Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with broad strain specificity [1]. Our screening campaign identified fragments that bind to the hydrophobic cleft with a high hit rate of 5%, which strongly suggests that a druggable pocket is present within the cleft. Progress in elaboration of promising fragments will be described [1,2].
Information on the binding sites and poses of elaborated fragments is critically important in the efficient progression of mM fragment hits to µM ligands and eventually nM leads. X-ray crystallography plays a key role, but rapid solution-state methods are also very valuable. We employed 19F NMR on AMA1 labelled with fluoro-Trp residues as a means of confirming binding to the target site on AMA1 and determining whether the bound ligand triggers conformational changes similar to those induced by peptide ligands for this site [3]. By introducing Trp residues at strategic sites within a ligand-responsive loop, we detect distinct conformational consequences when peptide and small-molecule ligands bind to AMA1. Our results also demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures.
[1] Lim SS, et al (2013) Development of inhibitors of Plasmodium falciparum apical membrane antigen 1 based on fragment screening. Australian Journal of Chemistry 66, 1530–1536.
[2] Devine SM, at al (2014) A critical evaluation of pyrrolo[2,3-d]pyrimidine-4-amines as Plasmodium falciparum apical membrane antigen 1 (AMA1) inhibitors. Submitted
[3] Ge X, et al (2014) Ligand-induced conformational changes in Plasmodium falciparum AMA1 detected using 19F NMR. Submitted