Plasmodium falciparum, the organism responsible for the most prevalent and most virulent cases of malaria in humans, poses a major burden to the developing world. The parasite is increasingly developing resistance to traditional therapies, such as chloroquine, so the need to determine novel drug targets is more prevalent than ever. One such method involves targeting proteins unique to the malarial proteome that do not have homologues in humans. An especially promising group of targets are protein kinases, which are involved in many different biochemical pathways within the cell. Eukaryotic cell cycle progression is moderated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). These kinases depend on the binding of a cognate regulatory unit (cyclin) in order to enter its activated state. Once activated, these cyclins then mediate phosphorylation events that are crucial to cell cycle advancement . Cyclin Dependent Kinases (CDKs) are common to most eukaryotes and are responsible for regulating the cell cycle of growth and proliferation. Proteins have been previously identified in Plasmodium that have sequence homology to traditional CDK and have a potential function to be classified as "CDK-like" kinases. Three kinases that fit this description are Plasmodium falciparum Kinase 5, 6, and mrk, or MO15- Related Kinase. These kinases are expected to have roles in both malarial growth and regulation of the cell cycle. Bacterial constructs were generated to express and purify recombinant forms of these kinases and potential substrates. Once the potential interactors were isolated, in vitro protein kinase assays were used to validate the interactions to the kinases as substrates. In summary, the study has identified substrates that are directly phosphorylated by PfPK6, and demonstrated that the identified proteins are not directly phosphorylated by PfPK5 and Pfmrk.

A novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7[micrometer]) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter of healthy RBCs decreases reversibly with pressure with an approximate slope of 0.015 [micrometer] / MPa. In infected cells, clear differences in the deformability and between the compression and decompression curves are observed. The results are discussed with respect to the elasticity of the phospholipid membrane and the spectrin molecular network. Employing micro-absorption spectroscopy with spatial resolution of 1.4 [micrometer] in the lateral and 3.6 [micrometer] in the axial direction the visible absorption spectrum of hemoglobin in a single red blood cell is measured under physiological conditions. The spectra of cells infected with the malaria parasite show changes in peak positions and relative intensities in the Soret and [alpha]- and [beta]- bands. These indicate hemoglobin degradation that can be correlated with the stages of the parasite multiplication cycle and can be used as a potential diagnostic marker. The research is further extended towards the understanding of pressure effects on the ligand binding kinetics to heme proteins. For a well characterized reaction at ambient pressure, CO binding to myoglobin in solution, we investigate the transient absorption following laser flash photolysis over eight decades in time at variable pressure and temperature. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier.

An estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial drug discovery. In an effort to discover novel therapeutics for malaria, we studied the antimalarial activities of a unique marine-derived peak fraction library provided by Harbor Branch Oceanographic Institute (HBOI). Within this unique library, we have screened 2,830 marine natural product (MNP) peak fractions through a medium throughput screening effort utilizing the SYBR Green-I fluorescence based assay, and have identified 253 fractions that exhibit antimalarial activity. From those inhibiting fractions we have identified twenty species of marine organisms that inhibit Plasmodium falciparum growth, from which thirty-five fractions were selected for further study. Among those thirty-five, eighty-three percent were also found to inhibit the chloroquine resistant strain of P. falciparum, Dd2. The most potent inhibitors were then screened for their cytotoxic properties using the MTT cell viability assay. Among the samples that exhibited potent inhibition of P. falciparum growth were fractions derived from a sponge of the genus Spongosorites sp.. This genus of sponge has been reported to contain the nortopsentin and topsentin class of bis-indole imidazole alkaloids. Nortopsentin A inhibited the parasite growth at the trophozoite stage with an IC50 value of 1.6 µM. This is the first report of antimalarial activity for this class of compound.

Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater. Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the planet, containing 34 of the 36 known phyla of life, and is expected to be a rich source of novel chemotypes. In collaboration with the Harbor Branch Oceanographic Institute in Ft. Pierce we have screened a library of over 2,800 marine macroorganism peak fractions against Plasmodium falciparum using the SYBR green I fluorescence-based assay. In this screening process we have identified six compounds from five novel chemical scaffolds all of which have low micromolar to submicromolar IC50 values and excellent selectivity indices. Additionally, one of these chemical scaffolds, the bis(indolyl)imidazole, was selected for further in vitro pharmacological and structure-activity relationship (SAR) profiling, key steps in the challenging process of identifying a new antimalarial drug lead compound.

Malaria is one of the most devastating infectious diseases causing 1-3 million fatalities a year. The majority of these cases occur amongst children in developing countries. Malarial strains in these areas are exhibiting increasing resistance to canonical treatments proving the importance of new drug targets for anti-malarials. Identification of new drug targets is dependent upon a better understanding of the molecular biology of the parasitic agent of malaria, Plasmodium. The regulation of Plasmodium's complex life cycle is still not well understood. Elucidation of signaling pathways involved in Plasmodium cell cycle regulation will provide insights into how the parasite thrives in human cells. A subset of kinases, referred to as cyclin-dependent kinases (CDKs), are crucial regulators of eukaryotic cell cycle progression. In silico studies show high homology between mammalian CDK's and a group of CDK-like Plasmodium kinases including PfPK5 (Plasmodium falciparum protein kinase 5). Plasmodium homologues to CDK regulators, cyclins, have also been identified. Understanding the role of PfPK5 in cell cycle regulation would require analysis of subcellular localization and cell cycle-dependent expression. Immunofluorescence assays demonstrate that PfPK5 is localized in the nucleus. PfPK5's expression profile, as determined by western blotting, shows highest expression in the schizont stage, the stage when the atypical multiple nucleated form of the parasite is observed. Possible PfPK5 interacting partners were detected by performing an anti-PfPK5 immunoprecipitation assay. Additionally, a hemagglutinin (HA)-tagged PfPK5 construct was made to increase the sensitivity of immunoprecipitation assay and identification of PfPK5 interacting partners. The characterization of PfPK5 and its interacting partners may prove useful in identification of novel drug targets in the future.

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