Validation of isoleucine utilization targets in Plasmodium falciparum

Abstract

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum . Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.

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

“…Their similar IC 50 values at both 48 and 96 h suggest that these compounds inhibit cytosolic ARS. Natural product ARS inhibitors were also screened for antimalarial activity (Table 1 (17) and consistent with its high selectivity toward bacterial-type enzymes (33,34), such as the apicoplast-targeted isoleucyl-tRNA synthetase (IleRS-2). This phenomenon was observed even when mupirocin was removed from the culture after the first cycle of incubation.…”

“…Although underexploited for many years, recent work has shown that plasmodial ARS are not only druggable enzymes (17,18) but also that selective inhibition of these enzymes versus their human homologs is feasible (16). In this work, we evaluated and tested a series of known ARS inhibitors on P. falciparum cell cultures and explored their ability to inhibit parasite replication in vitro.…”

“…In this latter work, cladosporin, a fungal secondary metabolite, was found to target the cytosolic lysyl-tRNA synthetase (LysRS) of the malaria parasite. Antimalarial ARS-directed drug design has also been applied satisfactorily against apicoplastic and cytosolic isoleucyl-tRNA synthetase (IleRS) (17) and apicoplastic LysRS (18). However, all previously identified antimalarial drugs targeting ARS either lacked potency (18) did not show in vivo antimalarial activity (17) or showed poor oral bioavailability (16).…”

“…An important example of the clinical application of an ARS inhibitor is provided by the antibiotic mupirocin (pseudomonic acid; marketed as Bactroban, GlaxoSmithKline), which selectively inhibits bacterial isoleucyl-tRNA synthetase without inhibiting its human homolog. Although proven antibacterial drug targets (12)(13)(14)(15), these enzymes have only recently been highlighted as antimalarial drug targets (16)(17)(18).…”

The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment

“…Interestingly, mupirocin was also shown to inhibit blood stage of P. falciparum in the nanomolar range 8 through the inhibition of apicoplastic IleRS. However, mupirocin failed to protect mice from systemic P. berghei infection, probably due to its in vivo instability and its high binding to serum.…”