: 2402-2403Crossref PubMed Scopus (58) Google Scholar) were prepared by solid-phase peptide synthesis as described previously. Peptide concentrations were established by quantitative amino acid analysis (for θ-defensins) or by A280 measurement (for HNPs). Table 1 contains the sequence of every peptide used in this study.TABLE 1Sequences of the peptides used in this studyName/laboratory IDSequenceHNP-1ACYCRIPACIAGERRYGTCIYQGRLWAFCCRC-100cyclic[GICRCICGRGICRCICGR]Chirality and orderRC-110cyclic[RGCICRCIGRGCICRCIG] (all-D)RC-111cyclic[RGCICRCIGRGCICRCIG]RC-112cyclic[GICRCICGRGICRCICGR] (all-D)Arginine substitutionsRC-100cyclic[GICRCICGRGICRCICGR]RC-107Gcyclic[GICRCICGGGICRCICGR]RC-107GGcyclic[GICRCICGGGICRCICGG]RC-107G2H2cyclic[GICHCICGGGICHCICGG]RC-107G2Hacyclic[GICRCICGGGICHCICGG]RC-107G2Hbcyclic[GICHCICGGGICRCICGG]RC-101cyclic[GICRCICGKGICRCICGR]Retrocyclins versus RTDsRC-100cyclic[GICRCICGRGICRCICGR]RC-100bcyclic[GICRCICGRRICRCICGR]RC-100ccyclic[RICRCICGRRICRCICGR]RTD-1cyclic[GFCRCLCRRGVCRCICTR]RTD-2cyclic[GVCRCLCRRGVCRCICRR]RTD-3cyclic[GFCRCICTRGFCRCICTR]Cyclic backbone and SS bondsNoncyclic RC-100GICRCICGRGICRCICGRReduced and alkylated RC-100cyclic[GICRCICGRGICRCICGR]Protegrin PG-1RGGRLCYCRRRFCVCVGR-amide Open table in a new tab Spore PreparationB. anthracis (Sterne strain 7702) spores were prepared as described elsewhere (33Banks D.J. Barnajian M. Maldonado-Arocho F.J. Sanchez A.M. Bradley K.A. Cell Microbiol. 2005; 7: 1173-1185Crossref PubMed Scopus (77) Google Scholar). Briefly, B. anthracis was grown in Trypticase soy broth medium (T8907, Sigma) at 30 °C with constant shaking at 250 rpm for 5-7 days until sporulation. The culture was centrifuged at 6000 × g for 20 min at 4 °C. The pellet was resuspended in sterile water, and cultured at 30 °C for two more days with constant shaking to promote further sporulation and bacterial lysis. Complete spore formation was confirmed by light microscopy. Spores were centrifuged at 6000 × g for 20 min at 4 °C and washed five times with sterile water. Before use, the spores were heated at 65 °C for 30 min to kill any germinated or germinating spores. No intact bacilli were present at this stage. Serial dilutions of the spore preparation were plated on Trypticase soy agar plates to determine the concentration of colony forming units.Radial Diffusion AssayTwo-stage radial diffusion assays were used to test the antimicrobial activity of peptides against B. anthracis spores and vegetative cells (34Lehrer R.I. Rosenman M. Harwig S.S. Jackson R. Eisenhauer P. J. Immunol. Methods. 1991; 137: 167-173Crossref PubMed Scopus (583) Google Scholar).Stage 1—1-4 × 106 colony forming units were dispersed in a thin 1% agarose underlay gel containing 10 mm phosphate buffer (pH 7.4), 100 mm NaCl, and 1% (v/v) Trypticase soy broth. A 6 × 6 array of wells, each with a 3-mm diameter and 9-μl capacity, was punched. Serially diluted peptide solutions containing 250, 79, 25, 7.9, 2.5, and 0.79 μg/ml peptides (8 μl each) were added to each set of 6 wells. The plate was incubated at 37 °C for 3 h to allow the peptides to diffuse into the underlay gel.Stage 2—An overlay gel containing 60 mg/ml Trypticase soy broth powdered medium plus 1% agarose was poured over the underlay gel, and the plate was incubated overnight to allow surviving bacteria to form micro-colonies. The clear zones around each well were measured 18-24 h later. To determine the minimal effective concentration (MEC), a linear regression function relating the adjusted diameter (zone diameter minus the well diameter) to the log10 peptide concentration was calculated. The X-intercept defined by this function gives the MEC. Typically, the correlation coefficient (r2) was >0.98.Enzymatic AssayThe enzymatic activity of anthrax lethal factor (LF), a zinc metalloprotease, was measured by monitoring cleavage of a specific substrate by fluorescence resonance energy transfer. The substrate, purchased from Calbiochem, was an internally quenched, N-acetylated, C-7-amino-4-methylcoumarin derivative of a 14-mer mitogen-activated protein kinase/extracellular signal-regulated-kinase-2 (MEK-2) peptide. Its cleavage by recombinant LF (Calbiochem) resulted in increased fluorescence that was monitored kinetically with an fmax fluorescence microplate reader (Molecular Devices, Sunnyvale, CA), with excitation set at 360 nm and emission at 460 nm. Unless otherwise noted, 100 nm LF was incubated for 30 min at room temperature with the specified amount of θ-defensin peptide before 50 μm substrate (final concentration) was added.Murine Macrophage IntoxicationMurine macrophage-like RAW 264.7 cells were seeded in 384-well plates at 4000 cells per well and incubated overnight at 37 °C. The medium was replaced by 20 μl of fresh Dulbecco's modified Eagle's medium containing 25 mm HEPES, 2 mm glutamine, 100 μg (each) penicillin and streptomycin, and 1% fetal bovine serum. The α- and θ-defensin peptides were serially diluted into the same medium, and 20 μl was added to appropriate wells. Peptide concentrations ranged from 0 to 60 μg/ml. LeTx (20 μl) diluted in fresh media was added to each well giving final concentrations of 100 ng/ml PA and 100 ng/ml LF. Cells were incubated overnight at 37 °C. Cell viability was assayed with the CellTiter-Glo® luminescent cell viability kit per the manufacturer's (Promega, Madison, WI) instructions. IC50 values were obtained with GraphPad Prism Software.UltracentrifugationSedimentation equilibrium runs were performed at 25 °C in 12-mm path length double sector cells on a Beckman Optima XL-A analytical ultracentrifuge. Absorption was monitored at 228 nm for 0.1 mg/ml samples and 260 nm for 1.0 mg/ml samples. Peptide samples were in 100 mm NaCl, 10 mm Tris, pH 7.4, and sedimentation equilibrium profiles were measured at 40,000 and 50,000 rpm. The data were initially fitted with a nonlinear least-squares exponential fit for a single ideal species using the Beckman Origin-based software (version 3.01). Preliminary analysis of the association behavior used the global analysis software (the "multifit" option of the abovementioned software) to analyze four scans simultaneously, corresponding to protein at 0.1 mg/ml at 40,000 and 50,000 rpm and protein at 1.0 mg/ml at 40,000 and 50,000 rpm. The partial specific volume (0.711 of RC-1 was calculated from its amino acid composition (35Cohn E.J. Edsall J.T. Cohn E.J. Edsall J. T Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. Reinhold Publishing Corporation, New York1943: 374-377Google Scholar).Surface Plasmon Resonance StudiesSPR experiments were performed on a BIAcore 3000 system (BIAcore AB, Uppsala, Sweden). Proteins were immobilized on a BIAcore CM5 sensor chip using the BIAcore amine-coupling protocol. Analytes were introduced into the flow cells in a running buffer containing 10 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm EDTA. The running buffer also contained 0.005% polysorbate-20 to reduce nonspecific binding. Values were corrected for background binding to the CM5 chip. Data were analyzed with BIAevaluation 3.1 software, and curve fitting was done with an assumption of 1:1 binding.SPR results are expressed in resonance units (RUs). To calibrate the instrument, we synthesized [14C]RC-2, which contained [14C]glycine (Sigma) and purchased 125I-bovine serum albumin (BSA) from MP Biomedicals (Irvine, CA). The [14C]RC-2 had a specific activity of 21.6 μCi/mg, and the 125I-BSA had a specific activity of 987 μCi/mg. To calibrate the system for BSA, we immobilized a mouse anti-BSA monoclonal antibody (U.S. Biological, Swampscott, MA) on a CM5 biosensor chip. After measuring the binding of 125I-BSA (1 μg/ml) to the biosensor chip, the bound analyte was recovered using the BIAcore Analyte Recovery Wizard®x program, and its radioactivity was measured in a Beckman liquid scintillation spectrometer. From seven such measurements, we determined that 1 RU of BSA was equivalent to 13.35 ± 0.55 pg (mean ± S.E.). We used this value to estimate the amount of immobilized LF on the biosensor chip in experiments with retrocyclins. To obtain a conversion factor for RC-2, we used a biosensor that contained immobilized recombinant gp120 (BioDesign International, Saco, ME). From these experiments, we determined that 1 RU corresponded to 4.41 ± 0.15 pg of RC-2 (mean ± S.E., n = 3). We used these values to estimate the stoichiometry with which retrocyclins bound to LF.Computational MethodsDocking was accomplished with the AutoDock 3.06 suite of programs (36Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (8960) Google Scholar) which assumes that the macromolecule is rigid, while the ligand is allowed torsional flexibility. A Lamarckian genetic algorithm (LGA) searches the conformational space of the ligand in the vicinity of the macromolecule and ranks the docked molecules on the basis of its binding energy. Also available are two local search methods based on the method of Solis and Wets (37Solis F.J. Wets R.J.B. Math. Operation Res. 1981; 6: 19-30Crossref Scopus (1260) Google Scholar) and an empirical free energy function that estimates the binding free energy (36Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (8960) Google Scholar). In the present work all protein and ligand hydrogen atoms were explicitly modeled, with polar and nonpolar atoms being assigned Lennard-Jones 12-10 and 12-6 parameters, respectively. They were added to the native and ligand complexed forms of anthrax lethal factor (Protein Data Bank designations 1J7N (38Pannifer A.D. Wong T.Y. Schwarzenbacher R. Renatus M. Petosa C. Bienkowska J. Lacy D.B. Collier R.J. Park S. Leppla S.H. Hanna P. Liddington R.C. Nature. 2001; 414: 229-233Crossref PubMed Scopus (350) Google Scholar), 1PWW (39Turk B.E. Wong T.Y. Schwarzenbacher R. Jarrell E.T. Leppla S.H. Collier R.J. Liddington R.C. Cantley L.C. Nat. Struct. Mol. Biol. 2004; 11: 60-66Crossref PubMed Scopus (175) Google Scholar), and 1YQY) (40Shoop W.L. Xiong Y. Wiltsie J. Woods A. Guo J. Pivnichny J.V. Felcetto T. Michael B.F. Bansal A. Cummings R.T. Cunningham B.R. Friedlander A.M. Douglas C.M. Patel S.B. Wisniewski D. Scapin G. Salowe S.P. Zaller D.M. Chapman K.T. Scolnick E.M. Schmatz D.M. Bartizal K. MacCoss M. Hermes J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7958-7963Crossref PubMed Scopus (144) Google Scholar) and the ligand (L2) of 1PWW using the WHAT IF web interface (41Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3353) Google Scholar).The NMR structures of RTD-1 and retrocyclin (RC)-2 (1HVZ and 2ATG) already had hydrogen atoms. RC-1 was generated by mutating RC-2 in silico with Pymol. 4W. L. Delano (2002) The Pymol Molecular Graphics System, www.pymol.org. All water molecules were removed during docking. Partial charges were assigned to the protein atoms using all-atom charges of the AMBER force field (43Cornell W.D. Bayli C.I. Gould I.R. Merz K.M.J. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11449) Google Scholar). Atomic solvation parameters and atomic fragmental volumes were added with the AddSol program of AutoDock 3.06. The Lennard-Jones parameters used for Zn2+, taken form the work of Stote and Karplus (44Stote R.H. Karplus M. Proteins. 1995; 23: 12-31Crossref PubMed Scopus (316) Google Scholar), successfully reproduced the crystal structures of L1 and L2 (Table 4). Hydrogen atoms for the hydroxamate ligand of 1YQY (L1) were added with BABEL, 5P. Walters and M. Stahl (1992) BABEL, smog.com/chem/babel/. and partial charges were generated with GAMESS (46Schmidt M.W. Baldridge K.K. Boatz J.A. Elbert S.T. Gordon M.S. Jensen J.H. Koseki S. Matsunaga N. Nguyen K.A. Su S.J. Windus T.L. Dupuis M. Montgomery J.A. J. Comput. Chem. 1993; 14: 1347-1363Crossref Scopus (19029) Google Scholar). Ligand rotatable bonds for all docked ligands were defined using the AutoTors module of AutoDock.TABLE 4Results of the computational docking studyDocked moleculeSource ligandPDB receptorSearch typeaL refers to a local search, and G refers to an LGA-based global search.No. of atomsTorsionsDocked energyr.m.s.d.No. of docksInternalIntermolecularOverallkcal/molA°L11YQY1YQYL428−17.65−101.15−118.800.75200L11YQY1YQYG428−19.44−104.23−123.670.86100L21PWW1PWWL19358−72.14−202.18−274.320.57200L21PWW1PWWG19358−109.47−145.24−254.715.97200RC-12ATGbRC-1 was generated by an in silico mutation on the RC-2 NMR structure.1J7NG26044cIncludes only side-chain torsional degrees of freedom. The circular peptide backbone was kept fixed during docking.−154.29−148.74−303.03—131RC-22ATG1J7NG27751cIncludes only side-chain torsional degrees of freedom. The circular peptide backbone was kept fixed during docking.−142.36−161.42−303.78—217RTD-11HVZ1J7NG28251cIncludes only side-chain torsional degrees of freedom. The circular peptide backbone was kept fixed during docking.−173.99−167.91−341.90—171a L refers to a local search, and G refers to an LGA-based global search.b RC-1 was generated by an in silico mutation on the RC-2 NMR structure.c Includes only side-chain torsional degrees of freedom. The circular peptide backbone was kept fixed during docking. Open table in a new tab Van der Waals and electrostatic energy grid maps were prepared using AutoGrid (36Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (8960) Google Scholar). These grid maps define the cubic space in the vicinity of receptor in which the search for the optimally binding ligand conformer is focused. The grid points were spaced 0.375 Å apart and based on the centers of L1 and L2, with the grid sized to allow a 5-Å clearance on either side of the ligands in the x, y, and z dimensions. For RC-2 and RTD-1 two grid sizes were used. The first grid was based on the N terminus MAPPK-2 peptide in complex with anthrax lethal factor (1JKY) (38Pannifer A.D. Wong T.Y. Schwarzenbacher R. Renatus M. Petosa C. Bienkowska J. Lacy D.B. Collier R.J. Park S. Leppla S.H. Hanna P. Liddington R.C. Nature. 2001; 414: 229-233Crossref PubMed Scopus (350) Google Scholar), which spans the entire active site. This grid was therefore centered on the MAPKK-2 peptide, also with a 5-Å clearance on either side in the x, y, and z dimensions and a grid spacing of 0.375 Å. Later, when it was found that the RTD-1 and RC-2 docked only in a cavity close to the C-terminal end of the MAPKK-2 peptide in active site, the grid size was reduced to cover only that volume of space to improve the search efficiency. RC-1 was therefore docked using the smaller grid alone. The force-field parameters of AutoDock 2.4 were used to evaluate nonbonded interaction energies instead of using the parameters of AutoDock 3.0, which estimates free energies. This was because the presence of a large number of conformational degrees of freedom in the docked ligands presented challenges with the estimation of the torsional free energy term (36Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (8960) Google Scholar). The reported binding energies are therefore representative of binding enthalpies and not binding free energies. Electrostatic interactions were evaluated using a distance-dependent dielectric constant to model solvent effects.For the global search using the LGA, the size of the initial random population was 200 individuals for the large grids and 50 individuals otherwise, the maximal number of energy evaluations was 2 × 107, the maximal number of generations was 500, the number of top individuals that survived into the next generation, the elitism, was 1, the probability that a gene would undergo a random change was 0.02, the crossover probability was 0.80, and the average of the worst energy was calculated over a window of ten generations.For a pure local search, the pseudo-Solis and Wets method was used, whereas the Solis and Wets method was used for the LGA part of the local search. The parameters used for local search in both cases were a maximum of 1000 iterations per local search, the probability of performing a local search on an individual was 1.0, the maximal number of consecutive successes or failures before doubling or halving the step size of the local search was 4, and the lower bound on the step size, 0.01, was the termination criteria for the local search. For RTD-1 and RC-2, a total of 50 dockings was performed using the large grid before switching to the smaller grid. For the root-mean squared deviation (r.m.s.d.) calculation of docked of L1 and L2, their crystal coordinates were used as reference.RESULTSActivity against Spores and Bacilli—We used the capsule-deficient Sterne strain of B. anthracis to examine the antimicrobial activity of retrocyclins. Radial diffusion and colony counting assays were performed in the presence of physiological NaCl concentrations. Table 2 summarizes the results of our radial diffusion assays, which showed that RC-1 and HNP-1 killed vegetative B. anthracis cells with an MEC < 1 μg/ml. Retrocyclin-1 also manifested this exceptional potency against B. anthracis spores, but HNP-1 did not (MEC 24.9 ± 0.49 μg/ml). Because RC-112, an all d-amino acid version of RC-1, and RC-110, a retroenantio version of RC-1 were as effective as the regular peptide, chiral interactions were apparently not required for antimicrobial activity. RC-111, the retro analog of RC-1, showed reduced activity against vegetative cells (MEC 13.6 ± 2.78 μg/ml) but