Toxoplasma gondii aspartic protease 5: structural basis of substrate binding and inhibition mechanism.

Toxoplasma gondii, a worldwide prevalent parasite is responsible for causing toxoplasmosis in almost all warm-blooded animals, including humans. Golgi-resident T. gondii aspartic protease 5 (TgASP5) plays an essential role in the maturation and export of the effector proteins those modulate the host immune system to establish a successful infection. Hence, inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. This is the first report of the detailed structural investigations of the TgASP5 mature enzyme using molecular modeling and an all-atom simulation approach which provide in-depth knowledge of the active site architecture of TgASP5. The analysis of the binding mode of the TEXEL (Toxoplasma EXport Element) substrate to TgASP5 highlighted the importance of the active site residues. Ser505, Ala776 and Tyr689 in the S2 binding pocket are responsible for the specificity towards Arg at the P2 position of TEXEL substrate. The molecular basis of inhibition by the only known inhibitor RRLStatine has been identified, and our results show that it blocks the active site by forming a hydrogen bond with a catalytic aspartate. Besides that, known aspartic protease inhibitors were screened against TgASP5 using docking, MD simulations and MM-PBSA binding energy calculations. The top-ranked inhibitors (SC6, ZY1, QBH) showed higher binding energy than RRLStatine. Understanding the structural basis of substrate recognition and the binding mode of these inhibitors will help to develop potent mechanistic inhibitors against TgASP5. This study will also provide insights into the structural features of pepsin-like aspartic proteases from other apicomplexan parasites for developing antiparasitic agents.Communicated by Ramaswamy H. Sarma.

Inhibition of Plasmodium falciparum plasmepsins by drugs targeting HIV-1 protease: A way forward for antimalarial drug discovery.

Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.