Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Abstract

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH(2)]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

Fig. 1.

Analysis of PfA-M1 expression and activity. (A) Western blot of transgenic parasites expressing the product of the inserted transgene encoding PfA-M1. The blot was probed with a monoclonal anti-c-myc primary antibody followed by horseradish peroxidase-conjugated anti-mouse immunoglobulin antibodies and visualized by enhanced chemiluminescence. (B) Indirect immunofluorescence of transgenic parasites stained with monoclonal anti c-myc primary antibody followed by anti-mouse cy2. (i) Bright field; (ii) anti-c-myc antibody; (iii) anti-c-myc/nuclear stain merged; (iv) merge of i and iii. The data show that the PfA-M1 transgenic protein is localized to the parasite cytosol. (C) Northern blot analysis of stage-specific parasite RNA reveals that the endogenous PfA-M1 is expressed by parasites at all developmental stages within the erythrocyte. Developmental stages are indicated at the top: R, ring stage parasites; ET, early trophozoite parasites; LT, late trophozoite parasites; and S, schizont stage parasites. Size difference observed between trophozoite and ring/schizont is due to different transcription initiation sites. (D) The pH optima for activity of rPfA-M1 (circles) and native PfA-M1 in soluble extracts of parasites (squares) measured by using the fluorogenic peptides substrate H-Arg-NHMec.

Fig. 2.

The structure of rPfA-M1. (A) Diagram of unbound rPfA-M1 colored by domain: I (blue), II (green), III (yellow), and IV (red). (B and C) Molecular surface of PfA-M1 (colored as in A) showing small (B) and large (C) openings to active site (active site cavity shown in magenta).

Fig. 3.

The inhibitory effect of the aminopeptidase inhibitor bestatin (squares), and Co4 (triangles) on P. falciparum clone 3D7 growth in culture compared with parasites grown in the absence of inhibitor (circles). Data are presented as mean ± SD of 3 independent, triplicate experiments. Parasites grown in the presence of bestatin or Co4 for 24 h exhibit cellular damage and stunted development compared with control parasites grown in the absence of drug (Inset).

Fig. 4.

Stereo diagram of inhibitors binding to active site of rPfA-M1. (A) 1.65-Å rPfA-M1-BES and (B) 2.0-Å rPfA-M1-Co4 active site showing inhibitors bound in the active site. Inhibitors (BES/Co4) are colored in magenta. Carbon atoms of residues are colored by domain: I (blue), II (green), IV (red), and Zn is shown as black sphere. Water molecules are shown as blue spheres. Hydrogen bonds are indicated (dashed lines). Electron density is a composite omit map contoured at 1.0 σ calculated by using a model containing only PfA-M1 atoms (no zinc, ligand, or water was included in the calculation).