Abstract
Apical membrane antigen-1 is a protein found in the merozoite stage of the malaria parasite Plasmodium falciparum and has been shown to be critical in the invasion of host erythrocytes. Using a random peptide library displayed on the surface of phage, a 20-residue peptide, R1 (VFAEFLPLFSKFGSRMHILK), was identified which specifically recognized and bound to P. falciparum AMA-1. Moreover, the peptide was found to competitively inhibit parasite invasion of red blood cells (RBC). In this study we report the synthesis and properties of the R1 peptide modified by comprehensive N-methyl scanning along the backbone of the peptide. The native R1 and eighteen R1 analogues containing single N-methyl substitutions were synthesized by manual solid-phase Fmoc peptide chemistry. The set of N-methylated peptides was assessed for relative binding affinity with Pf AMA-1 and RBC invasion inhibitory ability. N-methylation in positions 1, 8, and 14 in the R1 peptide produced analogues exhibiting stronger binding characteristics to Pf AMA-1. A tri N-methylated peptide was more than 10 times more active in the inhibition of RBC invasion assay. This peptide also exhibited enhanced serum stability which may be partially responsible for the increase in binding and bioactivity. We have therefore shown that modifications to a biologically active peptide can dramatically enhance activity and potentially provide a lead to a peptide therapeutic molecule with optimized pharmacokinetic properties.
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References
Anders RF, Crewther PE, Edwards S, Margetts M, Matthew ML, Pollock B, Pye D (1998) Immunisation with recombinant AMA-1 protects mice against infection by Plasmodium Chabaudi. Vaccine 16(2–3):240–247
Aurelio L, Box JS, Brownlee RTC, Hughes AB, Sleebs MM (2003) An efficient synthesis of N-methyl amino acids by way of intermediate 5-oxazolidinones. J Org Chem 68(7):2652–2667
Basco LK, Marquet F, Makler MM, Le Bras J (1995) Plasmodium falciparum and Plasmodium vivax: lactate dehydrogenase activity and its application for in vitro drug susceptibility assay. Exp Parasitol 80(2):260–271
Biron E, Chatterjee J, Ovadia O, Langenegger D, Brueggen J, Hoyer D, Schmid HA, Jelinek R, Gilon C, Hoffman A, Kessler H (2008) Improving oral bioavailability of peptides by multiple N-methylation: somatostatin analogues. Angew Chem Int Edit 47(14):2595–2599
Bruehlmeier M, Garayoa EG, Blanc A, Holzer B, Gergely S, Tourwe D, Schubiger PA, Blauenstein P (2002) Stabilization of neurotensin analogues: effect on peptide catabolism, biodistribution and tumor binding. Nucl Med Biol 29(3):321–327
Cranmer SL, Magowan C, Liang J, Coppel RL, Cooke BM (1997) An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 91(3):363–365
Eckert DM, Malashkevich VN, Hong LH, Carr PA, Kim PS (1999) Inhibiting HIV-1 entry: discovery of d-amino peptide inhibitors that target the gp41coiled-coil pocket. Cell 99(1):103–115
Hancock WS, Battersby JE (1976) A new micro-test for the detection of incomplete coupling reactions in solid phase peptide synthesis using 2, 4, 6-trinitrobenzenesulfonic acid. Anal Biochem 71(1):260–264
Harris KS, Casey JL, Coley AM, Masciantonio R, Sabo JK, Keizer DW, Lee EF, McMahon A, Norton RS, Anders RF, Foley M (2005) Binding hot spot for the binding inhibitory molecules of Plasmodium falciparum apical membrane antigen 1. Infect Immun 73(10):6981–6989
Hodder AN, Crewther PE, Anders RF (2001) Specificity of the protective antigen response to apical membrane antigen 1. Infect Immun 69(5):3286–3294
Kokkoni N, Stott K, Amijee H, Mason JM, Doig AJ (2006) N-methylated inhibitors of β-amyloid aggregation and toxicity. Optimization of the inhibitor structure. Biochemistry 45:9906–9918
Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65(3):418–420
Miller SC, Scanlan TS (1997) Site-selective N-methylation of peptides on a solid support. J Am Chem Soc 119(9):2301–2302
Mitchell GH, Thomas AW, Margos G, Dluzewski AR, Bannister LH (2004) Apical membrane antigen 1, a major malaria vaccine candidate, mediates a close cttachment of invasive merozoites with red blood cells. Infect Immun 72(1):154–158
Pedroso E, Grandas A, de las Heras X, Eritja R, Giralt A (1986) Diketopiperazine formation in solid phase synthesis using p-alkoxy ester resins and Fmoc-amino acids. Tetrahedron Lett 27(6):743–746
Rajeswaran WG, Hocart SJ, Murphy WA, Taylor JE, Coy DH (2001) Highly potent and subtype selective ligands derived by N-methyl scan of a somatostatin antagonist. J Med Chem 44(8):1305–1311
Ridley RG (2002) Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 415(6872):686–693
Vojkovsky T (1995) Detection of secondary amines on solid phase. Pept Res 8(4):236–237
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We thank Dr. Paul Donnelly for his editing and valuable input into this manuscript.
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Scanlon, D., Harris, K.S., Coley, A.M. et al. Comprehensive N-Methyl Scanning of a Potent Peptide Inhibitor of Malaria Invasion into Erythrocytes Leads to Pharmacokinetic Optimization of the Molecule. Int J Pept Res Ther 14, 381–386 (2008). https://doi.org/10.1007/s10989-008-9133-4
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DOI: https://doi.org/10.1007/s10989-008-9133-4