| Gliding and active host cell invasion are crucial malarial functions, driven by an actin/myosin motor located beneath the organism's plasma membrane. In Plasmodium merozoites and sporozoites, the motive force is transmitted---via the actin-binding, glycolytic enzyme, aldolase---to members of the Thrombospondin-Related Anonymous Protein (TRAP) family, which interact with host-cell surfaces, and enable the parasite to use this force to actively invade its target cells. During the gliding and invasive processes, TRAP is translocated from the anterior to the posterior end of the parasite and then cleaved within its transmembrane domain by rhomboid proteases, leaving its extracellular domain bound to the host cell. Presumably, TRAP's cytosplasmic tail must then disassociate from the aldolase tetramer to allow the enzyme to engage another TRAP protein and participate in the next round of motion.;The actin-aldolase-TRAP interaction thus constitutes the core of the malarial motor complex (glideosome) and is an attractive target for anti-malarial drug design. This dissertation describes a novel integration of heterogeneous data derived from the complex's sequence, crystallographic structure, biophysical measurements, biochemical measurements, and enzymology measurements to discover new data regarding the dynamic interplay between the components of the malarial motor complex, and to utilize these insights to develop small molecule inhibitors of malarial infection.;Prior to the work presented here, the co-crystal structure of Plasmodium aldolase bound to TRAP had been solved to 2.4 A resolution. However, the atomic interactions between aldolase and actin remained undefined. In collaboration with the Nussenzweig Lab at the NYU School of Medicine and the Bosch Lab at Johns Hopkins University, we used protein-protein docking and computational modeling techniques to model the interaction of Plasmodium falciparum aldolase with actin by homology to the interactions of aldolase with TRAP and the mammalian Band 3 protein. We also explored the nature of the actin-aldolase complex in vitro, using recombinant peptides, ELISA-based binding assays, and site-directed mutagenesis of both actin and aldolase, the results of which were in agreement with our computational predictions. Finally, guided by these modeling and mutagenesis studies, we obtained preliminary crystallographic data for the complete actin-aldolase-TRAP complex. Taken together, our computational, biochemical, and crystallographic results suggest that there are several sites on aldolase to which actin can bind, including the enzyme's active site and the aldolase dimeric interfaces, each of which may be masked or unmasked by aldolase's other binding partners within the glideosome, and each of which is in physiological equilibrium with the others.;In parallel with the above studies, we began a structure-based drug design effort targeting the co-crystallized aldolase-TRAP interface, adding the Sinnis Lab at NYU to our collaborative team. We have completed an initial computational screen of a library of 300,000+ chemicals, which identified 60 small molecules that may disrupt the recycling of aldolase and continued movement of the parasite by preventing the disassociation of the cytoplasmic TRAP tail from the glideosome. Importantly, stabilizing the TRAP-aldolase interaction should also specifically inhibit the catalytic activity of the malarial enzyme---while sparing its mammalian host---thereby disrupting the glycolytic cycle upon which Plasmodium parasites are exquisitely dependent for ATP generation.;To date, at least 16 of these hits have been validated in vitro as potential TRAP-aldolase enhancers through thermal shift and aldolase catalytic activity assays. Additionally, preliminary crystallographic data obtained for these compounds and the Plasmodium TRAP-aldolase complex suggests that some of these chemicals do, in fact, cross-link the two proteins. The compounds with the greatest in vitro activity were further analyzed for their specificity for the parasite and for their anti-malarial activity in vivo. Significantly, none of these compounds were toxic to human hepatocytes, and at least two of them markedly disrupted the gliding ability of Plasmodium parasites at micromolar concentrations. If ultimately successful, these compounds would represent a new class of anti-malarial agents, and---to our knowledge---the first set of drugs designed to enhance, rather than inhibit, a protein-protein interface. |
