Structural Insights Into Key Plasmodium Proteases as Therapeutic Drug Targets

Abstract

Malaria, caused by protozoan of genus Plasmodium, remains one of the highest mortality infectious diseases. Malaria parasites have a complex life cycle, easily adapt to their host's immune system and have evolved with an arsenal of unique proteases which play crucial roles in proliferation and survival within the host cells. Owing to the existing knowledge of enzymatic mechanisms, 3D structures and active sites of proteases, they have been proven to be opportune for target based drug development. Here, we discuss in depth the crucial roles of essential proteases in Plasmodium life cycle and particularly focus on highlighting the atypical "structural signatures" of key parasite proteases which have been exploited for drug development. These features, on one hand aid parasites pathogenicity while on the other hand could be effective in designing targeted and very specific inhibitors for counteracting them. We conclude that Plasmodium proteases are suitable as multistage targets for designing novel drugs with new modes of action to combat malaria.

Keywords: Ca2+-dependent subtilase; aspartyl protease fold; drug targets; malaria; papain-family cysteine proteases; proteases; therapeutics.

FIGURE 1

Role of proteases in hemoglobin degradation. Host Hb degradation takes place within the digestive vacuole primarily by co-ordinated action of plasmepsins and falcipains. Small peptides are further converted into amino acids by aminopeptidases. Amino acids are transported to parasite cytosol by an ATP-dependent membrane transporter.

FIGURE 2

Role of proteases in invasion and egress. Subtilisin-like proteases (SUB1 and SUB2) are mainly involved in the egress and invasion process. SERA5 is involved in the destabilization of the PV through degradation of its membrane proteins and also involved in merozoite priming through regulation of erythrocyte binding ligand MSP. ROM1 catalyzes the intramembrane cleavage of various merozoite adhesins such as AMA1 and helps in invasion process.

FIGURE 3

Unique features of Plasmodium vivax SUB1 structure. Cartoon representation of overall structure of PvSUB1 (PDB: 4TR2). Overall structure comprises N-terminal insertion in prodomain comprising the “belt” domain (shown in yellow) followed by a classical bacterial-like prodomain (orange), and the C-terminal subtilisin-like catalytic domain (cyan). The catalytic triad (Asp316/His372/Ser549; Red/Gray/olive) is shown as spheres. The oxyanion hole residue (Asn464) is shown as blue sphere. Bound calcium ions are shown as green spheres. Out of four calcium binding sites, three (Ca1, Ca2, Ca3) are specific to parasite subtilases. Ca4 is conserved in all homologs of subtilisin. Bacillus subtilsin (PDB: 1SUP) is superimposed (shown in magenta) highlighting the unique insertions in parasite subtilases. All structure figures have been made in Pymol.

FIGURE 4

Overall structure of Plasmodium plasmepsins. (A) Cartoon representation of overall structure of Plasmepsin II (PDB: 1IF4). Flexible proline rich loop is depicted in yellow and catalytic aspartates Asp34 and Asp214 are shown as blue spheres. Residues defining the catalytic cavity Val78 (orange) and Leu292 (yellow) are shown as spheres. (B) Cartoon representation of overall structure of Plasmepsin V (PDB: 4ZL4). Free C140 (within the flap) and cysteine residues forming disulfide linkages are shown in yellow. Helix-turn-helix motif is unique to PlmV unlike all other plasmodial plasmepsins and aspartyl proteases. All structure figures have been made in Pymol.

FIGURE 5

Overall structure of Falcipains. Cartoon representation of Falcipain-2 (PDB: 2GHU) displaying two distinct domains and a prominent active site cleft. Active site residues Cys-42 (Red), His-174 (Gray), and Asn-204 (Magenta) are shown as spheres. Papain (PDB: 9PAP; shown in yellow) as a model papain-like cysteine protease is superimposed. N-terminal extension (shown in blue) and the hemoglobin binding hairpin (HBH- shown in orange) are specific to plasmodial falcipains and not observed in other papain-like cysteine proteases. A detailed view of side chains of residues in the HBH are shown in the inset. All structure figures have been made in Pymol.

FIGURE 6

Overall structure of Plasmodium M17-family leucyl aminopeptidase (PfM17LAP). Cartoon representation of the biologically functional hexamer of PfM17LAP (PDB: 3LQX). Monomers are colored by chain: A (Green); B (Salmon pink); C (Yellow); D (Blue); E (Red); F (Orange). In inset is shown a monomer of PfM17LAP comprising of N and C-terminal domains connected with the helical linker (shown in pale yellow) and a Zn²⁺ ion (magenta). All structure figures have been made in Pymol.