Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Tracking the invasion: dispersal of Hymenoscyphus fraxineus airborne inoculum at different scales.

Ash dieback is caused by an invasive pathogen Hymenoscyphus fraxineus, which emerged in Europe in the 1990s and jeopardizes the management of ash stands. Although the biological cycle of the pathogen is well understood, its dispersal patterns via airborne spores remain poorly described. We investigated the seasonal and spatial patterns of dispersal in France using both a passive spore-trapping method coupled with a real-time PCR assay and reports of ash dieback based on symptom observations. Spores detection varies from year to year with a detection ability of 30-47% depending on meteorological conditions, which affect both production of inoculum and efficiency of the trapping. Nevertheless, our results are consistent and we showed that the sporulation peak occurred from June to August and that spores were detected up to 50-100 km ahead of the disease front, proving the presence of the pathogen before any observation of symptoms. The spore dispersal gradient was steep, most of inoculum remaining within 50 m of infected ashes. Two dispersal kernels were fitted using Bayesian methods to estimate the mean dispersal distance of H. fraxineus from inoculum sources. The estimated mean distances of dispersal, either local or regional scale, were 1.4 km and 2.6 km, respectively, the best fitting kernel being the inverse power-law. This information may help to design disease management strategies.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

Structure and dynamics of apical membrane antigen 1 from Plasmodium falciparum FVO.

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.

Ligand-induced conformational change of Plasmodium falciparum AMA1 detected using 19F NMR.

We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5-fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein-protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.