Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin

Abstract

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

Graphical abstract

Figure 1

Haploinsufficiency Profiling of Cladosporin Identifies the KRS1 (A) Chemical structure of cladosporin. (B) HIP analysis of cladosporin at 110 μM. See also Figure S1. Heterozygous strains deleted for essential genes are represented by gray boxes, and strains deleted for nonessential genes are represented by black dots. The deletion strain corresponding to the heterozygous krs1/KRS1 strain is labeled. (C) Alignment of two additional, independent cladosporin HIP experiments showing reproducibility of the KRS1 strain hypersensitivity. (D) Cladosporin is the only substance among a diverse collection of 1,800 compounds tested by HIP profiling that significantly affects the heterozygous KRS1 strain. Sensitivity was calculated as a logarithmic ratio of the relative abundance of any given strain in the treated versus untreated samples and corrected for outliers. The z score couples the sensitivity score to a value proportional to the variation in sensitivity of any HIP strain across the 1,800 diverse compounds tested, thus allowing the identification of nonspecific frequent hitters. For more information, refer to the Supplemental Experimental Procedures.

Figure 2

Krs1 Target Validation by Overexpression Analysis and Chemical Mutagenesis of S. cerevisiae (A) Cladosporin specificity was evaluated in strains overexpressing the following aminoacyl-tRNA synthetase genes: lysine (KRS1), glutamine (GLN4), isoleucine (ILS1), and threonine (TRS1). KRS1 overexpression confers a 3-fold increase in cladosporin resistance, whereas no change in cladosporin potency was observed in strains overexpressing the other synthetases. (B) Schematic view of the S. cerevisiae lysyl-tRNA synthetase (ScKrs1) protein domain organization. Resistance-conferring mutations (asterisks) are labeled and frequency of mutation provided in parentheses. (C) The ATP/lysine binding pocket of the ScKrs1 homology model (gray) is shown. See also Figure S2. The distance (given in angstroms [Å]) between the three mutated residues (stick representations) and atoms in the ATP molecule is indicated next to the dashed lines. (D) Expression of the ScKrs1 mutants Thr340Ile (Krs1_T340I), Gly551Ser (Krs1_G551S), Ile567Val (Krs1_I567V) shifted cladosporin IC₅₀ values ∼5.3-fold compared to an empty vector control strain, whereas overexpression of wild-type ScKrs1 (Krs1_WT) showed a 3.3-fold shift (comparable shift to that in A).

Figure 3

P. falciparum Acquires Copy Number Variants in Lysyl-tRNA Synthetase and Has Protein Synthesis Defects in the Presence of Cladosporin (A) Whole-genome analysis of cladosporin-resistant P. falciparum clones on a high-density DNA tiling microarray revealed that a common gene locus on chromosome 13 was amplified in each clone. Six genes were shared by all amplification events: lysyl-tRNA synthetase (PF13_0262), small nuclear ribonucleoprotein (MAL13P1.253), a conserved Plasmodium protein of unknown function (PF13_0263), ubiquitin-activating enzyme E1 (PF13_0264), a conserved Plasmodium protein of unknown function (MAL13P1.254), and N6-adenine-specific methylase (MAL13P1.255). An enhanced probe-by-probe analysis of this locus is shown for each clone, and common genes are shaded green. The lysyl-tRNA synthetase gene (asterisk) is present in all amplification events. See also Table S3. (B) Mixed erythrocytic-stage parasites were treated for 1 hr with cladosporin, artemisinin (ART), mefloquine (MFQ), anisomycin, or cycloheximide over a five-log range of drug concentrations to determine their effect on ³⁵[S]-cysteine/methionine incorporation. Radioactive counts were normalized to untreated cells, and each data point was plotted as the mean of two experiments performed in triplicate. Error bars represent the standard deviation. (C) The specific time of action for cladosporin in erythrocytic-stage parasites was determined by treating double-synchronized parasites (6 hr interval) and monitoring the cultures over a 60 hr period. The morphology of untreated parasites (–) and parasites treated with 400 nM cladosporin (+) were monitored by Giemsa-stained thin blood smears for one complete 48 hr life cycle (12 hr, ring; 24 hr, trophozoite; 36 hr, early schizont; 48 hr, mature schizont) and the first 12 hr of the second generation. Representative images are shown for each time point. Controls are shown in Figure S3.

Figure 4

Cladosporin Is Highly Selective for PfKrs1, which Is Modulated in Part by Two Key Active Site Residues (A and B) Direct biochemical analysis of recombinant lysyl-tRNA synthetase reveals that cladosporin has low nanomolar inhibition (IC₅₀ = 61 nM) against PfKrs1 (A, squares) and high micromolar activity (IC₅₀ > 20 μM) against human Krs1 (B, squares). As a control, recombinant enzyme was assayed under the same conditions but in the absence of the tRNA^Lys substrate (circles). Enzymatic data were from at least two independent assays performed in triplicate and expressed as means ± SD. A nonlinear regression curve fit is shown for each. (C) Superimposed structures of cladosporin (green) and ATP (cyan) docked to the yeast homology model (gray). Oxygen and nitrogen atoms are colored red and blue, respectively, in all molecules. Cladosporin is predicted to bind in the ATP-binding pocket of the yeast homology model with the isocoumarin moiety located in the same region as the adenine of ATP. Also, cladosporin's pyrane moiety superimposes between the sugar and the phosphate of ATP and projects toward Gln324 and Thr340. The two hydroxy groups of cladosporin are predicted to form hydrogen bonds with Asn335 and Glu328 (dotted lines; bond length label given in Å). (D) ScKrs1-Gln324Val mutant (IC₅₀ = 28 μM; squares) and ScKrs1-Thr340Ser mutant (IC₅₀ = 16 μM; diamonds) mimic the differences in the Plasmodium ATP pocket and significantly increase the potency of cladosporin compared to wild-type S. cerevisiae strain (IC₅₀ = 163 μM; triangles). The double mutant, ScKrs1-Gln324Val/Thr340Ser (IC₅₀ = 4 μM; circles), which more closely resembles PfKrs1, is 41-fold more sensitive to cladosporin. See also Figure S4. Enzymatic data were from at least two independent assays performed in triplicate and expressed as means ± SD. A nonlinear regression curve fit is shown for each.

Figure 5

Yeast Cells Dependent on Chimeric Lysyl-tRNA Synthetases Exhibit Wild-Type Growth Kinetics but Show Differential Sensitivity to Cladosporin (A) The homology model of ScKrs1 with cladosporin docked into the ATP binding site (asterisk). Amino acids 1–220 corresponding to the tRNA-binding domain are colored black, whereas residues beyond 220, which correspond to the aminoacylation domain, are shown in color. (B) Growth curves of yeast cells solely dependent on heterologous, chimeric lysyl-tRNA synthetases with aminoacylation domains of the indicated species. (C) Cladosporin dose-response curves of yeast cells solely dependent on heterologous, chimeric lysyl-tRNA synthetases with aminoacylation domains of the indicated species. Data for (B) and (C) were collected using the optical density assay and are a representation of triplicate experiments. The independent data points for each experiment are shown. In (C), a nonlinear regression curve fit was performed on the means ± SD of these data.