Structural Characterization of Aspartic Proteases Involved in Invasion and Egress of Apicomplexan Pathogens P. Falciparum and T.gondii

The phylum apicomplexan includes a variety of obligate intracellular parasites responsible for causing different deadly diseases in humans. P. falciparum is the most lethal Plasmodium species causing human malaria. Additionally, Toxoplasma gondii causes Toxoplasmosis leading to miscarriages and birth defects. These diseases are global burden as they cause millions of deaths worldwide. There is a need for development of potent and long lasting drugs against these parasites due to emergence of drug-resistance. Invasion, egress, and growth of the parasites are important stages of their life cycle for their survival and dissemination of the infection in human. Pepsin-like aspartic proteases have been shown to be involved in the invasion and egress process of Plasmodium and Toxoplasma species, and are considered as excellent drug-targets. Therefore, blocking the function of these aspartic proteases would aid in development of effective therapeutic strategies to control these diseases. Plasmepsin IX (PfPMIX) and Plasmepsin X (PfPMX) are pepsin-like aspartic proteases from Plasmodium involved in the invasion and egress. Similarly, Toxoplasma gondii aspartic proteases III (TgASPIII) which is homologous to PfPMIX and PfPMX also acts as maturase. These proteases are involved in activation of downstream protein cascade of invasion and egress. Until now, no structural information is available for these proteases. Understanding the molecular basis of the functional properties of these proteases would lead to the development of effective chemotherapeutic agents to combat these diseases. Therefore, to unravel the molecular insights into the interaction of these proteins the homology models of the PfPMX and TgASPIII were built. Docking was done to screen known HIV-1 and BACE protease inhibitors. MD simulation studies were performed for enzyme bound inhibitors with highest affinity. The structures of PfPMX and TgASPIII have the typical pepsin-like aspartic proteases fold and the characteristics catalytic motifs DTGS in N-terminal and DTGT in Cterminal in PfPMX (DSGT in TgASPIII), required for optimal activity in acidic pH conditions; and a flap formed by the loop region of the beta-hairpin. The residues from the flap and the two conserved catalytic motifs form the active site. The flap is involved in regulating the opening and closing of the enzyme and thereby influencing substrate specificity. In comparison to other pepsin-like aspartic proteases, a phenylalanine mutation in place of tyrosine is observed in the flap of the PfPMX and TgASPIII. The PfSUB1 (SML/EVE) substrate cleavage site in PfPMX is oriented in a manner that the backbone carbonyl of the P1Leu-GluP1' lies in a catalytically favorable position of two active site aspartates (Asp29 and Asp220). Similar orientation of TgROP1 was observed in TgASPIII. All the screened molecules are well-stabilized in the pocket with proper hydrogen bonds and hydrophobic interactions; and their hydroxyl group pointing towards the catalytic aspartates. Our structural analysis provides detailed insights into the substrate binding pocket of the PfPMX and TgASPIII, hence would be helpful in designing potent inhibitors of these proteases. Biochemical studies of the identified molecules are in-progress and further optimization of the candidate molecules will be done to design molecule having crossreactivity towards both the proteases.