Assessment and Validation of the Malaria SYBR Green Based Fluorescence Assay to Monitor the Growth Effect of Malaria Parasites in the Presence of Synthesized Inhibitors

The malarial species P. falciparum threatens over half a million people every year, with most fatalities occurring in African children. The species enters the human host through the bite of an infected mosquito; its intraerythrocytic development cycle eventually leads to infection of red blood cells. It is during this cycle that the human host exhibits the symptoms associated with malaria. However, our understanding of parasite biochemistry during intraerythrocytic development remains incomplete. Previous work showed a metalloprotease, falcilysin, to be an essential protein for the parasites viability. One major focus of our research is to synthesize piperazine-based hydroxamic acid inhibitors against falcilysin. The first set of inhibitors were tested against cultured parasites and were shown to behave as an antimalarial. However, the question remains: are the parasites dying due to the inhibitors specifically targeting falcilysin or are they targeting other essential proteins of P. falciparum? To affirm whether inhibitors kill the parasite by specifically targeting falcilysin we generated a parasite strain that allows us to regulate falcilysin expression. Our current objective is to validate the malaria SYBR Green fluorescence assay (SGFA) for its ability to detect growth effects of the parasites in the presence of previously synthesized inhibitors. The SGFA detects, through a 96 well microplate reader, the quantity of malarial DNA in infected red blood cells. To use this assay we will determine the detection limits of the SGFA.Then, we will assay a set of conditions that will give us ideal growth conditions for the malaria parasites in the presence and absence of drugs. As a set of controls we will test a range of concentrations for known antimalarial drugs and determine the concentration at which fifty percent of parasite growth is inhibited compared to literature values. Overall, the results of the assay will allow us to establish a set of reliable conditions to move forward and answer our underlying question