Molecular insights into the interaction between Plasmodium falciparum apical membrane antigen 1 and invasion-inhibitory peptides
2019-06-12T22:53:38Z (GMT) by
Apicomplexan parasites share a conserved invasion mechanism involving the formation of an anchoring structure named moving junction (MJ) at the parasite-host cell interface. The formation of the MJ is triggered by the interaction between the rhoptry neck protein complex (RON2/4/5/8), which is secreted from parasite rhoptries into the host cell membrane, and apical membrane antigen 1 (AMA1), which is discharged by parasite micronemes and integrated into parasite cell membrane. Disruption of this critical protein-protein interaction (PPI) by synthetic molecules represents a promising avenue for antimalarial drug discovery. AMA1 has been shown to directly interact with a stretch of peptide from C-terminus of RON2 via its conserved hydrophobic cleft, which is therefore an attractive target site for the design of small-molecule inhibitors. As the hydrophobic cleft spans an extended surface area, it was unclear which part of the cleft is most suitable for binding synthetic molecules. This knowledge gap has prompted us to unravel the key binding hot spots in the hydrophobic cleft and design novel inhibitors to target these sites. The invasion-inhibitory peptide R1, which was identified from a phage-display library, has high binding affinity and makes extensive interactions with the hydrophobic cleft of AMA1. R1 is thus exploited in this work to probe the key interactions in the cleft. Truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 of R1 are the most important residues for high affinity binding to AMA1. These residues interact with two complementary binding hot spots on AMA1. Fragment-based computational solvent mapping reveals that the first of these hot spots, which is close to the one end of hydrophobic cleft, is more suitable for small-molecule targeting. In contrast, the other hot spot, which we have termed the “Arg pocket”, appears to be a difficult site to target with small molecules, although it serves as the key anchor point for several known inhibitory agents. Using NMR spectroscopy, we have confirmed that R1 in solution binds to AMA1 with 1:1 binding stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. This work provides a basis for designing high affinity inhibitors of the AMA1-RON2 interactions and potentially informs the elaboration of hits identified from our parallel fragment-based drug screening program. Next we sought to tackle the less druggable Arg pocket using a 13-residue β-hairpin based on the C-terminal loop of RON2. A series of β-hairpin peptides was synthesized and assessed for their binding affinity and invasion-inhibitory activity. The best analogue had KD values of 8 μM and 0.8 μM against 3D7 and FVO AMA1, respectively, and displayed an IC50 of 34 μM against transgenic parasites expressing the FVO AMA1 allele in vitro. We solved the crystal structures of several β-hairpin peptides in complex with FVO AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The optimized β-hairpin peptides represent a novel class of inhibitors of the AMA1-RON2 interaction and serve as excellent leads for the design of small-molecule mimetics. Overall, this study identified critical binding hot spots in the hydrophobic cleft of AMA1 and discovered novel strain-transcending inhibitors that target one of the hot spots. We anticipated that the work presented in this thesis would facilitate the development of new antimalarial drugs that are effective against the majority of P. falciparum strains.