title: Inhibition by flavonoids of amyloid-like fibril formation by Plasmodium falciparum merozoite surface protein 2.
abstract: Merozoite surface protein 2 (MSP2) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed abundantly on the surface of Plasmodium falciparum merozoites. The results of a phase 2 trial in Papua New Guinean children showed MSP2 to be a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils under physiological conditions. Oligomers containing beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites. As the propensity of MSP2 to form fibrils in solution also has the potential to impede its development as a vaccine candidate, finding an inhibitor that specifically inhibits fibrillogenesis may enhance vaccine development. In this study, we tested the ability of three flavonoids, EGCG, baicalein, and resveratrol, to inhibit MSP2 fibrillogenesis and found marked inhibition with EGCG but not with the other two flavonoids. The inhibitory effect and the interactions of the flavonoids with MSP2 were characterized using NMR spectroscopy, thioflavin T fluorescence assays, electron microscopy, and other biophysical methods. EGCG stabilizes soluble oligomers and blocks fibrillogenesis by preventing the conformational transition of MSP2 from a random coil to an amyloidogenic beta-sheet structure. Structural comparison of the three flavonoids indicates an association between their propensity for autoxidation and their fibril inhibitory activity; the activity of EGCG can be attributed to the vicinal hydroxyl groups present in this flavonoid and their ability to form quinones. The molecular mechanism of fibril inhibition by EGCG appears to be complex and involves noncovalent binding followed by covalent modification of the protein. Although the addition of EGCG appears to be an effective means of stabilizing MSP2 in solution, the covalent modification of MSP2 would most likely not be acceptable in a vaccine formulation. However, these small molecule inhibitors of MSP2 fibril formation will be useful as mechanistic probes in studying oligomerization and fibril assembly of MSP2.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Indu R
authors.initials: IR
authors.lastname: Chandrashekaran
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Adda
authors.forename: Christopher A
authors.initials: CA
authors.lastname: MacRaild
authors.forename: Robin F
authors.initials: RF
authors.lastname: Anders
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D000953:Antigens, Protozoan; D002392:Catechin / Q000031:analogs & derivatives; D002648:Child; D005419:Flavonoids / Q000276:immunology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D017780:Malaria Vaccines / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology; D052940:Merozoites / Q000276:immunology; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000276:immunology; D015800:Protozoan Proteins
chemical_list: D000682:Amyloid; D000953:Antigens, Protozoan; D005419:Flavonoids; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; C065974:merozoite surface protein 2, Plasmodium; D002392:Catechin; C045651:epigallocatechin gallate