4697362_monash_151157.pdf (11.84 MB)
Ligands for the malaria apical membrane antigen 1 as inhibitors of parasite invasion of host erythrocytes: screening, structure and dynamics
thesisposted on 2017-02-27, 05:26 authored by Lim, San Sui
Malaria is one of the most widespread infectious diseases, causing approximately 250 million clinical cases and claiming more than 650,000 lives each year. Although current artemisinin combination therapies (ACT) have been highly effective against Plasmodium parasites, signs of resistance have already emerged. Hence, there is an urgent need for drugs with new modes of action to combat this threat. Apical membrane antigen 1 (AMA1) interacts with rhoptry neck (RON) protein complex to form part of the moving junction (MJ) complex important for the invasion of human red blood cells by Plasmodium falciparum (Pf). AMA1 has a conserved hydrophobic cleft that is the site of interactions with the rhoptry neck (RON) protein complex. Peptides identified by phage display, such as R1, as well as monoclonal antibodies that target this site on AMA1, are able to inhibit red blood cell invasion, but usually in a strain-specific manner as numerous polymorphic residues are clustered at one end of the cleft. My goal is to design small molecule inhibitors of AMA1 that have broad strain specificity and we are pursuing this goal using a fragment-based approach. My project began with cocktail screening of a fragment library against AMA1 using saturation transfer difference (STD). The hits found from the cocktail screen were then evaluated in the STD and Carr-Purcell-Meiboom-Gill (CPMG) R1 competition assays to identify hits that bind to the AMA1 hydrophobic cleft. Thereafter, the binding affinities (Kᴅ) and ligand efficiencies (LE) of the R1-competing hits were determined using surface plasmon resonance (SPR). A total of 57 fragment hits were identified in the screening campaign which corresponds to a 5 % hit rate. The high hit rate observed strongly suggest that a druggable site is present within the cleft. Subsequently, my work focused on mapping the specific binding sites of the hits using ¹ H-¹⁵N HSQC perturbation studies of PfAMA1 backbone amide resonances. To perform this study, the backbone amide resonances were first assigned using a combination of 3D NMR and specific ¹⁵N-Lys labelled HSQC experiments. The HSQC perturbation experiments identified fragments that bind to a conserved region on the AMA1 hydrophobic cleft, and these compounds represent promising starting scaffolds for subsequent chemical elaborations. The first X-ray crystal structure of FVO PfAMA1 was determined to understand the impact of sequence diversity on AMA1 structure and facilitate the design of small-molecule inhibitors. The crystal structure of AMA1 from the P. falciparum 3D7 strain was also reproduced at higher resolution in an attempt to obtain binding poses of fragments bound to the antigenically diverse forms of AMA1 (FVO and 3D7). Currently we are working towards getting the crystal structures of the fragments bound to different forms of AMA1, which will allow more rational design of fragment analogues. In parallel, chemical modifications of the hits based on the structure-activity relationship (SAR) of the analogues are underway to improve their binding affinities.