Poster Presentation First Malaria World Congress 2018

Development of small molecule inhibitors of the AMA1/RON2 interaction using a paramagnetic peptide probe (#408)

Mansura Akter , Nyssa Drinkwater , Krishnarjuna Bankala , Chris MacRaild , Tony Wang , Simon Drew , Sheena McGowan , Raymond Norton

    Apical membrane antigen 1 (AMA1) protein is essential for the invasion of host cells by zoites of all apicomplexan parasites. Another key player, rhoptry neck protein 2 (RON2), is inserted into the host cell membrane during invasion and acts as a ligand for AMA1. Several studies indicate that the AMA1/RON2 interaction is essential to malaria invasion.1 A range of small molecule ligands for AMA1 has been identified in a fragment screen and shown to bind to the conserved hydrophobic cleft of PfAMA1.2 However, crystal structures of AMA1 with these ligands bound failed to identify their binding site(s). We have therefore pursued alternative approaches based on NMR to define their binding sites.

     A spin-labelled peptide was developed to probe the binding sites of compounds on PfAMA1. A thiol-specific stable nitroxide spin label was attached to a solvent-exposed cysteine of a backbone-cyclised peptide derived from PfRON2.3 Its Kd was 1 µM by SPR. A 1.6 Å resolution crystal structure of the complex shows that the peptide binds to PfAMA1 FVO at one end of the hydrophobic groove, leaving much of the binding site exposed and allowing elaborated fragments to bind without interference.

     Paramagnetic relaxation enhancement (PRE)-based NMR experiments were carried out to screen the fragment hits in the presence of the spin-labelled peptide. As PRE effects are strongly distance-dependent, their detection implies that the fragment is bound close to the spin label. Thus, PRE-derived distance constraints can be inferred in order to identify the binding sites of elaborated fragments and serve as a guide for antimalarial drug design.