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• W2110625254 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. 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. The fungal secondary metabolite Cladosporin inhibits liver- and blood-stage malaria parasites Cladosporin specifically targets lysyl-tRNA synthetase (Krs1) Cladosporin is >100-fold more potent against parasite Krs1 relative to the human enzyme Two amino acids in the Krs1 ATP-binding pocket confer species-selective inhibition Malaria is a significant health problem, with 225 million annual cases and nearly 3.2 billion people at risk (WHO, 2010WHOWorld Malaria Report 2010. World Health Organization, Geneva2010Google Scholar). Control and treatment of this disease is compounded by a lack of an effective vaccine. In addition, the emergence of multidrug-resistant parasites has compromised efficacy of many of the frontline chemotherapy treatments. Although there are many effective drugs (Burrows et al., 2011Burrows J.N. Chibale K. Wells T.N. The state of the art in anti-malarial drug discovery and development.Curr. Top. Med. Chem. 2011; 11: 1226-1254Crossref PubMed Scopus (146) Google Scholar), endoperoxides are the only drug class for which clinically significant resistance has not been reported (Eastman and Fidock, 2009Eastman R.T. Fidock D.A. Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria.Nat. Rev. Microbiol. 2009; 7: 864-874Crossref PubMed Scopus (386) Google Scholar). However, endoperoxides, like many antimalarials, are inactive against the asymptomatic malaria liver stages. To ensure continued malaria control, with an aim for eradication, next-generation antimalarials are required to be active against multidrug-resistant parasites and efficacious against liver- and blood-stage infections. Traditional drug discovery efforts have revolved around high-value targets identified for their essentiality in the parasite. However, finding targets that are essential for blood and liver malarial stages has been technically challenging, and few inhibitors with these desirable properties are known because of the difficulties associated with simultaneously demonstrating that a target is essential to support viability in both blood and exoerythrocytic stages. An alternative approach to target discovery is to first find compounds with promising activity in phenotypic cell-based screens (Gamo et al., 2010Gamo F.J. Sanz L.M. Vidal J. de Cozar C. Alvarez E. Lavandera J.L. Vanderwall D.E. Green D.V. Kumar V. Hasan S. et al.Thousands of chemical starting points for antimalarial lead identification.Nature. 2010; 465: 305-310Crossref PubMed Scopus (770) Google Scholar, Guiguemde et al., 2010Guiguemde W.A. Shelat A.A. Bouck D. Duffy S. Crowther G.J. Davis P.H. Smithson D.C. Connelly M. Clark J. Zhu F. et al.Chemical genetics of Plasmodium falciparum.Nature. 2010; 465: 311-315Crossref PubMed Scopus (460) Google Scholar, Plouffe et al., 2008Plouffe D. Brinker A. McNamara C. Henson K. Kato N. Kuhen K. Nagle A. Adrian F. Matzen J.T. Anderson P. et al.In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen.Proc. Natl. Acad. Sci. USA. 2008; 105: 9059-9064Crossref PubMed Scopus (366) Google Scholar) and to then determine their mechanism of action through the identification of their specific targets (reviewed in McNamara and Winzeler, 2011McNamara C. Winzeler E.A. Target identification and validation of novel antimalarials.Future Microbiol. 2011; 6: 693-704Crossref PubMed Scopus (31) Google Scholar). Successful validation of a target from this latter approach provides a proof of concept for small-molecule inhibition and supports continued drug discovery based on rational design of the hit compound. In order to discover targets for both blood and liver stages, we performed a screen to identify inhibitors of P. falciparum blood- and liver-stage proliferation with a natural product library. Cladosporin, a fungal secondary metabolite whose primary target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity in both blood and liver stages. A member of the isocoumarin class, cladosporin is produced by various fungal genera such as Cladosporium, Aspergillus, Eurotium, and Chaetomium (Scott et al., 1971Scott P.M. Van Walbeek W. MacLean W.M. Cladosporin, a new antifungal metabolite from Cladosporium cladosporioides.J. Antibiot. (Tokyo). 1971; 24: 747-755Crossref PubMed Scopus (82) Google Scholar). It has been previously reported to have antifungal (Scott et al., 1971Scott P.M. Van Walbeek W. MacLean W.M. Cladosporin, a new antifungal metabolite from Cladosporium cladosporioides.J. Antibiot. (Tokyo). 1971; 24: 747-755Crossref PubMed Scopus (82) Google Scholar), insecticidal (Grove and Pople, 1981Grove J.F. Pople M. The insecticidal activity of some fungal dihydroisocoumarins.Mycopathologia. 1981; 76: 65-67Crossref Scopus (25) Google Scholar), and antibacterial properties (Anke, 1979Anke H. Metabolic products of microorganisms. 184. On the mode of action of cladosporin.J. Antibiot. (Tokyo). 1979; 32: 952-958Crossref PubMed Scopus (26) Google Scholar) as well as plant growth regulatory effects (Springer et al., 1981Springer J.P. Cutler H.G. Crumley F.G. Cox R.H. Davis E.E. Thean J.E. Plant growth regulatory effects and stereochemistry of cladosporin.J. Agric. Food Chem. 1981; 29: 853-855Crossref Scopus (32) Google Scholar) and anti-inflammatory responses in mouse lung tissue (Miller et al., 2010Miller J.D. Sun M. Gilyan A. Roy J. Rand T.G. Inflammation-associated gene transcription and expression in mouse lungs induced by low molecular weight compounds from fungi from the built environment.Chem. Biol. Interact. 2010; 183: 113-124Crossref PubMed Scopus (69) Google Scholar). Using both traditional and systems biology approaches, we show here that cladosporin potently and specifically inhibits cytosolic lysyl-tRNA synthetase in Plasmodium spp. In addition, we show that cladosporin is highly selective for the parasite enzyme and that selectivity is in part conferred by the amino acid identity at two key residues in the ATP binding pocket. Small molecules with activity against P. falciparum blood-stage parasites were previously identified in a phenotypic screen against a natural product library (Plouffe et al., 2008Plouffe D. Brinker A. McNamara C. Henson K. Kato N. Kuhen K. Nagle A. Adrian F. Matzen J.T. Anderson P. et al.In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen.Proc. Natl. Acad. Sci. USA. 2008; 105: 9059-9064Crossref PubMed Scopus (366) Google Scholar). Out of the 12,000 natural products, 275 compounds inhibited parasite growth with 50% inhibitory concentration (IC50) values in the submicromolar range. These hits were further screened by a high-content image-based assay to determine their ability to block in vitro P. yoelii liver-stage development (Meister et al., 2011Meister S. Plouffe D. Kuhen K. Bonamy G. Barnes S. Bopp S. Borboa R. Bright A. Che J. Cohen S. et al.Exploring Plasmodium hepatic stages to find next-generation antimalarial drugs.Science. 2011; 334: 1372-1377Crossref PubMed Scopus (253) Google Scholar). Cladosporin (Figure 1A ) demonstrated exceptional growth-inhibitory activities against both blood- and liver-stage parasite forms (IC50 ∼40–90 nM) while having little effect on the growth or viability of HepG2-CD81 cells (>10 μM) or other human cell lines (Table 1). The high selectivity index of cladosporin against Plasmodium parasites compared to mammalian cells (IC50/CC50 ≥ 111), as well as its equipotent activity against a diverse collection of multidrug-resistant Plasmodium lines (Table 1) and cidal action (see Table S1 available online), suggested that further investigation was warranted.Table 1Cladosporin Activity against Plasmodium Blood and Liver Stages and Human Cell LinesStrain or Cell LinePlasmodium ActivityCell LineIC50 (nM)CC50 (nM)3D7aIC50 determined by the 72 hr SYBR Green cell proliferation assay.45.4 ± 6.0Camp RaIC50 determined by the 72 hr SYBR Green cell proliferation assay.77.9 ± 3.2D10aIC50 determined by the 72 hr SYBR Green cell proliferation assay.89.6 ± 11.4D6aIC50 determined by the 72 hr SYBR Green cell proliferation assay.72.1 ± 3.1K1aIC50 determined by the 72 hr SYBR Green cell proliferation assay.80.1 ± 10.3NF54aIC50 determined by the 72 hr SYBR Green cell proliferation assay.87.9 ± 5.3FCBaIC50 determined by the 72 hr SYBR Green cell proliferation assay.66.7 ± 4.9FCR3aIC50 determined by the 72 hr SYBR Green cell proliferation assay.57.4 ± 10.8HEp2bFifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.9,666 ± 2,200HeLabFifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.74,285 ± 18,830HepG2bFifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.43,568 ± 25,080Huh7bFifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.>100,000HepG2-CD81bFifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.>10,000P. yoelii liver schizontcIC50 based on the mean parasite area calculated in the high-content screen.39.1 ± 18.4Dd2 clone162.5 ± 3.5Cladosporin-RDd2 clone#1aIC50 determined by the 72 hr SYBR Green cell proliferation assay.377.1 ± 31.4Cladosporin-RDd2 clone#2aIC50 determined by the 72 hr SYBR Green cell proliferation assay.389.5 ± 39.6Cladosporin-RDd2 clone#3aIC50 determined by the 72 hr SYBR Green cell proliferation assay.374.7 ± 54.0a IC50 determined by the 72 hr SYBR Green cell proliferation assay.b Fifty percent cytotoxic inhibitory concentration (CC50) was determined by CellTiter-Glo viability assay.c IC50 based on the mean parasite area calculated in the high-content screen. Open table in a new tab We next sought to discover the target of cladosporin. Cladosporin demonstrated high micromolar inhibition against the model yeast, S. cerevisiae, although activity varied slightly due to compound batch and the growth media conditions (summarized in Table S2). In rich growth media, cladosporin displays a 30% inhibitory concentration (IC30) of 110 μM, making it a suitable compound for haploinsufficiency profiling (HIP) assay (Giaever et al., 1999Giaever G. Shoemaker D.D. Jones T.W. Liang H. Winzeler E.A. Astromoff A. Davis R.W. Genomic profiling of drug sensitivities via induced haploinsufficiency.Nat. Genet. 1999; 21: 278-283Crossref PubMed Scopus (468) Google Scholar). The HIP target discovery assay is based on a genome-wide collection of heterozygous knockout yeast strains, each of which contains a marked gene deletion (Giaever et al., 2002Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. et al.Functional profiling of the Saccharomyces cerevisiae genome.Nature. 2002; 418: 387-391Crossref PubMed Scopus (3238) Google Scholar, Winzeler et al., 1999Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. et al.Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.Science. 1999; 285: 901-906Crossref PubMed Scopus (3181) Google Scholar). It has been shown that heterozygous diploid strains that bear a deletion in one copy of a small molecule's target gene show increased sensitivity to that small molecule relative to those strains that have two copies of the gene (Giaever et al., 1999Giaever G. Shoemaker D.D. Jones T.W. Liang H. Winzeler E.A. Astromoff A. Davis R.W. Genomic profiling of drug sensitivities via induced haploinsufficiency.Nat. Genet. 1999; 21: 278-283Crossref PubMed Scopus (468) Google Scholar). This is a likely consequence of decreased expression of the target in the heterozygous diploid strain. Each of these strains also carries a distinct 20 base pair DNA barcode that can be PCR amplified and quantified in competitive growth assays using microarrays containing the barcode complements. As each barcode is linked to a specific strain, barcodes that disappear over time in the presence of a small molecule reveal the identity of the molecular target of the small molecule. Three independent HIP assays reproducibly identified the heterozygous lysyl-tRNA synthetase (krs1/KRS1) knockout strain as being the only strain in the collection of 5,803 total strains (covering ∼95% of the genome) that was hypersensitive to cladosporin (Figures 1B and 1C). Because strains that bear deletions in drug pumps or other proteins that attenuate the action of small molecules often also show haploinsufficiency, we also assessed whether the krs1/KRS1 strain is ubiquitously sensitive. Historical profiling of 1,800 compounds (our unpublished data) revealed that the krs1/KRS1 strain was exclusively sensitive to cladosporin (Figure 1D), indicating a high degree of selectivity for cladosporin. KRS1 is a nonredundant and essential gene in yeast and functions to load lysine onto the corresponding tRNA molecule for protein translation (Mirande and Waller, 1988Mirande M. Waller J.P. The yeast lysyl-tRNA synthetase gene. Evidence for general amino acid control of its expression and domain structure of the encoded protein.J. Biol. Chem. 1988; 263: 18443-18451Abstract Full Text PDF PubMed Google Scholar). To further test if cladosporin specifically inhibits lysyl-tRNA synthetase, KRS1 was placed downstream of a Gal1 promoter and transformed into wild-type yeast. The same was done for genes encoding isoleucyl-tRNA synthetase (IlS1), glutamyl-tRNA synthetase (GLN4), and threonyl-tRNA synthetase (THS1). These strains were subsequently tested for growth defects in the presence of serially diluted cladosporin concentrations using galactose as a sole carbon source to drive promoter activity. Only the transgenic strain harboring the plasmid with the KRS1 gene conferred increased cladosporin resistance (2.2-fold shift), whereas the transgenic strains expressing other aminoacyl-tRNA synthetases (AARSs) exhibited no change in their sensitivity to cladosporin (Figure 2A ). In the absence of cladosporin, all strains grew at an equal rate and concurrently reached the same final OD600, indicating the overexpression constructs did not have a discernible effect on growth. To further strengthen the observed genetic link between the S. cerevisiae lysyl-tRNA synthetase (ScKrs1) and cladosporin, we examined mutations that confer cladosporin resistance in yeast. Yeast cells were mutagenized with ethylmethanesulfonate and then selected against 50 μM cladosporin, a concentration known to completely inhibit growth of wild-type strains in synthetic complete media (Table S2). Direct sequencing of the KRS1 gene in 37 clones revealed nonsynonymous mutations in ten of these clones (Figure 2B and Figure S2A). To better understand the significance of these mutations, we undertook homology modeling. The crystal structures of lysyl-tRNA synthetase from human (Guo et al., 2008Guo M. Ignatov M. Musier-Forsyth K. Schimmel P. Yang X.L. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: implications for multisynthetase complex formation.Proc. Natl. Acad. Sci. USA. 2008; 105: 2331-2336Crossref PubMed Scopus (70) Google Scholar) and two bacteria (Sakurama et al., 2009Sakurama H. Takita T. Mikami B. Itoh T. Yasukawa K. Inouye K. Two crystal structures of lysyl-tRNA synthetase from Bacillus stearothermophilus in complex with lysyladenylate-like compounds: insights into the irreversible formation of the enzyme-bound adenylate of L-lysine hydroxamate.J. Biochem. 2009; 145: 555-563Crossref PubMed Scopus (12) Google Scholar) have been published, and comparison reveals a high degree of structural conservation (Figure S2B). We took advantage of this conservation and used the human crystal structure as a template to generate a ScKrs1 homology model. The ScKrs1 model predicted that the identified mutations, Ile567Val (one strain), Gly551Ser (two strains), and Thr340Ile (seven strains), map to or near the binding pocket for ATP in ScKrs1 (Figures 2B and 2C and Figure S2A). Backcrossing of the ten selected mutants to isogenic wild-type strains of the opposite mating type and subsequent resistance assays revealed that all mutations were dominant. Subcloning of each of the mutated KRS1 genes into a low copy plasmid and reintroduction into the wild-type strain was sufficient to confer increased resistance to cladosporin (∼5-fold increase), indicating that second-site mutations did not contribute to resistance. We conclude that the mutated KRS1 gene is responsible for resistance in S. cerevisiae and that the resistance phenotype is dominant to the wild-type allele (Figure 2D). To establish target conservation in P. falciparum, we evolved cladosporin-resistant parasites (Table 1 and Tables S3 and S4) with hopes that the parasites would acquire resistance mutations in the target gene. The genomes of the cladosporin-sensitive parental clone and cladosporin-resistant clones were compared using a microarray that has the capacity to reveal most genomic changes that are acquired by the resistant strains, including copy number variants, deletions, small insertion/deletion events, and single-nucleotide polymorphisms (Dharia et al., 2009Dharia N.V. Sidhu A.B. Cassera M.B. Westenberger S.J. Bopp S.E. Eastman R.T. Plouffe D. Batalov S. Park D.J. Volkman S.K. et al.Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.Genome Biol. 2009; 10: R21https://doi.org/10.1186/gb-2009-10-2-r21Crossref PubMed Scopus (120) Google Scholar). Since background mutations may appear at a low frequency, the selection was run in triplicate in order to distinguish resistance-conferring mutations from random mutations. Genome scanning revealed that each of the independent clones had acquired copy number variants that shared six genes in a syntenic region of chromosome 13: MAL13P1.253, PF13_0263, PF13_0264, MAL13P1.254, and MAL13P1.255 and PF13_0262, encoding the cytoplasmic lysyl-tRNA synthetase (PfKrs1), the ortholog of KRS1 (Figure 3A and Table S3). Careful examination of the hybridization data showed that none of the other ∼5,500 genes were mutated in all three clones and that the only genomic differences between the sensitive and mutant lines were the copy number variants that encompassed PfKrs1. Although the high amino acid sequence conservation between the fungal and plasmodial enzymes (Figure S2A) suggests that cladosporin is likely targeting cytoplasmic lysyl-tRNA synthetase in both species, it is possible that one of the other genes in the amplification event could encode the target. Because inhibition of cytoplasmic lysyl-tRNA synthetase is expected to impair protein biosynthesis, incorporation of radioactive amino acids was investigated in cladosporin-treated parasites (Figure 3B). Treatment with cladosporin, or known protein synthesis inhibitors, anisomycin and cycloheximide, caused a rapid, and comparable, drop-off in incorporation of radiolabeled amino acids and indicated protein synthesis inhibition. In contrast, mefloquine and artemisinin, antimalarial drugs with unrelated mechanisms of action, did not diminish radioisotope incorporation, as previously shown (Rottmann et al., 2010Rottmann M. McNamara C. Yeung B.K. Lee M.C. Zou B. Russell B. Seitz P. Plouffe D.M. Dharia N.V. Tan J. et al.Spiroindolones, a potent compound class for the treatment of malaria.Science. 2010; 329: 1175-1180Crossref PubMed Scopus (931) Google Scholar). Malaria parasites have two lysyl-tRNA synthetases, one of which is targeted to the apicoplast (PF14_0166), a specialized subcellular organelle involved in fatty acid and isoprenoid biosynthesis, while the other one is targeted to the cytoplasm (PF13_0262). The apicoplast is essential to parasite viability, and inhibition of protein synthesis in this organelle yields a characteristic “delayed death” phenotype in which parasite death is observed one generation after drug treatment. Known apicoplast inhibitors include mupirocin, which targets the apicoplast-targeted isoleucyl-tRNA synthetase (Istvan et al., 2011Istvan E.S. Dharia N.V. Bopp S.E. Gluzman I. Winzeler E.A. Goldberg D.E. Validation of isoleucine utilization targets in Plasmodium falciparum.Proc. Natl. Acad. Sci. USA. 2011; 108: 1627-1632Crossref PubMed Scopus (99) Google Scholar); tetracycline (Goodman et al., 2007Goodman C.D. Su V. McFadden G.I. The effects of anti-bacterials on the malaria parasite Plasmodium falciparum.Mol. Biochem. Parasitol. 2007; 152: 181-191Crossref PubMed Scopus (206) Google Scholar); azithromycin (Sidhu et al., 2007Sidhu A.B. Sun Q. Nkrumah L.J. Dunne M.W. Sacchettini J.C. Fidock D.A. In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin.J. Biol. Chem. 2007; 282: 2494-2504Crossref PubMed Scopus (125) Google Scholar); and the apicoplast 23S ribosomal RNA inhibitor clindamycin (Dharia et al., 2009Dharia N.V. Sidhu A.B. Cassera M.B. Westenberger S.J. Bopp S.E. Eastman R.T. Plouffe D. Batalov S. Park D.J. Volkman S.K. et al.Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.Genome Biol. 2009; 10: R21https://doi.org/10.1186/gb-2009-10-2-r21Crossref PubMed Scopus (120) Google Scholar). To distinguish between these two potential targets, P. falciparum parasites were synchronized to the ring stage, treated with cladosporin, and the maturation of the parasite monitored over two life cycles. Parasites treated with cladosporin did not exhibit a delayed death phenotype and quickly arrested in the metabolically active trophozoite stage (Figure 3C). The phenotype of cladosporin-treated parasites is indistinguishable from that of anisomycin, another eukaryotic protein synthesis inhibitor, and supports the notion that both have the same mechanism of action. In contrast, clindamycin, which specifically inhibits protein biosynthesis in the apicoplast, did not diminish growth until the end of the second blood-stage life cycle (Figure S3). Although these results do not preclude cladosporin as also targeting the apicoplast lysyl-tRNA synthetase, these data show that cladosporin is a fast-acting agent that shows the same characteristics as other known cytoplasmic protein synthesis inhibitors. To confirm that cladosporin is an inhibitor of PfKrs1, direct biochemical assays were performed on recombinant protein. PfKrs1 activity was assayed in the presence or absence of the corresponding lysine-tRNA (tRNALys) substrate, and synthetase activity was measured using a Transcreener adenosine monophosphate (AMP) assay. This assay is a far-red, competitive fluorescence polarization immunoassay that detects the reaction product, AMP. Cladosporin possessed a half inhibitory concentration of 61 nM against PfKrs1 (Figure 4A ), which is comparable to its activity in cellular screens (IC50 40–90 nM; Table 1). As expected, no activity was observed in the absence of exogenous tRNALys. The agreement between the biochemical and biological inhibition constants further supports the notion that lysyl-tRNA synthetase is the primary target within the cell. Our data show that cladosporin has relatively little activity on human cells, including the HepG2-CD81 cells used in the hepatic-stage screen (Table 1). To confirm that cladosporin is selective for PfKrs1, the human counterpart was assayed. Recombinant human lysyl-tRNA synthetase was assayed as described above. As predicted, cladosporin activity was only weakly detected at the high micromolar ranges for human Krs1 (Figure 4B). These data reinforce the notion that cladosporin specifically and selectively targets P. falciparum lysyl-tRNA synthetase. The S. cerevisiae and P. falciparum amino acid sequences share a high degree of identity to the human protein (yeast 58.3%, plasmodium 53.4%), and the homology model could accommodate both primary sequences into the human template with high confidence. According to the predicted models, 30 of 36 amino acids within a 5 Å radius of the ATP/lysine-binding site are conserved between S. cerevisiae and P. falciparum. Using an in silico docking approach it was possible to model cladosporin into the ATP-binding pocket in an energetically favorable mode, mimicking that of ATP (Figure 4C). Residues Asn335 and Glu328, which hydrogen bond to ATP, are also predicted to hydrogen bond with the hydroxyl groups of cladosporin. The accuracy of this prediction was validated biochemically. Characterization of the recombinant PfKrs1 revealed that lysine had no effect on cladosporin inhibition (Figure S4A), whereas increasing concentrations of ATP in the reaction buffer significantly reduced cladosporin inhibition (Figure S4B). This was corroborated by the inability of exogenous lysine in culturing media to affect cladosporin potency against yeast cells (Table S5). Taken together, these data support the notion that cladosporin interacts with the ATP-binding pocket of lysyl-tRNA synthetase and shares a similar mode of binding to ATP. To further understand the basis of cladosporin selectivity, the amino acid residues of the ATP-binding pocket were compared to those in Krs1 of other species (Figure S4C). While the majority of amino acid residues in the binding pocket were highly conserved, a clear divergence existed at two amino acid positions corresponding to S. cerevisiae residues Gln324 and Thr340 (Table 2). In Plasmodium spp., these positions are occupied by a valine and serine residue, respectively. Based on the identity of the Plasmodium amino acid residues, smaller side chains are favored, presumably due to decreased steric hindrance. Additionally, a hydrophobic residue at position 324 also appears favorable for cladosporin binding.Table 2Summary of Amino Acid Conservation at Key Residues in the ATP Pocket of Krs1 and Corresponding IC50 Value of CladosporinOrganismCladosporin InhibitionKey Active Site ResiduesaAmino acid identity at homologous positions to ScKrs1 residues 324 and 340 based on primary sequence alignments (see also Figure S4).IC50 (μM)MICbMinimal inhibitory concentrations of cladosporin reported by Anke (1979). (μg/ml)Position 324Position 340Plasmodium falciparumcSYBR Green proliferation assay of blood-stage parasites.0.04–0.08ValSerPlasmodium yoeliidHigh-content imaging of liver-stage parasites.0.04ValSerTrypanosoma brucei2.05ValThrLeishmania donovani2.56ValThrToxoplasma gondii2.63AsnAlaHomo sapiens>10GlnThrSaccharomyces cerevisiae30–110GlnThrEscherichia coli>100AsnMetBacillus stearothermophilus>100ValMeta Amino acid identity at homologous positions to ScKrs1 residues 324 and 340 based on primary sequence alignments (see also Figure S4).b Minimal inhibitory concentrations of cladosporin reported by Anke, 1979Anke H. Metabolic products of microorganisms. 184. On the mode of action of cladosporin.J. Antibiot. (Tokyo). 1979; 32: 952-958Crossref PubMed Scopus (26) Google Scholar.c SYBR Green proliferation assay of blood-stage parasites.d High-content imaging of liver-stage parasites. Open table in a new tab Despite being separated by 15 amino acids in the primary sequence, the homology model predicted that these residues were juxtaposed in the active site (Figure S4D). The docking model also predicts that the pyrane moiety of cladosporin points toward both of these residues and that Thr340 resides at an intimate distance of 3.7 Å (Figure 4C and Figure S4D). Extending the comparison to other eukaryotic and prokaryotic pathogens with Krs1 orthologs, a clear correla" @default.
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• W2110625254 title "Selective and Specific Inhibition of the Plasmodium falciparum Lysyl-tRNA Synthetase by the Fungal Secondary Metabolite Cladosporin" @default.
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