Recent advances in the biology and drug targeting of malaria parasite aminoacyl-tRNA synthetases

Plasmodium falciparum causes the most lethal form of malaria and is the world’s largest killer with ~ 438,000 deaths and more than 200 million infections annually [1]. While the 2015 Nobel prize in physiology celebrates the triumph over deadly malarial and worm parasites, drug resistance among pathogens of bacterial and eukaryotic origin, including malaria parasites and worms is inevitable. The current situation is worsened by the increasing drug resistance in malaria parasites, even to mainstream drugs in clinical use, such as artemisinins [1]. Vaccination programmes have not been successful yet, which makes it urgent to find new molecular scaffolds to design efficient anti-malarials [1]. The highly complex progression of the parasite through its life cycle depends on its varying its proteome to fit different cellular milieus of vector salivary gland, gut, human blood stream, hepatocytes and erythrocytes [2‐4]. A dynamic proteome presents problems for selecting multistage targets as reflected in the inefficacy of many drugs in clinical use on the liver stage. In this direction, housekeeping pathways, such as protein translation, are attractive drug targets as they are not only vital but also active in all stages [5].

The malaria parasite contains three genomes; nuclear, apicoplastic (a relic chloroplast) and mitochondrial and all three genomes require dedicated translational machineries to function [5]. Protein translation machinery provides a diverse collection of proteins to be targeted and malarial aminoacyl-tRNA synthetases (aaRSs) have received the most attention for drug targeting in the last half-decade [5, 6]. aaRSs catalyze the first reaction of protein biosynthesis by combining a specific amino acid to cognate tRNA molecules in a two-step reaction (Fig. 1) [7]. Generally, there are 20 different aminoacyl-tRNA synthetases in a protein translational compartment, specific to one of the twenty amino acids [7‐9]. Depending on the architecture of the active site and mode of tRNA binding, aaRSs are divided into two structural classes, with 10 enzymes in each class [7‐9]. aaRSs are one of the most ancient enzymes and over the course of evolution, have appended additional domains to their core structure to perform additional non-canonical functions [10, 11]. These functional expansions range from splicing, cytokine-like function to roles in DNA damage response. Molecular details, structures and a fundamental understanding of workings of aaRSs, including their moonlighting functions, are available in great detail and discussed in many reviews [7‐11].

Protein translation ensures a high fidelity by quality checks at several steps [12, 13]. Proofreading at the aminoacylation step to discriminate between cognate amino acid and isosteric substrates is performed by an editing pocket appended (cis) to many aaRSs and by trans-editing factors [12‐14]. Class I enzymes contain an insertion in their Rossmann fold called connective polypeptide 1 (CP1), which in some cases forms the editing pocket [14]. CP1 can catalyze the reversion of both pre- and post-transferred errors in aminoacylation. Class II aaRSs contain a distinct editing domain, which mostly hydrolyse the mischarged tRNAs (post-transfer). Trans-editing factors like AlaX and Ybak hydrolyse misacylated tRNAs [12‐14]. Enantiomeric selectivity is provided by the D-tyrosyl-tRNA deacylase (DTD) enzyme, which hydrolyses D-amino acids coupled to tRNA molecules [5, 15, 16].

Research on crucial malaria parasite aaRSs was majorly initiated with their genomic analysis and tabulation in 2008 by Bhatt et al. [17]. Their comprehensive analysis revealed that malaria parasite P. falciparum contains 37 aaRS genes in its nucleus, which can form 36 enzymes [17] (Table 1). Many interesting aspects about malaria aaRSs came to light through this study. For instance, compared to other organisms, malarial aaRSs constitute a much larger fraction of the overall proteome. Additionally, these aaRSs have an unusual domain architecture and contain additional domains [17]. Most intriguingly, it was, till recently, unclear how 36 aaRSs, instead of the theoretically required 60 aaRSs, provide charged tRNAs to three translational compartments; cytoplasm, mitochondrion and apicoplast (20 tRNAs per compartment being the theoretical requirement). Studies mainly focused on cellular distribution of aaRSs and import of cytoplasmically charged tRNA to mitochondrion have now revealed the scheme by which the malaria parasite efficiently utilizes a compromised array of 36 aaRSs to synthesize its proteome (Table 1) (Fig. 2). Localization studies combined with robust bioinformatics predictions have revealed that there are 16 aaRSs exclusive to cytoplasm and 15 nucleus-encoded aaRSs exclusively targeted to apicoplast (Table 1) [17‐26]. Four single copy aaRSs (alanyl-tRNA synthetase; AlaRS, threonyl-tRNA synthetase; ThrRS, cysteinyl-tRNA synthetase; CysRS and glycyl-tRNA synthetase; GlyRS) are shared between the apicoplast and cytoplasm by dual localizations, where mechanisms like alternative splicing (CysRS) and presumably, alternative translation initiation (AlaRS, ThrRS and GlyRS) occur (Table 1) (Fig. 2) [18‐20]. Moreover, since the apicoplast lacks glutaminyl-tRNA synthetase (GlnRS), a charged glutamine-specific tRNA is provided by the reactions of two apicoplastic enzymes; glutamyl-tRNA synthetase (GluRS) and a unique glutamyl-tRNA amidotransferase (GatAB) [27, 28]. Apicoplastic non-discriminating GluRS mischarges glutamine-specific tRNA with glutamic acid followed by tRNA-bound glutamic acid conversion into glutamine by the heterodimeric GatAB, thus providing a complete set of 20 charged tRNAs (Fig. 2) [27, 28].

Plasmodium falciparum mitochondrion was shown to harbour an enzymatically active mitochondrial phenylalanyl-tRNA synthetase (PheRS), which is unique to Plasmodium as it is absent in other apicomplexans [29]. Mitochondrial PheRS is the only aaRS present in parasite mitochondrion and its functional relevance remains unclear. The mitochondrion seem to be dependent on charged tRNA import for synthesizing its three respiratory chain associated genes; cytochrome c oxidase subunits I and III (COX1, COX3) and cytochrome b (Cytb) (Fig. 2) [5, 29, 30]. Recently, evidence for import of cytoplasmically charged phenylalanine and cysteine tRNAs was provided which suggest that the same is likely true for other tRNAs [29]. Similar studies on Toxoplasma have demonstrated the presence of an analogous translational setup in mitochondrion [31].

While the aminoacylation requirements of three translationally active compartments in P. falciparum are the same, it was shown that proofreading requirements at the aminoacylation level are not the same for apicoplast and cytoplasm [18]. The apicoplast seem to be tolerant for mischarged tRNAs as it only contains three aaRSs with editing pocket (Table 1) (Fig. 2). The same would not be true for the mitochondrion as it was shown that parasite mitochondrion import charged tRNAs from cytoplasm and hence fidelity would be similar to cytoplasm [29].

aaRSs can also form a highly efficient aminoacylation ensemble called a multi-synthetase complex, which consists of nine aaRSs tethered by scaffold proteins such as P43, P18 and P38 in higher eukaryotes [10]. Bioinformatics analysis of malarial aaRSs identified only one putative cytoplasmic adaptor protein, P43, that could participate in the formation of the multi-synthetase complex [17]. Plasmodium-related apicomplexan Toxoplasma gondii possesses a reduced multi-synthetase complex consisting of P43, methionyl- (MetRS), glutaminyl-, glutamyl-, and tyrosyl- (TyrRS) tRNA synthetases [32]. A similar reduced P43-dependent complex can be expected for malaria parasite.

aaRSs have not been comprehensively studied for their non-canonical functions in malaria parasite though studies suggest that malaria parasite aaRSs have evolved to meet parasite-specific needs [17‐26, 33].

Theoretically, each aaRS is vital for parasite survival and hence, a potential drug target [6, 37‐39]. Over the last half a decade, aaRSs from the malaria parasite have provided many lead inhibitor compounds that can be used to develop species-specific drugs [6, 21, 24, 40‐45]. High content screenings have provided aaRS inhibitors as lead anti-malarials [41, 45]. aaRSs are multidomain enzymes and thus provide the flexibility of designing intervention strategies against multiple sites, viz. aminoacylation pocket, editing site, tRNA binding region and additional domains of non-canonical functionalities. Structural studies of malaria parasite aaRSs by X-ray crystallography have hugely boosted the anti-malarial drug discovery programme. Reported anti-malarials that target aaRSs are listed in Table 2 and key targets and their inhibition are discussed below.

As clear from the above report, malaria parasite aaRSs are not only intriguing for fundamental research, but are also validated drug targets. The apicoplast and mitochondrial translational setups are equally druggable as the cytoplasmic counterpart, and require more studies targeted at exploring their structures and mechanisms. Available inhibitors of bacterial-type organellar aaRSs suggest that their targeting is feasible. Many of the cytoplasmic aaRSs remain to be explored for their structure and physiological roles. Previous studies have hinted at parasite specific adaptations in housekeeping aaRS enzymes, making the predicted extra domains in non-characterized aaRSs, fascinating to study. Moreover, aaRSs are conserved enzymes and thus repurposing of drugs developed against malarial aaRSs can be used to target other eukaryotic pathogens and hence be of much value.