High-resolution NMR studies of malaria proteins as a basis for designing better drugs and vaccines
2017-03-02T23:29:05Z (GMT) by
Malaria is a life-threatening disease caused by Plasmodium spp., particularly Plasmodium falciparum (Pf). Although current therapies have been very successful in controlling malaria, resistant parasites have already emerged. No vaccine is currently available for complete prevention of the disease. Therefore, the design of drugs with new modes of action to combat this threat and developing a vaccine for disease prevention remain high priorities. Antigenic polymorphism is a major limiting factor in the development of drugs and vaccines for treating malaria. This thesis focuses on the development of small molecule inhibitors for P. falciparum apical membrane antigen 1 (PfAMA1) as new antimalarials and an optimized merozoite surface protein 2 (MSP2)-based vaccine construct to achieve a strain-transcending immune response. PfAMA1 is an essential component of the moving junction (MJ) complex that forms between erythrocytes and parasites during the invasion by P. falciparum. PfAMA1 has a conserved hydrophobic cleft that is the binding site for rhoptry neck protein 2 (RON2). As anti-AMA1 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 new antimalarials. The first part of the thesis describes the determination of the nuclear magnetic resonance (NMR) assignments for two variants (3D7 and FVO) of PfAMA1 in order to facilitate the screening and identification of small molecules that bind to the hydrophobic cleft. Additionally, the first crystal structure of FVO PfAMA1 was determined to understand the role of polymorphism on AMA1 structure and to facilitate the design of small molecule inhibitors. The binding sites for small molecules that bind to the hydrophobic cleft were identified using [15N-1H]-TROSY perturbation experiments. Among the small molecules tested, the binding sites for benzimidazoles on both FVO and 3D7 PfAMA1 were similar, suggesting that these compounds are promising starting scaffolds for the development of potential PfAMA1 inhibitors. The second part of my PhD involves the design and development of an optimized MSP2-based vaccine construct for malaria. MSP2 is an intrinsically disordered glycosylphosphatidylinositol-anchored antigen expressed on the merozoite surface. Vaccine trials with recombinant MSP2 showed protection against P. falciparum. However, the implications of MSP2 disorder in producing a protective immune response are unknown. Therefore, the conformational and dynamic properties of two allelic forms of MSP2 (3D7 and FC27) were studied using NMR spectroscopy and it was found that these properties were identical in conserved regions but significantly different between the variable regions. It was observed that the conformationally restricted regions are more antigenic than the more flexible regions, suggesting that the immunogenic efficacy of disordered antigens can be improved by increasing the conformational order of their flexible regions. The immune response to MSP2 provides protection, but the protection is strain-specific owing to the polymorphic nature of MSP2. In order to achieve a strain-transcending immune response to MSP2, a series of chimeric MSP2 constructs was designed, characterized and used in immunization experiments in mice. Overall, a robust immune response to both conserved and variable regions of all constructs was generated in mice. Among the constructs tested, V3D7VFC27C chimera (V3D7: 3D7 variable region, VFC27: FC27 variable region and C: conserved C-terminal region) was able to induce a uniform immune response to both conserved and variable region epitopes of 3D7 and FC27 MSP2 epitopes. Total IgG levels to this construct were equivalent to that obtained from immunizing with co-administered 3D7+FC27, and generated stronger IgG2b and IgG2c responses. Constructs lacking the conserved regions showed a reduced immune response, suggesting the importance of these regions in generating a robust immune response. Structural information on antibody-bound MSP2 epitopes would help to understand the native MSP2 conformation as well as the strain-specific protection of MSP2. Additionally, such conformations could be incorporated into either full-length or chimeric MSP2 to increase their conformational order, which would lead to improved immunogenicity. 6D8 is a monoclonal antibody that recognizes a conserved epitope in recombinant MSP2 but cannot recognize native MSP2. The crystal structure of 6D8 scFv-bound 6D8 epitope was determined and compared with the structure of the lipid-bound epitope. When bound to lipid the conserved residue Arg22 is part of a helix and unable to make interactions with 6D8 that are required for recognition. This suggests a mechanism by which native MSP2 escapes antibody recognition. Moreover, NMR binding experiments identified transient and strain-specific interactions between 6D8 and the variable regions of 3D7 and FC27 MSP2 that lead to different affinities for 6D8. In summary, small molecules that bind to the PfAMA1 hydrophobic cleft were identified and chemical elaboration of these molecules could generate high affinity strain-transcending PfAMA1 inhibitors. On the other hand, chimeric constructs of MSP2 can be used to induce a strain-transcending immune response to P. falciparum and represent viable choices to include in a future malaria vaccine. Additionally, the role of conformational order in determining antigenicity of MSP2 was investigated and, together with the information from the antibody-bound MSP2 epitopes, could have important implications for the design of a better MSP2 vaccine.