Inhibition of Protein Synthesis and Malaria Parasite Development by Drug Targeting of Methionyl-tRNA Synthetases

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS cyt and PfMRS api . Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS cyt ) or proteobacteria/primitive bacteria (PfMRS api ). We show that PfMRS cyt localizes in parasite cytoplasm, while PfMRS api localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with 35 S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. P lasmodium falciparum is the most virulent form of Plasmodium and a causative agent of malaria. The World Health Organization (WHO) estimates that there are ϳ0.62 million deaths due to malaria per year (1). The P. falciparum genome is AT-rich (81%) and codes for ϳ5,300 proteins, with unusual distributions of several residues (2). Almost 60% of encoded proteins appear to be unique to the parasite, reflecting great evolutionary distance between the parasite and the genomes of known eukaryotes (3). The malaria parasite (and the related apicomplexan Toxoplasma gondii) has three translationally active compartments, i.e., cytoplasm, apicoplasts, and mitochondria (4-8). All malaria parasite proteins involved in the protein synthesis machinery are encoded by the nuclear genome. Either these proteins are transported to target organelles or their modified/activated substrates are transported across the organelles to perform required functions (4-9). The primary enzymes responsible for translating genetic code into polypeptide chains are aminoacyl-tRNA synthetases (aaRSs). The canonical function of aaRSs is to ligate a specific amino acid to its cognate tRNA, which is then employed in protein synthesis. The aaRSs are an ancient class of essential enzymes, and their catalytic domains are conserved in all kingdoms (bacteria, archaebacteria, and eukaryotes). On the basis of catalytic domain architecture, aaRSs are classified into two categories, with each class containing ϳ10 aaRSs (10). Although aaRSs perform the basic function of charging tRNA molecules for protein synthesis, a...

Screening of quinoline, 1,3-benzoxazine, and 1,3-oxazine-based small molecules against isolated methionyl-tRNA synthetase and A549 and HCT116 cancer cells including an in silico binding mode analysis

“…Step 2: Preparation of 2-(2-butyl-4-chloro-1-(4-phenoxybenzyl)-1H-imidazol-5-yl)-5-(4-methoxyphenyl)-1-oxa-3-azaspiro- [5.5]undecane: this compound was obtained from AA (1 mmol), 2-butyl-4-chloro-1-(4-phenoxybenzyl)-1H-imidazole-5-carbaldehyde (1 mmol), and K 2 CO 3 (2.5 mmol) in methanol as a brown crystalline solid, yield: 84%; melting point: 55-57°C; elemental analysis calculated for C 36 [1,3]oxazine (8). Compound 8 was obtained in two steps.…”

“…Research in the previous decades explored numerous scaffolds including quinolines, pyrrolines and chloramphenicol derivatives as inhibitors of bacterial MRS, and most of them displayed high specificity towards bacterial MRS and failed in inhibiting human MRS. 8,9 Quinoline derivatives in particular have been extensively presented as inhibitors of bacterial MRS, and more recently the quinoline-4-one based small molecule REP8839 was reported as a novel inhibitor of MRS in methicillin-resistant Staphylococcus aureus as well as Streptococcus pyogenes. 10 Furthermore, benzoxaborole derivatives were shown to have significant leucyl-tRNA inhibitory activity.…”

Side chain rotameric changes and backbone dynamics enable specific cladosporin binding in Plasmodium falciparum lysyl‐tRNA synthetase

“…While eukaryotes and most prokaryotes contain class II KRSs, some bacteria and archaea contain class I KRSs . Drug targeting of aaRSs presents an opportunity to stall global protein synthesis and this aspect makes aaRSs worthy of attention for development of anti‐infectives . The type II Pf KRS exists as a homodimer and each monomer consists of 17 β‐strands and 17 α‐helices along with an N‐terminal OB‐fold anticodon‐binding and a C‐terminal catalytic domain .…”

“…[10][11][12] Drug targeting of aaRSs presents an opportunity to stall global protein synthesis and this aspect makes aaRSs worthy of attention for development of anti-infectives. [13][14][15] The type II PfKRS exists as a homodimer and each monomer consists of 17 β-strands and 17 α-helices along with an N-terminal OB-fold anticodon-binding and a C-terminal catalytic domain. 3 In order to understand the structural underpinnings of CLD binding to KRSs, we studied four sets of apo/holo KRS-CLD complexes where crystal structures are available.…”

Aminoacyl tRNA Synthetases as Malarial Drug Targets: A Comparative Bioinformatics Study

“…This reaction takes place in two steps; first ATP activates the amino acid through formation of aminoacyl-adenylate intermediate, while the second step involves ligation of the adenylate intermediate to the cognate tRNA molecule through a covalent bond generating AMP [8,9,18]. Although the canonical function of these enzymes is to add amino acids to tRNA for translation and they are highly conserved in their catalytic domains, in general aaRSs show sequence, structural and functional diversity across organisms [19]. Furthermore, in some organisms, aaRSs have evolved to perform non-canonical functions such as angiogenesis, RNA splicing, signaling events, transcription regulation, apoptosis and immune responses [20–22].…”