Malaria, which is caused by Plasmodium parasites that are transmitted to humans through the bites of infected mosquitoes, leads to about 1 million deaths per year (1). The increasing incidence of drug resistance, absence of an effective vaccine and lack of diversity amongst compounds in current clinical development renders this old disease a re-emerging global health threat. New anti-malaria drugs with novel modes of action are urgently required to confront these challenges.

Erythrocyte invasion by malaria parasites relies on the interaction between Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck protein (RON) complex (2). As this invasion mechanism is highly conserved in all apicomplexan parasites, chemical intervention in this protein-protein interaction represents a promising therapeutic approach to combat malaria infection. A well-defined, highly conserved hydrophobic cleft on AMA1, which was identified as a RON2 binding site (3), is favourable for designing small-molecule inhibitors to bind and block the erythrocyte invasion. In addition, targeting conserved residues by small molecules will be more likely to produce an anti-malarial with broad strain specificity and less susceptibility to drug resistance.

The development of small-molecule inhibitors can be aided by a molecular probe, which enables the detailed characterization of inhibitory interactions in the conserved hydrophobic cleft and serves as a pharmacophore for structure-based drug design. A 20-mer peptide designated R1, which displays high binding affinity for P. falciparum 3D7 AMA1 and inhibits erythrocyte invasion in vitro strain-specifically (4), is investigated in this study. We have established an expression and purification protocol for isotopically-labelled R1 and AMA1, and investigated the AMA1-R1 interaction by NMR spectroscopy. Using SPR, we have also identified the minimal binding construct of R1 by a series of truncations, and revealed the key AMA1-interacting residues along this part of peptide using an alanine scan. The study provides a starting point for small-molecule inhibitor design and potentially a new class of anti-malarial drug.