Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Abstract

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

Keywords: aminoacyl-tRNA synthetase; borrelidin; drug design; malaria; plasmodium.

Fig. 1.

Antimalarial drug design strategy and pipeline used for identifying potent and selective ARS inhibitors.

Fig. 2.

Library of borrelidin analogs tested on cell-based assays of P. falciparum cultures. The structure of borrelidin is shown with the three substituents modified in the analog library circled in red. The structural variation of the analogs is color coded red according to the substitutions explored: (i) the nitrile group was substituted for a carboxylic acid moiety (cyan); (ii) the cyclopentane moiety was substituted for a cyclobutane (yellow) or aliphatic side chain (red); and (iii) the carboxylic acid group was esterified (pink) or amidated (green). Further, the new ester and amide moieties included a range of smaller substituent side chains including carboxylate isosteres (gray) or heterocycles (orange), both aromatic and nonaromatic in nature.

Fig. 3.

Antimalarial activity of the library of borrelidin analogs. In vitro antimalarial activities of borrelidin derivatives tested at 100 nM. Borrelidin was included as positive control. Compounds inhibiting over 80% at 100 nM (13 among the 30 compounds tested) were considered to be active and were selected for IC₅₀ determination in both P. falciparum iRBC (nanomolar) and HEK293T cultures (micromolar). The fold selectivity comparison of the 13 selected borrelidin analogs compared with borrelidin is shown. See also Table S1.

Fig. 4.

In vitro and in vivo activities of BC196 and BC220. (A) Chemical structure of BC196 and BC220 compared with borrelidin. (B) In vivo mice survival of P. yoelii-infected mice treated with the selected subset of borrelidin analogs. Percentage of mice survival of P. yoelii-infected mice, measured over 20 d after drug treatment. Chloroquine (Cq) has been used as positive control (Table 3). (C) Comparison of the inhibitory activity of the aminoacylation reaction catalyzed by human cytosolic ThrRS. (D) Homology model of P. falciparum ThrRS. Residues have been colored by its conservation with the human cytosolic ThrRS: conserved -same residue- (yellow), not conserved -different residue- (white). Borrelidin is shown in magenta, whereas AMP is shown in cyan. (E) Predicted binding mode of BC196 (green) and BC220 (cyan), superimposed with the predicted binding mode of borrelidin (magenta) in Homo sapiens. Predicted binding modes are identical to those found in P. falciparum ThrRS (not shown in figure).

Fig. 5.

HPLC-PDA analysis of borrelidin and BC220 stability in P. falciparum-infected RBC cultures. HPLC-PDA traces of extracted metabolites from P. falciparum-infected RBC treated with BC220 (cyan, magenta, yellow) and borrelidin (blue, green, brown), respectively. The traces do not vary as incubation time increases. The peak corresponding to BC220 is highlighted with a green asterisk, and the peak corresponding to borrelidin is highlighted with a red asterisk. See also Table S2 and Fig. S3.