Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m808762200
ISSN1083-351X
AutoresKaren S. Harris, Joanne L. Casey, Andrew M. Coley, John A. Karas, Jennifer K. Sabo, Yen Yee Tan, Olan Dolezal, Raymond S. Norton, Andrew B. Hughes, Denis B. Scanlon, Michael Foley,
Tópico(s)Chemical Synthesis and Analysis
ResumoApical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters. Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters. According to World Health Organization reports, almost half of the world human population is at risk of contracting malaria and approximately one million people are killed by this disease each year, most of which are young children. No reliable vaccine currently exists against Plasmodium falciparum, the parasite that causes the most severe form of malaria, and parasites are becoming increasingly resistant to previously effective pharmaceuticals (1Ridley R.G. Nature. 2002; 415: 686-693Crossref PubMed Scopus (707) Google Scholar). Thus, novel strategies to combat this disease are urgently required. Proteins involved in the erythrocytic stage of the parasite life cycle represent attractive targets for anti-malarial therapeutics, because this stage is responsible for the clinical symptoms of malaria. Apical membrane antigen 1 (AMA1) 3The abbreviations used are: AMA1, apical membrane antigen 1; MTBD, 7-methyl-1,5,7-triazabicyclo-[4.4.0]-dec-5-ene; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PBS, phosphate-buffered saline; NOE, nuclear Overhauser effect; mAb, monoclonal antibody; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate; DMF, dimethylformamide; DCM, dichloromethane; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SPR, surface plasmon resonance. is an integral membrane protein common to all Plasmodium species that is thought to be important for parasite invasion of host red blood cells and hepatocytes (2Silvie O. Franetich J.F. Charrin S. Mueller M.S. Siau A. Bodescot M. Rubinstein E. Hannoun L. Charoenvit Y. Kocken C.H. Thomas A.W. van Gemert G.J. Sauerwein R.W. Blackman M.J. Anders R.F. Pluschke G. Mazier D. J. Biol. Chem. 2004; 279: 9490-9496Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 3Triglia T. Healer J. Caruana S.R. Hodder A.N. Anders R.F. Crabb B.S. Cowman A.F. Mol. Microbiol. 2000; 38: 706-718Crossref PubMed Scopus (256) Google Scholar). The molecular mechanisms of the role of AMA1 in parasite invasion are unknown, but recent evidence suggests that it is involved in stabilizing the binding of the invasive form of the parasite to the erythrocyte (3Triglia T. Healer J. Caruana S.R. Hodder A.N. Anders R.F. Crabb B.S. Cowman A.F. Mol. Microbiol. 2000; 38: 706-718Crossref PubMed Scopus (256) Google Scholar, 4Mitchell G.H. Thomas A.W. Margos G. Dluzewski A.R. Bannister L.H. Infect. Immun. 2004; 72: 154-158Crossref PubMed Scopus (182) Google Scholar). In animal models, immunization with recombinant AMA1 induces protection from subsequent parasite challenge (5Anders R.F. Crewther P.E. Edwards S. Margetts M. Matthew M.L. Pollock B. Pye D. Vaccine. 1998; 16: 240-247Crossref PubMed Scopus (188) Google Scholar, 6Collins W.E. Pye D. Crewther P.E. Vandenberg K.L. Galland G.G. Sulzer A.J. Kemp D.J. Edwards S.J. Coppel R.L. Sullivan J.S. Morris C.L. Anders R.F. Am. J. Trop. Med. Hyg. 1994; 51: 711-719Crossref PubMed Scopus (165) Google Scholar), and it appears to be an essential protein in the parasite life-cycle as AMA1 genetic knockouts are not viable (3Triglia T. Healer J. Caruana S.R. Hodder A.N. Anders R.F. Crabb B.S. Cowman A.F. Mol. Microbiol. 2000; 38: 706-718Crossref PubMed Scopus (256) Google Scholar, 7Hehl A.B. Lekutis C. Grigg M.E. Bradley P.J. Dubremetz J.F. Ortega-Barria E. Boothroyd J.C. Infect. Immun. 2000; 68: 7078-7086Crossref PubMed Scopus (137) Google Scholar). AMA1 has therefore received much attention as a potential vaccine candidate or drug target. Although lacking the major structural diversity of some other P. falciparum antigens, AMA1 exhibits allelic variation that is a result of point mutations. Because immunization with AMA1 induces inhibitory antibodies that are less effective against heterologous parasite lines (8Healer J. Murphy V. Hodder A.N. Masciantonio R. Gemmill A.W. Anders R.F. Cowman A.F. Batchelor A. Mol. Microbiol. 2004; 52: 159-168Crossref PubMed Scopus (140) Google Scholar, 9Hodder A.N. Crewther P.E. Anders R.F. Infect. Immun. 2001; 69: 3286-3294Crossref PubMed Scopus (275) Google Scholar, 10Kennedy M.C. Wang J. Zhang Y.L. Miles A.P. Chitsaz F. Saul A. Long C.A. Miller L.H. Stowers A.W. Infect. Immun. 2002; 70: 6948-6960Crossref PubMed Scopus (221) Google Scholar), overcoming this diversity is a key hurdle in the development of AMA1-based anti-malarials. An immune response or drug would therefore ideally target a conserved region that is required for function. In addition to polyclonal and mAbs that bind to AMA1 and block parasite invasion in vitro (9Hodder A.N. Crewther P.E. Anders R.F. Infect. Immun. 2001; 69: 3286-3294Crossref PubMed Scopus (275) Google Scholar, 11Dutta S. Haynes J.D. Barbosa A. Ware L.A. Snavely J.D. Moch J.K. Thomas A.W. Lanar D.E. Infect. Immun. 2005; 73: 2116-2122Crossref PubMed Scopus (55) Google Scholar, 12Kocken C.H. van der Wel A.M. Dubbeld M.A. Narum D.L. van de Rijke F.M. van Gemert G.J. van der Linde X. Bannister L.H. Janse C. Waters A.P. Thomas A.W. J. Biol. Chem. 1998; 273: 15119-15124Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), peptides that specifically recognize this antigen and demonstrate similar inhibitory activity have been isolated from random peptide libraries displayed on the surface of phage (13Harris K.S. Casey J.L. Coley A.M. Masciantonio R. Sabo J.K. Keizer D.W. Lee E.F. McMahon A. Norton R.S. Anders R.F. Foley M. Infect. Immun. 2005; 73: 6981-6989Crossref PubMed Scopus (88) Google Scholar, 14Li F. Dluzewski A. Coley A.M. Thomas A. Tilley L. Anders R.F. Foley M. J. Biol. Chem. 2002; 277: 50303-50310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The most potent peptide inhibitor of invasion was the recently identified 20-residue peptide, R1 (13Harris K.S. Casey J.L. Coley A.M. Masciantonio R. Sabo J.K. Keizer D.W. Lee E.F. McMahon A. Norton R.S. Anders R.F. Foley M. Infect. Immun. 2005; 73: 6981-6989Crossref PubMed Scopus (88) Google Scholar). Peptides that recognize functional sites and act as biological agonists or antagonists can be useful for the design of small molecule mimics, or as therapeutics per se. The R1 peptide is an important lead compound for drug development, because its ability to block parasite growth indicates that it targets a site critical for AMA1 function. However, of the four parasite lines tested, the R1 peptide was only effective against the 3D7 and D10 strains, and not HB3 or W2mef, limiting its utility as a therapeutic agent in its current form. A further barrier to the general use of peptides as pharmaceuticals is that they are rapidly degraded by peptidases and proteases in biological fluids, leading to poor bioavailability (15Egleton R.D. Davis T.P. Peptides. 1997; 18: 1431-1439Crossref PubMed Scopus (157) Google Scholar). It was therefore of interest to modify the R1 peptide to improve its pharmaceutical potential and AMA1-binding properties. The methylation of backbone amides (N-methylation) is one strategy of peptide modification that has been employed to confer superior characteristics on a lead peptide. This involves the replacement of residues within a peptide sequence with the corresponding N-methyl amino acid, subtly changing the peptide backbone. The introduction of a methyl group into a peptide has the advantage of substantially improving a number of pharmacokinetically useful parameters, including membrane permeability, conformational flexibility, and proteolytic stability (16Fairlie D.P. Abbenante G. March D.R. Curr. Med. Chem. 1995; 2: 654-686Google Scholar). Additionally, it results in a loss of hydrogen bonding potential at the affected site, reducing the role of mainchain hydrogen bonds at a binding interface and potentially altering binding properties (17Bergseng E. Xia J. Kim C.Y. Khosla C. Sollid L.M. J. Biol. Chem. 2005; 280: 21791-21796Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). There are a number of examples of N-methyl amino acid containing peptides with a range of bioactivities, including antibiotic (18Ebata M. Takahashi Y. Otsuka H. Bull. Chem. Soc. Jpn. 1966; 39: 2535-2538Crossref PubMed Scopus (29) Google Scholar), anti-viral (19Pettit G.R. Kamano Y. Herald C.L. Fujii Y. Kizu H. Boyd M.R. Boettner F.E. Doubek D.L. Schmidt J.M. Chapuis J.C. Michel C. Tetrahedron. 1993; 49: 9151-9170Crossref Scopus (168) Google Scholar), anti-cancer (20Ramanjulu J.M. Ding X.B. Joullie M.M. Li W.R. J. Org. Chem. 1997; 62: 4961-4969Crossref Scopus (24) Google Scholar), and immunosuppressant activities (21Wenger R.M. Helv. Chim. Acta. 1984; 67: 502-525Crossref Scopus (161) Google Scholar). Importantly, modification of biologically active peptides by the inclusion of N-methyl amino acids into an original sequence has been shown to enhance the potency (22Dechantsreiter M.A. Planker E. Matha B. Lohof E. Holzemann G. Jonczyk A. Goodman S.L. Kessler H. J. Med. Chem. 1999; 42: 3033-3040Crossref PubMed Scopus (753) Google Scholar), change the receptor subtype selectivity (23Rajeswaran W.G. Hocart S.J. Murphy W.A. Taylor J.E. Coy D.H. J. Med. Chem. 2001; 44: 1305-1311Crossref PubMed Scopus (55) Google Scholar), and stabilize a peptide toward proteolytic degradation in a biological system (24Bruehlmeier M. Garayoa E.G. Blanc A. Holzer B. Gergely S. Tourwe D. Schubiger P.A. Blauenstein P. Nucl. Med. Biol. 2002; 29: 321-327Crossref PubMed Scopus (63) Google Scholar). This approach therefore offers the potential to optimize multiple parameters simultaneously. In this study, we systematically N-methylate residues within the previously identified AMA1-binding peptide, R1, and describe the synthesis and altered properties of these mono-, di-, and tri-N-methylated R1 derivatives. To introduce an N-methyl group into residues for which an N-methyl analogue is not commercially available, we used on-resin N-methylation (25Miller S.C. Scanlan T.S. J. Am. Chem. Soc. 1997; 119: 2301-2302Crossref Scopus (216) Google Scholar), facilitating a comprehensive N-methyl scan of the R1 peptide. Analysis of each of these R1 derivatives revealed that inclusion of a single N-methyl group was sufficient to dramatically improve AMA1 affinity, bioactivity, and peptide stability without altering the global peptide structure. Moreover, the introduction of multiple N-methyl groups further increased AMA1 affinity and slightly improved recognition of AMA1 derived from different parasite lines. This represents a substantial step forward in the design of an effective anti-malarial compound able to bind to AMA1 and inhibit malarial parasite growth. Furthermore, this work may contribute to the development of a rational strategy for modulating and optimizing the activity of bioactive peptides. Materials-Chlorotrityl resin, N,N-diisopropylethylamine, piperidine, hydroxybenzotriazole, HBTU were all obtained from GLBiochem, Shanghai, China. DMF, DCM, diethyl ether, and 2-mercaptoethanol were obtained from Ajax Chemicals (Melbourne, Australia). MTBD, 1,8-diazabicyclo[5.4.0]undec-7-ene, methyl triflate, o-nitrobenzenesulfonyl chloride, chloranil, and TNBS were obtained from Sigma-Aldrich. The N-methyl amino acids Fmoc-NMe-Val-OH, Fmoc-NMe-Phe-OH, Fmoc-NMe-Ala-OH, Fmoc-NMe-Leu-OH, Fmoc-NMe-Ile-OH, Fmoc-NMe-Met-OH, and Fmoc-Ser-OH were obtained from Peptide Solutions (Melbourne, Australia). Fmoc-NMe-Arg(Mtr)-OH was obtained from Chem Impex (Wood Dale, IL). Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Glu(OtBu)-OH were obtained from GLBiochem and incorporated into the sequence as N-methyl residues by the "on-resin" method of Miller and Scanlan (25Miller S.C. Scanlan T.S. J. Am. Chem. Soc. 1997; 119: 2301-2302Crossref Scopus (216) Google Scholar). Derivatization of the Resin-Chlorotrityl resin (1.0 g, 1 mmol) was suspended in dry DCM (10 ml). Fmoc-Lys(Boc)-OH (1 mmol) was dissolved in dry DCM (10 ml), and the solution was added to the resin. N,N-diisopropylethylamine (4 mmol, 680 μl) was added, and the solution was stirred for 2 h. After this time methanol (1 ml) was added, and the solution was stirred for another 30 min. The resin was washed sequentially with DCM and ether and then air-dried. The loading of Fmoc-Lys(Boc) onto the resin was estimated by the increase in resin weight and was typically 0.5–0.6 mmol/g. Solid-phase Synthesis Protocol-The scale for synthesis was typically 0.5 g and 0.25 mmol. Resin (0.5 g) was washed with DMF (4 × 10 ml) and then treated with a solution of 25% piperidine/DMF (containing 0.1 m hydroxybenzotriazole) for 10 min. After this time the resin was washed with DCM (4 × 10 ml), then DMF (4 × 10 ml), and then a small sample (∼5 mg) was subjected to a TNBS test (26Hancock W.S. Battersby J.E. Anal. Biochem. 1976; 71: 260-264Crossref PubMed Scopus (261) Google Scholar) for the presence of primary amine. The Fmoc-amino acid (1 mmol and 4-fold excess) was activated by dissolution in DMF (2 ml) followed by the addition of a solution of HBTU in DMF (2 ml, 0.5 m) to which was added N,N-diisopropylethylamine (340 μl, 2 mmol). This solution was sonicated for 2 min and then added to the resin. This resin-coupling reaction mixture was agitated for 1 h and then drained and washed with DMF (4 × 10 ml). A resin sample was then tested for the presence of residual amine using the TNBS test (26Hancock W.S. Battersby J.E. Anal. Biochem. 1976; 71: 260-264Crossref PubMed Scopus (261) Google Scholar). If the test was positive then another aliquot of the Fmoc-amino acid was activated, and the coupling procedure was repeated. Where an N-methyl was to be incorporated into the peptide sequence following the piperidine treatment, o-nitrobenzenesulfonyl chloride (0.75 mmol, 3 eq) was dissolved in dry DCM (4 ml), and the solution was added to the resin together with 2,4,6-collidine (1.25 mmol, 5 eq). This reaction mixture was stirred for 2 h. The N terminus was then methylated by treatment with a solution of methyl triflate (0.75 mmol, 3 eq) and the base MTBD (2 eq, 77 mg) in DMF (4 ml), and the mixture was stirred for 30 min. After this period the resin was drained, washed with DMF (4 × 10 ml), and then treated repeatedly with a solution of 2-mercaptoethanol/1,8-diazabicyclo[5.4.0]undec-7-ene/DMF (200 μl/200 μl/4 ml). Usually this procedure involved 3 × 30 min treatments until there was no more yellow color evident in the solution (due to the o-nitrobenzenesulfonyl group). The resin was then tested for the presence of a secondary amine due to the N-methyl group on the N terminus. The next amino acid was then activated and coupled in the normal manner (HBTU). Note that the TNBS test could not be used to detect the secondary amine of proline and the N-methyl amino acids: in this case the chloranil test (27Vojkovsky T. Pept. Res. 1995; 8: 236-237PubMed Google Scholar) was used. Peptide Cleavage from Resin and Purification-Upon completion of the required peptide synthesis cycles the N-terminal amino group was deprotected and the resin was washed with DCM (5 × 10 ml) and lastly ether. The resin (typically 0.8–1.0 g) was air-dried overnight and then treated with a solution of trifluoroacetic acid/dithiothreitol/triisopropylsilyl/H2O (90/5/2.2/2.5, 10 ml) for 2 h. The mixture was then filtered, and cold ether (40 ml) was added to the solution, which resulted in the precipitation of the peptide. The peptide slurry was centrifuged (3500 rpm, 3 min) and the crude peptide was isolated by decanting the ethereal supernatant. The peptide was then dissolved in 30% acetonitrile/water, and the solution was lyophilized. The lyophilized peptides were purified on a Gemini C18 5 μm semi-prep high-performance liquid chromatography column (Phenomenex: dimension, 50 × 21.2 mm; flow, 6 ml/min). Collected fractions were analyzed on an Absorbosphere HD C18 5 μm analytical high-performance liquid chromatography column (Alltech: dimension, 150 × 3.2 mm; flow, 0.7 ml/min). Pure fractions were pooled and lyophilized, and the molecular weight was confirmed by electrospray mass spectrometry. Production of M13 bacteriophage displaying the R1 peptide was carried out as described elsewhere (28Casey J.L. Coley A.M. Foley M. Zachariou M. Affinity Chromatography Methods and Protocols. 2nd Ed. Humana Press, Totowa, NJ2007: 111-136Google Scholar). Briefly, K91 Escherichia coli cells harboring the modified phage genome fUSE5, which included DNA encoding the R1 peptide, were incubated in broth culture to produce phage. Phage were harvested by precipitation with polyethylene glycol/NaCl (30% (w/v) polyethylene glycol 8000 (Sigma), 2.6 m NaCl) followed by centrifugation and resuspension of the phage pellet in phosphate-buffered saline (PBS). Phage concentration was determined in colony-forming units per milliliter (cfu/ml) as previously described (28Casey J.L. Coley A.M. Foley M. Zachariou M. Affinity Chromatography Methods and Protocols. 2nd Ed. Humana Press, Totowa, NJ2007: 111-136Google Scholar). The 3D7 parasite line was cultured essentially as described previously (29Cranmer S.L. Magowan C. Liang J. Coppel R.L. Cooke B.M. Trans. R. Soc. Trop. Med. Hyg. 1997; 91: 363-365Abstract Full Text PDF PubMed Scopus (168) Google Scholar, 30Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6184) Google Scholar), and parasites were synchronized by sorbitol lysis of all but ring stage parasites (31Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2839) Google Scholar). R1 peptide and methylated R1 analogues were assayed for their ability to inhibit parasite growth as previously described (13Harris K.S. Casey J.L. Coley A.M. Masciantonio R. Sabo J.K. Keizer D.W. Lee E.F. McMahon A. Norton R.S. Anders R.F. Foley M. Infect. Immun. 2005; 73: 6981-6989Crossref PubMed Scopus (88) Google Scholar). Briefly, peptides were diluted in PBS, and 50 μl was added in triplicate to sterile flat-bottomed microtiter wells (Nunc). Complete culture media (50 μl) and synchronized schizont-stage parasites (100 μl) were added to each well (4% hematocrit, 0.3% parasitemia) and plates were incubated for 40–42 h at 37 °C in a moist atmosphere of 94% N2, 1% O2, 5% CO2. After washing with ice-cold PBS, parasites were frozen, then thawed, and relative parasitemia levels were determined by assaying for parasite lactate dehydrogenase activity (10Kennedy M.C. Wang J. Zhang Y.L. Miles A.P. Chitsaz F. Saul A. Long C.A. Miller L.H. Stowers A.W. Infect. Immun. 2002; 70: 6948-6960Crossref PubMed Scopus (221) Google Scholar). Absorbance was measured at 650 nm (A650 nm), and the percent inhibition of invasion was calculated as: 100 - [(A650 nm peptide sample - A650 nm red blood cells only)/(A650 nm no peptide control - A650 nm red blood cells only) × 100]. 96-well microtiter plates (Maxisorp, Nunc) were coated with 2 μg/ml recombinant AMA1. The antigen was diluted in coating buffer (15 mm Na2CO3, 34 mm NaHCO3, pH 9.6), and plates were incubated overnight at 4 °C. Unbound protein was removed by washing with PBS, and any unbound surfaces were blocked with 10% skim milk powder in PBS. R1 peptide or N-methylated R1 analogues were applied to the wells in duplicate in the presence of a constant concentration of R1 phage (∼2 × 109 cfu/ml). Following incubation for 1 h at room temperature, with shaking, unbound phage was removed by washing with PBS 0.05% Tween 20 (Sigma). Bound phage was detected with horseradish peroxidase-conjugated anti-M13 antibodies (1:5000, Amersham Biosciences). Binding was visualized using tetramethylbenzidine (Sigma), and the absorbance was read at 450 nm. A Biacore T100 biosensor instrument (32Papalia G.A. Baer M. Luehrsen K. Nordin H. Flynn P. Myszka D.G. Anal. Biochem. 2006; 359: 112-119Crossref PubMed Scopus (44) Google Scholar) was used to measure the kinetics of the interaction of R1 peptides and their N-methylated derivatives with AMA1 proteins from different strains of P. falciparum. All immobilization and binding experiments were performed at 25 °C using HBS-EP+ buffer (10 mm HEPES, 150 mm NaCl, 3.4 mm EDTA, and 0.05% surfactant P20, pH 7.4) as the running buffer. AMA1 was immobilized on CM5 sensor chips using standard amine-coupling chemistry. The carboxymethyl dextran surface was activated with a 7-min injection at 10 μl/min of a 1:1 ratio of 0.4 m 1-ethy-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/0.1 m N-hydroxy succinimide. Coupling of AMA1 to the chip surface was achieved using a manual control approach whereby small volumes (1–15 μl) of AMA1 diluted to 12.5 μg/ml in 10 mm sodium acetate, pH 4.5, were injected over the activated surface at 5 μl/min until immobilization of ∼1500–2500 relative units was reached (1 relative units = 1 pg of protein/mm2). The immobilization procedure was completed by a 7-min injection (10 μl/min) of 1 m ethanolamine (pH 8.5) to deactivate residual reactive sites. Final immobilized AMA1 levels achieved were as follows: 3D7 = 1530, HB3 = 2705 relative units and W2mef = 2499 relative units. In each experiment at least one of the flow cells on the chip was used as "mock surface" for referencing purposes. This was prepared by subjecting a surface to the amine coupling procedure with no protein present. All biosensor binding experiments were performed in triplicate in HBS-EP+ running buffer at 25 °C. To generate the binding data, a series of peptide concentrations, typically ranging from 5 to 320 nm for 3D7 and from 0.32 to 40.96 μm for W2mef and HB3, were injected over immobilized AMA1 at a constant flow rate of 60 μl/min for 1 min. Peptide dissociation was then monitored by injecting HBS-EP+ running buffer for 5 min, after which time all of the peptide completely dissociated from the AMA1 surface and thus no further regeneration was required. All Biacore sensorgrams were processed using Scrubber software (Version 2, available from Biologic Software, Australia). Sensograms were first zeroed on the y axis and then x-aligned at the beginning of the injection. Bulk refractive index changes were removed by subtracting the reference flow cell responses. The average response of all blank injections was subtracted from all analyte injections and blank sensorgrams to remove systematic artifacts in the experimental and reference flow cells. Scrubber analysis software was used to determine ka and kd from the processed data sets by globally fitting to a 1:1 biomolecular binding model that included the mass transport term (33Myszka D.G. Jonsen M.D. Graves B.J. Anal. Biochem. 1998; 265: 326-330Crossref PubMed Scopus (110) Google Scholar). The KD was calculated from the quotient kd/ka. Alternatively, for rapidly dissociating interactions, affinity (KD) estimates were derived using steady-state affinity algorithm available within Scrubber. Samples were prepared for NMR analysis by dissolving peptides in 500 μl of 95% H2O/5% 2H2O containing 10 mm sodium acetate to a final concentration of ∼1.7 mm. The pH was adjusted to 4.6–4.7. 1H NMR spectra of most peptides showed a single major set of resonances, indicating that any impurities were non-peptidic (the amide and aromatic regions of one-dimensional spectra are shown in supplemental Fig. S1). Additional peaks with intensities 90% pure, these additional peaks must have arisen from minor conformers. The most likely origin of this conformational heterogeneity is cis-trans isomerism around the peptide bond involving the N-methyl group at Leu-8; this has been observed previously in other N-methylated peptides (34Goodman M. Chen F. Lee C.Y. J. Am. Chem. Soc. 1974; 96: 1479-1484Crossref PubMed Scopus (15) Google Scholar, 35Trzepalka E. Kowalczyk W. Lammek B. J. Pept. Res. 2004; 63: 333-346Crossref PubMed Scopus (10) Google Scholar). Two different preparations of N-Me-Leu-8 showed identical ratios of major to minor species. Two-dimensional homonuclear total correlation spectra with a spin-lock time of 60 ms and double quantum filtered correlation NMR spectra were acquired at 600 MHz on a Bruker DRX-600 spectrometer. Two-dimensional nuclear Overhauser enhancement (NOESY) spectra with a mixing time of 250 ms were also acquired at 600 MHz. For N-Me-Leu-8 and N-Me-Val-1/N-Me-Leu-8/N-Me-Ser-14, nuclear Overhauser enhancement spectra with a mixing time of 250 ms were also acquired at 800 MHz on a Bruker Avance 800. Water was suppressed using the WATERGATE pulse sequence (36Piotto M. Saudek V. Sklenal V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3527) Google Scholar). All spectra were collected at 5 °C unless otherwise stated and were referenced to an impurity peak at 0.15 ppm or to the water resonance. Translational diffusion measurements were performed for N-Me-Leu-8, N-Me-Leu-8/N-Me-Ser-14, and N-Me-Val-1/N-Me-Leu-8/N-Me-Ser-14 using a pulsed field gradient longitudinal eddy-current delay pulse sequence (37Gibbs S.J. Johnson C.S. J. Magn. Reson. 1991; 93: 395-402Google Scholar, 38Dingley A.J. Mackay J.P. Chapman B.E. Morris M.B. Kuchel P.W. Hambly B.D. King G.F. J. Biomol. NMR. 1995; 6: 321-328Crossref PubMed Scopus (121) Google Scholar) as implemented by Yao et al. (39Yao S. Howlett G.J. Norton R.S. J. Biomol. NMR. 2000; 16: 109-119Crossref PubMed Scopus (112) Google Scholar). Spectra were processed using XWINNMR (Version 3.5, Bruker Biospin) and TOPSPIN (Version 1.3, Bruker Biospin) and analyzed using XEASY (Version 1.3.13) (40Bartels C. Xia T.H. Billeter M. Guntert P. Wuthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1604) Google Scholar). Chemical shift assignments for the major conformation of the peptides are tabulated in the supplemental material and have been deposited in the BioMagResBank (41Seavey B.R. Farr E.A. Westler W.M. Markley J.L. J. Biomol. NMR. 1991; 1: 217-236Crossref PubMed Scopus (346) Google Scholar). 3JHNHA coupling constants were measured from double quantum filtered correlation spectra at 600 MHz and converted to dihedral restraints as follows: 3JHNHα > 8 Hz, φ =-120 ± 40°; 3JHNHα < 6 Hz, φ =-60 ± 30°. If a positive φ angle could be excluded on the basis of NOE data (42Ludvigsen S. Poulsen F.M. J. Biomol. NMR. 1992; 2: 227-233Crossref PubMed Scopus (86) Google Scholar), φ angles were restricted to the range -180 to 0°. No χ1 angle or hydrogen bond restraints were included for N-Me-Leu-8 and N-Me-Val-1/N-Me-Leu-8/N-Me-Ser-14. Intensities of NOE cross-peaks were measured in XEASY and calibrated using the CALIBA macro of the program CYANA (version 1.0.6) (43Herrmann T. Guntert P. Wuthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1329) Google Scholar). NOEs providing no restraint or representing fixed distances were removed. For structure calculations of N-Me-Leu-8 and N-Me-Val-1/N-Me-Leu-8/N-Me-Ser-14, the constraint list resulting from the CALIBA macro of CYANA was used directly in XPLOR-NIH (44Schwieters C.D. Kuszewski J.J. Tjandra N. Marius Clore G. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1869) Google Scholar) to calculate a family of 100 structures using the simulated annealing script. The library files supplied with XPLOR-NI
Referência(s)