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Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development

Open AccessPublished:December 31, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102860

      Abstract

      Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium) and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start antiparasitic drug development.

      Keywords

      Introduction

      Multiple eukaryotic parasites are responsible for the prevalence and continuous spread of more than a million infections worldwide, thus burdening public health initiatives and the economy (
      • Bloom D.E.
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      • Pham J.S.
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      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ). Effective treatment and control of parasitic diseases needs the development of novel drugs as this process is further impeded by the periodic and inevitable development of drug resistance in parasites and insecticide resistance in vectors. Such barriers to effective treatment could permit the resurgence of parasitic diseases, so there is an urgent need for novel antiparasitic drug scaffolds.
      Eukaryotic parasites can cause diverse diseases of varying severity in hosts, including both animals and humans. The parasites Plasmodium, Toxoplasma gondii, and Cryptosporidium of the phylum Apicomplexa are responsible for causing malaria, toxoplasmosis, and cryptosporidiosis, respectively (
      • Bloom D.E.
      • Cadarette D.
      Infectious Disease Threats in the Twenty-First Century: Strengthening the Global Response.
      ,
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ,
      • Seeber F.
      • Steinfelder S.
      Recent advances in understanding apicomplexan parasites.
      ). Plasmodium species are responsible for the most acute forms of infection in humans after the proliferation and killing of red blood cells, with estimated 241 million malaria cases in 85 endemic countries (
      • Bloom D.E.
      • Cadarette D.
      Infectious Disease Threats in the Twenty-First Century: Strengthening the Global Response.
      ,
      • Pham J.S.
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      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ,
      • Seeber F.
      • Steinfelder S.
      Recent advances in understanding apicomplexan parasites.
      ) (Fig. 1). On the other hand, P. vivax is more geographically widespread and is responsible for causing severe infections and relapses. Toxoplasma gondii, an intracellular parasite, infects animals; however, they are also pathogenic in immunocompromised humans and can cause infections via food-borne illnesses (
      • Webster
      • Dubey J.P.
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      ). This parasite is estimated to persist chronically in 25-30% of the global population (
      • Webster
      • Dubey J.P.
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      ). After the human hosts ingest cysts, sporozoites are released. These sporozoites infect epithelial cells of the intestine, where the sporozoites develop into tachyzoites which multiply and infect more cells (Fig 1). These stages together account for severe symptoms of the disease (
      • Webster
      • Dubey J.P.
      J.P. Toxoplasmosis of Animals and Humans.
      ) (Fig. 1), and limited drugs are available for the treatment of toxoplasmosis. Cryptosporidium affects bovine calves by infecting epithelial cells of the intestine, causing gastrointestinal disease leading to severe and chronic diarrhoea. It can further cause direct or indirect human contamination and have debilitating effects, especially in immunocompromised individuals (
      • Tzipori S.
      • Widmer G.
      A hundred-year retrospective on cryptosporidiosis.
      ). C. hominis and C. parvum are known to cause intestinal infections in humans (Fig. 1).
      Figure thumbnail gr1
      Figure 1Life cycle of eukaryotic parasites in the human host. Trypanosoma brucei gambiense: The infected tsetse fly injects metacyclic trypomastigotes into the host’s bloodstream, where they transform into bloodstream trypomastigotes. The trypomastigotes multiply through binary fission in blood, lymph and other body fluids and are ingested by the tsetse flies that bite the infected human host. Plasmodium spp.: The infected female Anopheles mosquito, while taking a blood meal, injects sporozoites into the human host. The sporozoites infect the liver cells, where they mature into schizonts. Upon the rupture of these schizonts, merozoites are released into the bloodstream. The merozoites infect the red blood cells and mature into trophozoites and, ultimately, schizonts which rupture and release more merozoites into the bloodstream. Some merozoites mature into male and female gametocytes, which the mosquito ingests when it takes bloodmeal from an infected host. Leishmania spp.: The female sandfly injects promastigote into the human bloodstream while taking a blood meal. The promastigotes are phagocytosed by immune cells like macrophages. Once inside the macrophage, the promastigote matures into amastigote. The amastigote multiplies inside these cells through division and, upon rupturing of the cells, infects other cells. The amastigotes are ingested by sandflies when they take blood meal from an infected human. Brugia spp.: The third-stage Brugia larvae enter the human host via bites of Mansonia and Aedes mosquitoes. The larvae develop into adult worms in the human lymphatic system. The adult worms produce microfilariae which enter the bloodstream and are ingested by mosquitoes when they take a blood meal. Toxoplasma gondii: Humans are an intermediate host for Toxoplasma gondii. They are infected by the ingestion of T. gondii cysts, mostly through contaminated food. Upon ingestion, the excystation occurs, and sporozoites are released. These sporozoites infect the intestinal epithelial cells, where the sporozoites develop into tachyzoites. The tachyzoites undergo asexual reproduction and go on to infect more cells. Some of these tachyzoites invade tissue systems, forming bradyzoites through encystation. The bradyzoites can remain undetected in the human host for a long time, becoming active only when the host’s immune status is compromised. Cryptosporidium spp.: The sporulated oocyst of Cryptosporidium can enter the human host through the ingestion of fecally contaminated water or food. The oocyst releases sporozoites in the gastrointestinal (GI) tract. The parasites infect the epithelial cells in the GI tract and undergo asexual (producing schizonts and merozoites) and sexual (producing and macro- and microgametes) life cycles. Following the fertilization of gametes, the zygote produces two kinds of oocysts: thick-walled and thin-walled. The thin-walled oocysts continue infecting cells within the host, whereas the thick-walled oocysts are transmitted into the environment through faeces. Giardia spp.: The Giardia cysts enter human hosts through the oral ingestion of contaminated food or water. The cysts shed their external hard cover in the small intestine and release trophozoites which remain in the lumen. The trophozoites replicate through longitudinal binary fission. Some of them form cysts which are infectious and are passed in the stool.
      Similarly, trypanosomatid parasites Trypanosoma brucei and Leishmania of phylum Euglenozoa are two important parasites that cause severe human diseases (
      • Golding N.
      Review of “Trypanosomes and Trypanosomiasis” by Stefan Magez and Magdalena Radwanska (Editors).
      ,
      • Mann S.
      • Frasca K.
      • Scherrer S.
      • Henao-Martínez A.F.
      • Newman S.
      • Ramanan P.
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      A Review of Leishmaniasis: Current Knowledge and Future Directions.
      ). Trypanosoma brucei causes African trypanosomiasis (sleeping sickness), where they proliferate inside the bloodstream and the human lymphatic system and subsequently affect the central nervous system leading to fatality (Fig. 1). Trypanosoma cruzi causes the chronic and fatal Chagas disease which affects 6-7 million people worldwide. Leishmaniasis is one of the neglected tropical diseases (
      • Mann S.
      • Frasca K.
      • Scherrer S.
      • Henao-Martínez A.F.
      • Newman S.
      • Ramanan P.
      • Suarez J.A.
      A Review of Leishmaniasis: Current Knowledge and Future Directions.
      ). The two Leishmania parasites L. major and L. donovani are responsible for different human infections (Fig. 1) of varying severity, causing cutaneous leishmaniasis and visceral leishmaniasis (kala-azar) with an estimated 700,000 to 1 million new cases annually. Cutaneous leishmaniasis causes skin sores, while visceral leishmaniasis, the serious form of the disease, causes injury to internal organs (
      • Mann S.
      • Frasca K.
      • Scherrer S.
      • Henao-Martínez A.F.
      • Newman S.
      • Ramanan P.
      • Suarez J.A.
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      ). There is a lack of effective and non-toxic drugs for these Trypanosomatid parasites, compounded by the threat of the emergence of parasite resistance to available drugs. Other eukaryotic parasites with anaerobic metabolism, like Giardia (giardiasis), Trichomonas (trichomoniasis), and Entamoeba (amebiasis), are also a public health problem (
      • Cai W.
      • Ryan U.
      • Xiao L.
      • Feng Y.
      Zoonotic giardiasis: an update.
      ). Although nitroimidazole drugs can be used, persistent resistance remains a significant issue. The helminth parasite Brugia malayi causes lymphatic filariasis (elephantiasis) in humans, which is triggered by the immune system’s reaction to adult worms of Brugia malayi and Brugia timori and can lead to permanent disability (

      Newman TE, Juergens AL. Filariasis. [Updated 2022 May 3]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK556012/

      ) (Fig. 1). The treatment of such diverse parasitic diseases urgently requires the identification of robust drug targets and the continued development and design of novel drugs in order to tackle drug resistance. One such family of essential enzymes, the aminoacyl-tRNA synthetases (aaRSs), which tend to be conserved within different parasites, hold promise as a target for antiparasitic drug development and design.

      Aminoacyl-tRNA synthetase (aaRSs) as antiparasitic drug targets

      The aminoacyl-tRNA synthetase (aaRSs) family of enzymes (also known as aminoacyl-tRNA ligases) are ubiquitous since they catalyze the linking of cognate amino acid that corresponds to the tRNA anticodon triplet (
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      , 10 11,
      • Kwon N.H.
      • Fox P.L.
      • Kim S.
      Aminoacyl-tRNA synthetases as therapeutic targets.
      ,
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ) (Fig. 2). The enzymatic reaction comprises two steps; first, aaRS utilizes an ATP molecule to activate the cognate amino acid to generate an active aminoacyl-adenylate intermediate (amino acid-AMP) releasing pyrophosphate (PPi). Second, the cognate tRNA binds to the enzyme, which transfers the amino acid to the 3’ end of tRNA, releasing AMP (
      • Bloom D.E.
      • Cadarette D.
      Infectious Disease Threats in the Twenty-First Century: Strengthening the Global Response.
      ,
      • Cusack S.
      Aminoacyl-tRNA synthetases.
      ,
      • Rubio Gomez M.A.
      • Ibba M.
      Aminoacyl-tRNA synthetases.
      ,
      • Kwon N.H.
      • Fox P.L.
      • Kim S.
      Aminoacyl-tRNA synthetases as therapeutic targets.
      ,
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ). The resulting aminoacylated tRNA, an important substrate for protein translation, is then transported by elongation factors to the ribosome to carry out protein synthesis (Fig. 2). Aminoacyl-tRNA synthetases also contain editing domains that ensure high fidelity of tRNA charging (
      • Kwon N.H.
      • Fox P.L.
      • Kim S.
      Aminoacyl-tRNA synthetases as therapeutic targets.
      ,
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ). The aaRSs reduce errors by hydrolysing misactivated amino acids (pre-transfer to the tRNA) (Fig. 2) and misacylated tRNAs utilizing separate post-transfer editing domains (
      • Rubio Gomez M.A.
      • Ibba M.
      Aminoacyl-tRNA synthetases.
      ). Aminoacyl-tRNA synthetases are thus essential enzymes for protein synthesis (i) for providing aminoacylated-tRNA with the cognate amino acid, and (ii) for ensuring the accuracy of protein translation (Fig. 2). The aaRSs are also important to several other cellular processes beyond their catalytic role - including regulation of transcription, biosynthesis of signal molecules and mitochondrial RNA cleavage (
      • Cusack S.
      Aminoacyl-tRNA synthetases.
      ,
      • Rubio Gomez M.A.
      • Ibba M.
      Aminoacyl-tRNA synthetases.
      ,
      • Kwon N.H.
      • Fox P.L.
      • Kim S.
      Aminoacyl-tRNA synthetases as therapeutic targets.
      ,
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ). The aminoacyl-tRNA synthetases are categorized into two classes, I and II, on the basis of their structure, where class I aaRSs are mostly monomeric and contain the Rossman fold catalytic domain (
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ,
      • Kwon N.H.
      • Fox P.L.
      • Kim S.
      Aminoacyl-tRNA synthetases as therapeutic targets.
      ,
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ) (Fig. 3). On the other hand, class II has a characteristic anti-parallel beta-sheet fold surrounded by alpha-helices. Aminoacyl tRNA synthetases are prominently conserved in their catalytic domain due to their specific function; however, their sequence, structure, and function are seen to be diverse across species. Structural and experimental data show that eukaryotic parasite aaRSs enzymes are excellent drug targets with multiple druggable sites; an ATP-binding pocket, the adjoining amino acid binding pocket, and a tRNA recognition site (210, 11, 12, 13) (Fig. 4). The editing domains that are present on some aaRSs are additional targets for drugs. Some parasite aaRSs are localised to the cytosol (also simply referred to as the cytoplasm) and another subcellular organelle, apicoplast, a vestigial non-photosynthetic plastid. The apicoplast is essential for parasite survival as it plays a crucial role in lipid metabolism in malaria parasites. Additionally, one aaRS from P. falciparum is localised to the mitochondria (
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ). These parasites are dependent on the apicoplast and on the mitochondria and some aaRSs are dual localized in the cytosol and the apicoplast (
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ). Thus, aaRSs in multiple organelles are potential drug targets in parasites.
      Figure thumbnail gr2
      Figure 2The critical role of aminoacyl-tRNA synthetases (aaRSs) in protein synthesis. Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with the corresponding amino acids. These aminoacylated tRNAs bind to elongation factors for transport to the ribosome. At the A-site of ribosome, aminoacylated tRNAs bind and recognize the codon on the mRNA by base pairing. Subsequently, the nascent peptide chain gets transferred from a tRNA which is at the ribosome's P-site, adding one amino acid to the chain. Finally, the A-site then frees up as the ribosomal subunits temporarily dissociate and migrate on the mRNA, removing the tRNA from the P-site and placing the tRNA formerly in the A-site. They now now carry the nascent protein chain in the P-site. Finally, a stop codon at the A-site triggers a release factor which leads to dissociation of the ribosome and release of the completed protein chain.
      Figure thumbnail gr3
      Figure 3Classification of aminoacyl-tRNA synthetases (aaRSs) into class 1 and II and subclasses a, b, c and d and inhibitors known for each. The parasites for which inhibitors have been developed against specific aaRSs are listed; Cp: Cryptosporidium parvum, Tb: Trypanosoma brucei, Tc: Trypanosoma cruzi, Pf: Plasmodium falciparum, Pv: Plasmodium vivax, Tg: Toxoplasma gondii, Gl: Giardia lamblia, Bm: Brugia malayi, Lm: Leishmania major, Ld: Leishmania donovani.
      Figure thumbnail gr4
      Figure 4The potential druggable sites on aminoacyl-tRNA synthetases (aaRSs). The sites for likely interaction between aaRS (belonging to either class I and II) and a compound/inhibitor/drug which can inhibit the enzyme activity are highlighted; 1. ATP binding site, 2. anticodon-binding site, 3. amino-acid binding site, 4. editing site, and 5. auxiliary site.
      Aminoacyl-tRNA synthetases are well-established drug targets for antibacterial and antifungal activities (
      • Bouz G.
      • Zitko J.
      Inhibitors of aminoacyl-tRNA synthetases as antimycobacterial compounds: An up-to-date review.
      ,
      • Nakama T.
      • Nureki O.
      • Yokoyama S.
      Structural Basis for the Recognition of Isoleucyl-Adenylate and an Antibiotic, Mupirocin, by Isoleucyl-TRNA Synthetase.
      ,
      • Rock F.L.
      • Mao W.
      • Yaremchuk A.
      • Tukalo M.
      • Crépin T.
      • Zhou H.
      • Zhang Y.-K.
      • Hernandez V.
      • Akama T.
      • Baker S.J.
      • et al.
      An Antifungal Agent Inhibits an Aminoacyl-TRNA Synthetase by Trapping TRNA in the Editing Site.
      ). Several inhibitors have been developed, of which an antibiotic, the IleRS inhibitor mupirocin, and the LeuRS inhibitor tavaborole, which is an antifungal, are approved for clinical treatment of methicillin-resistant Staphylococcus aureus (MRSA) and fungal-infective onychomycosis (
      • Bouz G.
      • Zitko J.
      Inhibitors of aminoacyl-tRNA synthetases as antimycobacterial compounds: An up-to-date review.
      ,
      • Nakama T.
      • Nureki O.
      • Yokoyama S.
      Structural Basis for the Recognition of Isoleucyl-Adenylate and an Antibiotic, Mupirocin, by Isoleucyl-TRNA Synthetase.
      ,
      • Rock F.L.
      • Mao W.
      • Yaremchuk A.
      • Tukalo M.
      • Crépin T.
      • Zhou H.
      • Zhang Y.-K.
      • Hernandez V.
      • Akama T.
      • Baker S.J.
      • et al.
      An Antifungal Agent Inhibits an Aminoacyl-TRNA Synthetase by Trapping TRNA in the Editing Site.
      ). As antiparasitic drug targets, aaRSs have promising potential because the parasite, like all life forms, is essentially reliant on protein translation. Moreover, due to the specific requirement of active and fast proliferation, parasites are sensitive towards disruption in the critical machinery of protein translation.

      Inhibitors against parasite aminoacyl-tRNA synthetases

      In this work, we summarize advancements in exploring parasitic aminoacyl-tRNA synthetases as drug targets by consolidating experimental data on biochemical characterization, validation, inhibitor development, and three-dimensional structural dissections for all aminoacyl-tRNA synthetases (in alphabetical order starting from alanyl-tRNA synthetase) from seven eukaryotic pathogens Brugia spp., Cryptosporidium spp., Giardia spp., Leishmania spp., Plasmodium spp., Toxoplasma gondii and Trypanosoma spp. This work will facilitate research integration and provide new directions for anti-pathogen drug discovery.

      Alanyl-tRNA synthetase (AlaRS)

      A single nuclear gene in the parasite Plasmodium encodes for alanyl-tRNA synthetase (AlaRS), giving two proteins with different localizations, i.e., the cytosol and the apicoplast (
      • Jackson K.E.
      • Pham J.S.
      • Kwek M.
      • De Silva N.S.
      • Allen S.M.
      • Goodman C.D.
      • McFadden G.I.
      • Ribas de Pouplana L.
      • Ralph S.A.
      Dual targeting of aminoacyl-tRNA synthetases to the apicoplast and cytosol in Plasmodium falciparum.
      ,
      • Khan S.
      • Sharma A.
      • Jamwal A.
      • Sharma V.
      • Pole A.K.
      • Thakur K.K.
      • Sharma A.
      Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P.
      ). Plasmodium AlaRS also contains a second active site with editing activity since glycine and serine are the most common mischarging events due to their similar size (
      • Sokabe M.
      • Okada A.
      • Yao M.
      • Nakashima T.
      • Tanaka I.
      Molecular basis of alanine discrimination in editing site.
      ). AlaRS presents an opportunity to target aminoacylation and the editing activities occurring in two distinct parasite compartments. Several potential P. falciparum AlaRS (PfAlaRS) inhibitors were screened in silico using homology models, revealing one compound A5, (4-{2-nitro-1-propenyl}-1,2-benzenediol,), that was validated to inhibit parasite growth at micromolar levels while producing sparse cytotoxicity (Table 1) (Fig. 3, 4, 5) (
      • Khan S.
      • Sharma A.
      • Jamwal A.
      • Sharma V.
      • Pole A.K.
      • Thakur K.K.
      • Sharma A.
      Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P.
      ). In another study, a pre-validated MNP library (marine natural product; a specific group of bacterial extract pre-fractions with demonstrated activity against Leishmania) was used and four potential Leishmania major AlaRS inhibitors decreased the overall tRNA-AlaRS aminoacylation activity (
      • Kelly P.
      • Hadi-Nezhad F.
      • Liu D.Y.
      • Lawrence T.J.
      • Linington R.G.
      • Ibba M.
      • Ardell D.H.
      Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens.
      ,
      • Schulze C.J.
      • Bray W.M.
      • Woerhmann M.H.
      • Stuart J.
      • Lokey R.S.
      • et al.
      Function-first" lead discovery: mode of action profiling of natural product libraries using image-based screening.
      ). The three promising mixes (1881C, 2059D, and 2096D) affected aminoacylation with inhibition ranging from 80% to 99% (Table 1). Interestingly, cross-reactivity is also seen with Trypanosoma cruzi AlaRS, which indicates a broad-spectrum potential and no effect on the human homolog (
      • Kelly P.
      • Hadi-Nezhad F.
      • Liu D.Y.
      • Lawrence T.J.
      • Linington R.G.
      • Ibba M.
      • Ardell D.H.
      Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens.
      ). AlaRS is yet to be explored in four of the seven pathogens Brugia, Cryptosporidium, Giardia, and Toxoplasma, discussed in this review (
      • Kelly P.
      • Hadi-Nezhad F.
      • Liu D.Y.
      • Lawrence T.J.
      • Linington R.G.
      • Ibba M.
      • Ardell D.H.
      Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens.
      ). The first three-dimensional structure of parasitic AlaRS remains to be determined; however, similar to Plasmodium, homology-modelled structures from bacteria and fungi could be explored via in silico docking.
      Table 1Inhibitors developed for aminoacyl-tRNA synthetases (aaRSs) against eukaryotic parasites uptill June 2022. #Predicted binding mechanism: The inhibitor has been experimentally validated to inhibit aminoacylation/pre-transfer editing activity of the aaRS enzyme and thus is predicted to bind at the active/pre-transfer-editing site. However, structural interpretation or validation is not available
      aaRSsInhibitor(s)ParasiteBinding mechanismReference
      AlaRSA3; A5Plasmodium falciparumActive site #Khan et al., 2011
      Natural marine product library (1881C, 2059D and 2096D)Leishmania major

      Trypanosoma cruzi
      Active site #Kelly et al., 2020
      ArgRSheminPlasmodium falciparumNot knownJain et al., 2016
      AsnRSVariolin BBrugia malayiActive site #Sukuru et al., 2006
      Natural product extracts (L-aspartate-B-hydroxomateBrugia malayiPre-transfer editing site #Danel et al., 2011
      TAM B (from Streptomyces sp. 17944 extracts)Brugia malayiPre-transfer editing site #Yu et al., 2011
      WS936D (from Streptomyces sp. 9078 extracts)Brugia malayiPre-transfer editing site #Yu et al., 2012
      Adipostatins A-D (from Streptomyces sp. 4875 extracts)Brugia malayiPre-transfer editing site #Rateb et al., 2015
      IleRSMupirocinPlasmodium falciparumActive siteIstvan et al., 2011
      ThiaisoleucinePlasmodium falciparumActive site #Istvan et al., 2011
      NSC70422 (Ile-AMP analog)Trypanosoma bruceiActive site #Cestari and Stuart 2013
      HisRS15 fragmentsTrypanosoma cruziAuxiliary siteKoh et al., 2015
      LeuRSBenzoxaborole derivativesTrypanosoma bruceiPost-transfer editing siteDing et al., 2011
      2-Pyrrolinone derivativesTrypanosoma bruceiActive site (predicted; 3D model of synthetic active site)Zhao et al., 2012
      N-(4-sulfamoylphenyl)thioureas derivativesTrypanosoma bruceiActive site (predicted; 3D model of synthetic active site)Zhang et al., 2013
      3,5-dicaffeoylquinic acid and derivativesGiardia lambliaActive site #Zhang et al., 2012
      AN2690Leishmania donovaniActive site #Minhas et al., 2018, Tandon et al., 2020
      Benzoxaborole derivatives (AN6426, AN8432)Plasmodium falciparumEditing active siteSonoiki et al., 2016
      Benzoxaborole derivative (AN6426)Cryptosporidium parvumEditing active sitePalencia et al., 2016
      Toxoplasma gondiiEditing active sitePalencia et al., 2016
      series of α-phenoxy-N sulfonylphenyl acetamides (Compound 28g)Trypanosoma bruceiActive site (predicted; 3D model of synthetic active site)Xin et al., 2020
      Amides (Compound 74 and 91)Trypanosoma bruceiActive site (predicted; 3D model of synthetic active site)Li et al., 2021
      LysRSCladosporinPlasmodium falciparumActive siteKhan et al., 2013
      Compound 5Plasmodium falciparumActive siteBaragana et al., 2019
      Cryptosporidium parvumActive site
      ASP3026 (anaplastic lymphoma kinase inhibitor)Plasmodium falciparumActive siteZhou et al., 2020
      Cladosporin derivatives, CL-2Plasmodium falciparumActive siteBabbar et al., 2021a
      Cladosporin derivatives, Cla-B, Cla-CPlasmodium falciparumActive siteBabbar et al., 2021b
      LysRS2 ApicoplastM-26, M-37Plasmodium falciparumActive site #Hoen et al., 2013
      MetRSAminoquinolone derivatives (Compound 1)Trypanosoma bruceiActive site (predicted model)Shibata et al., 2011
      Urea-based inhibitor series (Compound 2 and 26)Trypanosoma bruceiActive site (predicted model)Shibata et al., 2012
      Series of urea-based inhibitors (UBIs)Trypanosoma bruceiActive siteKoh et al., 2014
      MSMLR library of small molecules: 12 compoundsTrypanosoma bruceiActive site #Pedro-Rosa et al., 2015
      a fluro-imidazopyridine (Compound-1717)Giardia intestinalis

      Giardia lamblia
      Not knownRanade et al., 2015

      Michaels et al., 2020
      REP3123 and REP8839,

      C1, C2, C3
      Plasmodium falciparumActive site (predicted; in silico)Hussain et al., 2015
      Ursolic acidantileishmanial

      antitrypanosomal
      Active site (predicted; in silico)Labib et al., 2016
      imidazopyridine-containing compounds (2093, 2114, 2259)Cryptosporidium parvum

      Cryptosporidium hominis
      Active site #Buckner et al., 2019
      Compound 1 and 26Trypanosoma bruceiActive siteZhang et al., 2020
      DDD806905Leishmania majorAllosteric ligand binding siteTorrie et al., 2020
      PheRSBicyclic azetidines

      BRD7929, BRD8494
      Cryptosporidium parvumActive site and an auxiliary siteFunkhouser-Jones et al., 2020
      Bicyclic azetidine (BRD7929)Cryptosporidium parvumActive site #Vinayak et al., 2020
      Bicyclic azetidine (BRD1389)Plasmodium vivaxActive siteSharma et al., 2020
      Bicyclic azetidine (BRD7929)Toxoplasma gondiiActive siteRadke et al., 2022
      Plasmodium falciparumActive siteSharma et al., 2022
      ProRSHalofuginonePlasmodium falciparumActive siteKeller et al., 2012, Zhou et al., 2013
      Halofuginol (derivative of halofuginone)Plasmodium falciparum

      Plasmodium berghei
      Active siteHerman et al., 2015
      HalofuginoneToxoplasma gondiiActive siteJain et al., 2015
      Febrifugine and Halofuginone derivativesPlasmodium falciparumActive siteJain et al., 2017
      1-(pyridin-4-yl) pyrrolidin-2-one derivativesPlasmodium falciparumOkaniwa et al., 2021
      Double drugging: halofuginone and ATP analog L95Toxoplasma gondiiActive siteYogavel et al., 2022
      ThrRSBorrelidinPlasmodium falciparumActive site #Otoguro et al., 2003
      Plasmodium yoeliiActive site #
      Borrelidin analogsPlasmodium falciparumActive site #Suguwara et al., 2013
      T1-T11Plasmodium falciparumActive site #Khan et al., 2011
      BorrelidinTrypanosoma bruceiActive site #Kalidas et al., 2014
      BorrelidinLeishmania donovaniActive site #Chadha et al., 2018
      Natural marine product library (1438C, 1758C, 2059D and 2096B)Leishmania majorActive site #Kelly et al., 2020
      TrpRS apicoplastindolmycinPlasmodium falciparumActive site #Pasaje et al., 2016
      TyrRSFisetinLeishmania major

      Leishmania donovani
      Active siteLarson et al., 2011

      Anand et al., 2016
      ML901Plasmodium falciparumActive siteXie et al., 2022
      Figure thumbnail gr5
      Figure 5Inhibitors developed and three-dimensional structures of aminoacyl-tRNA synthetases (aaRSs) from eukaryotic parasites (January 2006 till June 2022). The cytoplasmic aaRSs are shown in orange boxes where AlaRS corresponds to Alanyl-tRNA synthetase. The apicoplast aaRSs are shown in purple boxes. Green denotes where three-dimensional structure is known in complex with an inhibitor.

      Arginyl-tRNA synthetase (ArgRS)

      P. falciparum cytoplasmic arginyl-tRNA synthetase (PfArgRS) is a class I monomeric enzyme. Hemin, an iron-containing porphyri, binds PfArgRS and inhibits its aminoacylation activity with IC50 of ∼2μM (Table 1) (
      • Jain V.
      • Yogavel M.
      • Sharma A.
      Dimerization of Arginyl-tRNA Synthetase by Free Heme Drives Its Inactivation in Plasmodium falciparum.
      ). IC50, a half-maximal inhibitory concentration, measures the potency of a compound in inhibiting enzyme activity. Hemin induced a dimeric form of PfArgRS, making it inactive and thus incapable of recognizing the cognate tRNAArg. Increased levels of hemin, particularly in chloroquine-treated malaria parasites, led to decreased levels of tRNAArg. At the same time, the human ArgRS can recognize the tRNAArg even in the presence of hemin. However, the binding site of hemin on the three-dimensional structure of PfArgRS is unknown. ArgRS has been explored only in P. falciparum and is among the least studied aaRSs. The structure of PfArgRS can be utilized for homology modelling and docking in the other six pathogens discussed, and aminoacylation activity inhibition can be tested using established approaches (
      • Jain V.
      • Yogavel M.
      • Sharma A.
      Dimerization of Arginyl-tRNA Synthetase by Free Heme Drives Its Inactivation in Plasmodium falciparum.
      ).

      Asparaginyl-tRNA synthetase (AsnRS)

      The cytoplasmic asparaginyl-tRNA synthetase (AsnRS) was first characterized as a significant druggable target in the nematode Brugia malayi (
      • Kron M.
      • Petridis M.
      • Milev Y.
      • Leykam J.
      • Härtlein M.
      Expression, localization and alternative function of cytoplasmic asparaginyl-tRNA synthetase in Brugia malayi.
      ). BmAsnRS catalyzes the production of diadenosine triphosphate and binding studies involving BmAnsRS show a possible role of AsnRS in modulating immune cell function (
      • Kron M.A.
      • Wang C.
      • Vodanovic-Jankovic S.
      • Howard O.M.
      • Kuhn L.A.
      Interleukin-8-like activity in a filarial asparaginyl-tRNA synthetase.
      ,
      • Ramirez B.L.
      • Howard O.M.
      • Dong H.F.
      • Edamatsu T.
      • Gao P.
      • Hartlein M.
      • Kron M.
      Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2.
      ). The solution structure of Brugia malayi AsnRS revealed a lysine-rich region in its N-terminus, which interacts with tRNA (
      • Crepin T.
      • Peterson F.
      • Haertlein M.
      • Jensen D.
      • Wang C.
      • Cusack S.
      • Kron M.
      A hybrid structural model of the complete Brugia malayi cytoplasmic asparaginyl-tRNA synthetase.
      ).
      In silico docking followed by experimental testing of docked compounds against B. malayi AsnRS revealed 45 compounds with mid-micromolar IC50s (
      • Sukuru S.C.
      • Crepin T.
      • Milev Y.
      • Marsh L.C.
      • Hill J.B.
      • Anderson R.J.
      • Morris J.C.
      • Rohatgi A.
      • O'Mahony G.
      • Grøtli M.
      • Danel F.
      • Page M.G.
      • Härtlein M.
      • Cusack S.
      • Kron M.A.
      • Kuhn L.A.
      Discovering new classes of Brugia malayi asparaginyl-tRNA synthetase inhibitors and relating specificity to conformational change.
      ). A marine natural product called Variolin B inhibited ∼50% of Brugia AsnRS activity at 50μM concentration (Table 1) (
      • Sukuru S.C.
      • Crepin T.
      • Milev Y.
      • Marsh L.C.
      • Hill J.B.
      • Anderson R.J.
      • Morris J.C.
      • Rohatgi A.
      • O'Mahony G.
      • Grøtli M.
      • Danel F.
      • Page M.G.
      • Härtlein M.
      • Cusack S.
      • Kron M.A.
      • Kuhn L.A.
      Discovering new classes of Brugia malayi asparaginyl-tRNA synthetase inhibitors and relating specificity to conformational change.
      ). BmAsnRS can recognize and edit misacylation before the transfer to tRNA, and thus a “pre-transfer assay” identifies compounds and allows for screening inhibitors. Natural product extracts (L-aspartate-B-hydroxamate, an asparagine analog) were identified using this approach (Table 1) (
      • Danel F.
      • Caspers P.
      • Nuoffer C.
      • Härtlein M.
      • Kron M.A.
      • Page M.G.
      Asparaginyl-tRNA synthetase pre-transfer editing assay.
      ). TAM B (isolated from Streptomyces sp 17944 extracts), belonging to the tirandamycins class of compounds was shown to kill adult B. malayi parasites (
      • Yu Z.
      • Vodanovic-Jankovic S.
      • Ledeboer N.
      • Huang S.X.
      • Rajski S.R.
      • Kron M.
      • Shen B.
      Tirandamycins from Streptomyces sp. 17944 inhibiting the parasite Brugia malayi asparagine tRNA synthetase.
      ) (Fig. 1). Another study from the same group revealed a compound WS936D from Streptomyces sp 9078 extracts (WS9326A derivatives) that inhibits B. malayi AsnRS aminoacylation, thereby killing adult B. malayi parasites at low nanomolar concentrations (
      • Yu Z.
      • Vodanovic-Jankovic S.
      • Kron M.
      • Shen B.
      New WS9326A congeners from Streptomyces sp. 9078 inhibiting Brugia malayi asparaginyl-tRNA synthetase.
      ). These compounds did show selectivity, as no notable cytotoxicity was observed in human hepatic cells (
      • Yu Z.
      • Vodanovic-Jankovic S.
      • Kron M.
      • Shen B.
      New WS9326A congeners from Streptomyces sp. 9078 inhibiting Brugia malayi asparaginyl-tRNA synthetase.
      ). Another study discovered four novel alkylresorcinols called adipostatins A, B, C, and D (from active strain Streptomyces sp. 4875), which all inhibit B. malayi AsnRS with apparent IC50s estimated at 15μm for adipostatins A, B and C and 30μM for adipostatin D (
      • Rateb M.E.
      • Yang D.
      • Vodanovic-Jankovic S.
      • Yu Z.
      • Kron M.A.
      • Shen B.
      Adipostatins A-D from Streptomyces sp. 4875 inhibiting Brugia malayi asparaginyl-tRNA synthetase and killing adult Brugia malayi parasites.
      ). They also kill adult parasites in vitro without any notable general cytotoxicity in host cells (Table 1) (
      • Rateb M.E.
      • Yang D.
      • Vodanovic-Jankovic S.
      • Yu Z.
      • Kron M.A.
      • Shen B.
      Adipostatins A-D from Streptomyces sp. 4875 inhibiting Brugia malayi asparaginyl-tRNA synthetase and killing adult Brugia malayi parasites.
      ). AsnRS remains to be explored in six of the seven pathogens discussed, and only apo structure is known from Brugia. The efficient high-throughput screening platform with recombinant B. malayi could be reoriented for other pathogens (
      • Rateb M.E.
      • Yang D.
      • Vodanovic-Jankovic S.
      • Yu Z.
      • Kron M.A.
      • Shen B.
      Adipostatins A-D from Streptomyces sp. 4875 inhibiting Brugia malayi asparaginyl-tRNA synthetase and killing adult Brugia malayi parasites.
      ).

      Histidyl-tRNA synthetase (HisRS)

      The first structures of eukaryotic histidyl-tRNA synthetase (HisRS) were determined from Trypanosoma cruzi and T. brucei (
      • Merritt E.A.
      • Arakaki T.L.
      • Gillespie J.R.
      • Larson E.T.
      • Kelley A.
      • Mueller N.
      • Napuli A.J.
      • Kim J.
      • Zhang L.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Buckner F.S.
      • van Voorhis W.C.
      • Hol W.G.
      Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.
      ). While the T. cruzi structure was apo, T. brucei HisRS is a complex with L-His and histidyladenylate, wherein the binding interactions are vastly distinct from bacterial or human homologs. Upon L-His binding, a rearrangement occurs in the active site, which was not significant during the formation of the first product histidyladenylate after L-His reacts with ATP (
      • Merritt E.A.
      • Arakaki T.L.
      • Gillespie J.R.
      • Larson E.T.
      • Kelley A.
      • Mueller N.
      • Napuli A.J.
      • Kim J.
      • Zhang L.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Buckner F.S.
      • van Voorhis W.C.
      • Hol W.G.
      Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.
      ). Fifteen newly identified fragments (from a library of 680) are structurally bound in a new “fragment-binding pocket” in T. cruzi HisRS, which is essentially a narrow groove proximal to the bound L-His (Table 1) (
      • Koh C.Y.
      • Siddaramaiah L.K.
      • Ranade R.M.
      • Nguyen J.
      • Jian T.
      • Zhang Z.
      • Gillespie J.R.
      • Buckner F.S.
      • Verlinde C.L.
      • Fan E.
      • Hol W.G.
      A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.
      ,
      • Verlinde C.L.
      • Fan E.
      • Shibata S.
      • Zhang Z.
      • Sun Z.
      • Deng W.
      • et al.
      Fragment-based cocktail crystallography by the medical structural genomics of pathogenic protozoa consortium.
      ). It is suggested that the fragments likely compete for binding with ATP or the product HAMP or possibly both, causing inhibition of HisRS. This pocket can potentially achieve the desired “selectivity” since this pocket is absent in human HisRS. However, low affinities of these fragments warranted very high concentrations, which is not desirable; thus, enzyme inhibition has to be considered with caution (
      • Koh C.Y.
      • Siddaramaiah L.K.
      • Ranade R.M.
      • Nguyen J.
      • Jian T.
      • Zhang Z.
      • Gillespie J.R.
      • Buckner F.S.
      • Verlinde C.L.
      • Fan E.
      • Hol W.G.
      A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.
      ). Nevertheless, these fragments can be utilized as a starting point for developing inhibitors of trypanosomatid HisRS and for the other six pathogens discussed, in which HisRS has not been explored as a drug target yet. Available structures of Trypanosoma cruzi and T. brucei HisRS can be explored to address the significance of the “fragment binding site”.

      Isoleucyl-tRNA synthetase (IleRS)

      Mupirocin, an established drug against bacterial isoleucyl-tRNA synthetase (IleRS), inhibits the P. falciparum growth in the blood stage in a potent nanomolar range (
      • Istvan E.S.
      • Dharia N.V.
      • Bopp S.E.
      • Gluzman I.
      • Winzeler E.A.
      • Goldberg D.E.
      Validation of isoleucine utilization targets in Plasmodium falciparum.
      ) (Fig. 1). This study analyzed P. falciparum parasites resistant to mupirocin that have mutations in their apicoplast IleRS, validating it as a drug target (
      • Istvan E.S.
      • Dharia N.V.
      • Bopp S.E.
      • Gluzman I.
      • Winzeler E.A.
      • Goldberg D.E.
      Validation of isoleucine utilization targets in Plasmodium falciparum.
      ). Also, the cytoplasmic Plasmodium IleRS was inhibited by isoleucine analog thiaisoleucine. Both mupirocin and thiaisoleucine showed the elimination of cultured parasites in vivo (Table 1) (
      • Istvan E.S.
      • Dharia N.V.
      • Bopp S.E.
      • Gluzman I.
      • Winzeler E.A.
      • Goldberg D.E.
      Validation of isoleucine utilization targets in Plasmodium falciparum.
      ). Twenty small molecules were identified from compounds available from National Cancer Institute that were similar to the intermediate Ile-AMP. These could kill T. brucei forms in the bloodstream (Table 1) (
      • Cestari I.
      • Stuart K.
      Inhibition of isoleucyl-tRNA synthetase as a potential treatment for human African Trypanosomiasis.
      ) (Fig. 1). Compound NSC70422 notably showed good selectivity against mammalian cells and cured T. brucei-infected mice with low cell toxicity as it acted as a competitive inhibitor of the TcIleRS (
      • Cestari I.
      • Stuart K.
      Inhibition of isoleucyl-tRNA synthetase as a potential treatment for human African Trypanosomiasis.
      ). IleRS is yet to be explored in five of seven pathogens. The three-dimensional structure of parasitic IleRS is unavailable, but structures of bacterial and fungal IleRS can be used for in silico docking.

      Leucyl-tRNA synthetase (LRS)

      A modelled structure of T. brucei CP1 (Connective Polypeptide 1) (editing) domain based upon C. albicans LeuRS was utilized to develop several benzoxaborole compounds since AN2690 (5-fluro-1.3-dihydro-1-hydroxy-2,1-benzoxaborole) has been used as an antifungal successfully against Candida albicans (
      • Ding D.
      • Meng Q.
      • Gao G.
      • Zhao Y.
      • Wang Q.
      • Nare B.
      • Jacobs R.
      • Rock F.
      • Alley M.R.
      • Plattner J.J.
      • Chen G.
      • Li D.
      • Zhou H.
      Design, synthesis, and structure-activity relationship of Trypanosoma brucei leucyl-tRNA synthetase inhibitors as antitrypanosomal agents.
      ). These compounds also inhibited T. brucei LeuRS aminoacylation activity by targeting the LeuRS editing site. Further, ex vivo growth was inhibited at low micromolar IC50s with negligible host toxicity (Table 1) (
      • Ding D.
      • Meng Q.
      • Gao G.
      • Zhao Y.
      • Wang Q.
      • Nare B.
      • Jacobs R.
      • Rock F.
      • Alley M.R.
      • Plattner J.J.
      • Chen G.
      • Li D.
      • Zhou H.
      Design, synthesis, and structure-activity relationship of Trypanosoma brucei leucyl-tRNA synthetase inhibitors as antitrypanosomal agents.
      ). Similarly, the CP1 domain of Leishmania donovani LeuRS was critical for editing the mischarged tRNA and aminoacylation activity (
      • Tandon S.
      • Manhas R.
      • Tiwari N.
      • Munde M.
      • Vijayan R.
      • Gourinath S.
      • Muthuswami R.
      • Madhubala R.
      Deciphering the interaction of benzoxaborole inhibitor AN2690 with connective polypeptide 1 (CP1) editing domain of Leishmania donovani leucyl-tRNA synthetase.
      ). AN2690 also had a low-to-moderate affinity to LdLeuRS (Kd = 30μM) as it inhibits parasite growth in vitro and in vivo in BALB/c mice while exhibiting negligible toxicity in host cells (
      • Manhas R.
      • Tandon S.
      • Sen S.S.
      • Tiwari N.
      • Munde M.
      • Madhubala R.
      Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase.
      ). Zhao et al., 2012 revealed a novel set of compounds with a 2-pyrrolinone scaffold by in silico screening (SPECS chemical library) of modelled TbLeuRS synthetic active site (Table 1) (
      • Zhao Y.
      • Wang Q.
      • Meng Q.
      • Ding D.
      • Yang H.
      • Gao G.
      • Li D.
      • Zhu W.
      • Zhou H.
      Identification of Trypanosoma brucei leucyl-tRNA synthetase inhibitors by pharmacophore- and docking-based virtual screening and synthesis.
      ). Another novel class of TbLeuRS inhibitors (N-(4-sulfamoylphenyl)thioureas), similarly targeting a 3D in silico model of the synthetic active site, were identified by screening and then modifying the small, targeted library of potential aaRSs inhibitors (
      • Zhang F.
      • Du J.
      • Wang Q.
      • Hu Q.
      • Zhang J.
      • Ding D.
      • Zhao Y.
      • Yang F.
      • Wang E.
      • Zhou H.
      Discovery of N-(4-sulfamoylphenyl)thioureas as Trypanosoma brucei leucyl-tRNA synthetase inhibitors.
      ). They mimic the intermediate aminoacyl-AMP; however, most compounds have poor permeability and poor inhibitions except compound 59, which had IC50 = 1.1μM. In a separate study, 3,5-dicaffeoylquinic acid and its derivatives, including 3,5-dicaffeoylquinic acid propyl ester (viz., compounds 2, 3 and 4), were good at killing the G. lamblia parasites with IC₅₀ values of 1.79, 5.51 and 2.56 μM respectively (Table 1) (
      • Zhang Y.H.
      • Xue M.Q.
      • Bai Y.C.
      • Yuan H.H.
      • Zhao H.L.
      • Lan M.B.
      3,5-Dicaffeoylquinic acid isolated from Artemisia argyi and its ester derivatives exert anti-leucyl-tRNA synthetase of Giardia lamblia (GlLeuRS) and potential anti-giardial effects.
      ) and the derivatives notably exhibited reduced toxicity and enhanced activity. These were an aqueous ethanol extract of dicaffeoylquinic acids containing Artemisia argyi (
      • Zhang Y.H.
      • Xue M.Q.
      • Bai Y.C.
      • Yuan H.H.
      • Zhao H.L.
      • Lan M.B.
      3,5-Dicaffeoylquinic acid isolated from Artemisia argyi and its ester derivatives exert anti-leucyl-tRNA synthetase of Giardia lamblia (GlLeuRS) and potential anti-giardial effects.
      ). Similarly, in a separate study, two 3-aminomethyl compounds (termed AN6426 and AN8432) were potent against multidrug-resistant P. falciparum W2 strain with 50% inhibitory concentration (IC50s: 310 nM and 490 nM) (Table 1) (
      • Sonoiki E.
      • Palencia A.
      • Guo D.
      • Ahyong V.
      • Dong C.
      • Li X.
      • Hernandez V.S.
      • Zhang Y.K.
      • Choi W.
      • Gut J.
      • Legac J.
      • Cooper R.
      • Alley M.R.
      • Freund Y.R.
      • DeRisi J.
      • Cusack S.
      • Rosenthal P.J.
      Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.
      ). The treatment was effective against P. berghei infection after oral administration once a day for four days in a murine model.
      AN6426 also inhibits growth in human cells for C. parvum and T. gondii. Similar inhibition activity is seen against Cryptosporidium and Toxoplasma parasites as it targets the LeuRS editing site (Table 1) (
      • Palencia A.
      • Liu R.J.
      • Lukarska M.
      • Gut J.
      • Bougdour A.
      • Touquet B.
      • Wang E.D.
      • Li X.
      • Alley M.R.
      • Freund Y.R.
      • Rosenthal P.J.
      • Hakimi M.A.
      • Cusack S.
      Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.
      ). In T. gondii, it prevents the proliferation of Toxoplasma parasites in human fibroblasts at mid-micromolar concentrations. Also, their activity intensifies in the presence of amino acid norvaline which can be mischarged to the end of tRNALeu and is a substrate for post-transfer editing by LeuRS. AN6426 and tRNALeu form a covalent adduct in the enzyme’s editing site, either blocking aminoacylation if it interacts with the tRNALeu acceptor end or blocking post-transfer editing if it interacts, for example, with ATP (
      • Palencia A.
      • Liu R.J.
      • Lukarska M.
      • Gut J.
      • Bougdour A.
      • Touquet B.
      • Wang E.D.
      • Li X.
      • Alley M.R.
      • Freund Y.R.
      • Rosenthal P.J.
      • Hakimi M.A.
      • Cusack S.
      Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.
      ). Recent studies identified a series of α-phenoxy-N sulfonylphenyl acetamides as inhibitors of T. brucei LeuRS using a 3D in silico model of the synthetic active site. Compound 28g was the most potent, with an IC50 of 0.70 μM, and potency higher by 250-fold than the starting hit, i.e. compound 1 (Table 1) (
      • Xin W.
      • Li Z.
      • Wang Q.
      • Du J.
      • Zhu M.
      • Zhou H.
      Design and synthesis of α-phenoxy-N-sulfonylphenyl acetamides as Trypanosoma brucei Leucyl-tRNA synthetase inhibitors.
      ). In a subsequent study, utilizing the initial hit compound thiourea ZCL539, a follow-up series of amides were designed and synthetized and proven effectual against T. brucei LeuRS. Compounds 74 and 91 were the most potent compounds with IC50 of 0.24 and 0.25 μM (about 700-fold higher potent than the starting hit) (Table 1) (
      • Li Z.
      • Xin W.
      • Wang Q.
      • Zhu M.
      • Zhou H.
      Design and synthesis of N-(3-sulfamoylphenyl)amides as Trypanosoma brucei leucyl-tRNA synthetase inhibitors.
      ). LeuRS and its inhibitors that target the editing active site are well-studied in all seven pathogens discussed except Brugia and apo structure is available from P. falciparum for in silico approaches.

      Lysyl-tRNA synthetase (LysRS)

      The natural product cladosporin, a fungal secondary metabolite, is active against blood and liver stage growth of P. falciparum at a nanomolar range. Cladosporin targets the cytosolic lysyl-tRNA synthetase (LysRS), as parasites that overexpress LysRS are resistant to cladosporin (
      • Hoepfner D.
      • McNamara C.W.
      • Lim C.S.
      • Studer C.
      • Riedl R.
      • Aust T.
      • McCormack S.L.
      • Plouffe D.M.
      • Meister S.
      • Schuierer S.
      • Plikat U.
      • Hartmann N.
      • Staedtler F.
      • Cotesta S.
      • Schmitt E.K.
      • Petersen F.
      • Supek F.
      • Glynne R.J.
      • Tallarico J.A.
      • Porter J.A.
      • Fishman M.C.
      • Bodenreider C.
      • Diagana T.T.
      • Movva N.R.
      • Winzeler E.A.
      Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.
      ). The apo structure of LysRS from Entamoeba histolytica in a complex with small ligands shows that conformational changes occur upon lysine binding in the catalytic domain, similar to the earlier reports on bacterial LysRS structures (
      • Bonnefond L.
      • Castro de Moura M.
      • Ribas de Pouplana L.
      • Nureki O.
      Crystal structures of Entamoeba histolytica lysyl-tRNA synthetase reveal conformational changes upon lysine binding and a specific helix bundle domain.
      ). Three-dimensional structures of PfLysRS in apo form and complex with substrates show cladosporin interacting with the ATP binding site as it mimics the natural substrate adenosine (
      • Khan S.
      • Garg A.
      • Camacho N.
      • Van Rooyen J.
      • Kumar Pole A.
      • Belrhali H.
      • Ribas de Pouplana L.
      • Sharma V.
      • Sharma A.
      Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.
      ,
      • Khan S.
      • Sharma A.
      • Belrhali H.
      • Yogavel M.
      • Sharma A.
      Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.
      ). In silico docking revealed two potent compounds, M-26 and M-37, showing delayed death inhibition by inhibiting aminoacylation activity by recombinant P. falciparum apicoplast LysRS (Table 1) (
      • Hoen R.
      • Novoa E.M.
      • López A.
      • Camacho N.
      • Cubells L.
      • Vieira P.
      • Santos M.
      • Marin-Garcia P.
      • Bautista J.M.
      • Cortés A.
      • Ribas de Pouplana L.
      • Royo M.
      Selective inhibition of an apicoplastic aminoacyl-tRNA synthetase from Plasmodium falciparum.
      ). Baragana et al., 2019 reported selective inhibitors of the apicomplexan LysRS, of which compound 5, when given at a low oral dosage (1.5 mg/kg once daily for four days), reduced parasitemia by over 90% in a malaria mouse model as it also inhibits C. parvum LysRS and growth of C. parvum parasites in vitro (Table 1) (
      • Baragaña B.
      • Forte B.
      • et al.
      Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.
      ). In addition, compound 5 reduced the parasite burden by almost two times when given orally for seven days in two separate mouse models of cryptosporidiosis (
      • Baragaña B.
      • Forte B.
      • et al.
      Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.
      ). This study also reported the 3D structure of C. parvum LysRS in complex with cladosporin and L-Lys. LysRS-1 (KRS-1) from Leishmania donovani is an essential gene (
      • Chadha S.
      • Mallampudi N.A.
      • Mohapatra D.K.
      • Madhubala R.
      Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.
      ).
      Cladosporin, due to its poor bioavailability and high metabolic instability, is unable to progress toward being a drug candidate. It became appropriate to explore analogs of cladosporin against PfLysRS with slight stereochemical or functional modifications. Four sets of analogs designed by making point modifications in the scaffold of cladosporin were assessed in enzyme and parasite assays (Table 1) (
      • Babbar P.
      • Das P.
      • Manickam Y.
      • Mankad Y.
      • Yadav S.
      • Parvez S.
      • Sharma A.
      • Reddy D.S.
      Design, Synthesis, and Structural Analysis of Cladosporin-Based Inhibitors of Malaria Parasites.
      ). The most potent compound, CL-2, performed better than cladosporin, and additional H-bonds were seen along with increased aqueous solubility. CL-2 binds to the adenosine pocket itself. The IC50 for CL-2 was 0.1 μM in the ATP hydrolysis assay, and the EC50 values for CL-2 were 0.08 and 4.0 μM (
      • Babbar P.
      • Das P.
      • Manickam Y.
      • Mankad Y.
      • Yadav S.
      • Parvez S.
      • Sharma A.
      • Reddy D.S.
      Design, Synthesis, and Structural Analysis of Cladosporin-Based Inhibitors of Malaria Parasites.
      ). EC50 measures the concentration of the compound to obtain a 50% killing in a cell-based assay. The same group later reported another set of derivatives of cladosporin, Cla-B and Cla-C, where the tetrahydropyran (THP) frame was replaced with a piperidine ring having functional implications (
      • Babbar P.
      • Sato M.
      • Manickam Y.
      • Mishra S.
      • Harlos K.
      • Gupta S.
      • Parvez S.
      • Kikuchi H.
      • Sharma A.
      Inhibition of Plasmodium falciparum Lysyl-tRNA Synthetase via a Piperidine-Ring Scaffold Inspired Cladosporin Analogues.
      ). Complex structures with Cla-B and Cla-C reveal similar binding orientations as PfLysRS with cladosporin bound (Table 1) (
      • Babbar P.
      • Sato M.
      • Manickam Y.
      • Mishra S.
      • Harlos K.
      • Gupta S.
      • Parvez S.
      • Kikuchi H.
      • Sharma A.
      Inhibition of Plasmodium falciparum Lysyl-tRNA Synthetase via a Piperidine-Ring Scaffold Inspired Cladosporin Analogues.
      ). However, the orientation of the piperidine ring varies from that of the THP ring of the cladosporin. Screening of 1215 bioactive compounds led to the discovery of ASP3026, an anaplastic lymphoma kinase inhibitor, as a PfLysRS inhibitor with nanomolar potency and > 80-fold more effective than the human LysRS (Table 1) (
      • Zhou J.
      • Huang Z.
      • Zheng L.
      • Hei Z.
      • Wang Z.
      • Yu B.
      • Jiang L.
      • Wang J.
      • Fang P.
      Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor.
      ). ASP3026 occupies the same site as cladosporin, with few structural adjustments. ASP3026 is already used in clinical trials against B-cell lymphoma and solid tumours (
      • Zhou J.
      • Huang Z.
      • Zheng L.
      • Hei Z.
      • Wang Z.
      • Yu B.
      • Jiang L.
      • Wang J.
      • Fang P.
      Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor.
      ). LysRS is among the well-studied aaRSs from P. falciparum and C. parvum thus providing a robust platform to explore LysRS in the other pathogens.

      Methionyl-tRNA synthetase (MetRS)

      T. brucei methionyl-tRNA synthetase (MetRS) is an essential enzyme as knockout showed growth defects (
      • Shibata S.
      • Gillespie J.R.
      • Kelley A.M.
      • Napuli A.J.
      • Zhang Z.
      • Kovzun K.V.
      • Pefley R.M.
      • Lam J.
      • Zucker F.H.
      • Van Voorhis W.C.
      • Merritt E.A.
      • Hol W.G.
      • Verlinde C.L.
      • Fan E.
      • Buckner F.S.
      Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.
      ). Further, several effective compounds showed more than 95% inhibition of aminoacylation activity at 50nM concentration (
      • Shibata S.
      • Gillespie J.R.
      • Kelley A.M.
      • Napuli A.J.
      • Zhang Z.
      • Kovzun K.V.
      • Pefley R.M.
      • Lam J.
      • Zucker F.H.
      • Van Voorhis W.C.
      • Merritt E.A.
      • Hol W.G.
      • Verlinde C.L.
      • Fan E.
      • Buckner F.S.
      Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.
      ). Compound 1 was most effective in T. brucei mouse model with delivery at 25mg/kg/day for three days showing high parasite suppression and delayed death, and low mammalian cell toxicity (Table 1) (Fig. 1). Subsequently, Leishmania major MetRS complexed with products methionyladenylate and pyrophosphate showed significant rearrangements in the overall structure of LmMetRS and/or tRNA (as compared to bacterial MetRS) that are vital to enable tRNAMet to access the methionyladenylate intermediate at the active site (
      • Larson E.T.
      • Kim J.E.
      • Zucker F.H.
      • Kelley A.
      • Mueller N.
      • Napuli A.J.
      • Verlinde C.L.
      • Fan E.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Merritt E.A.
      • Hol W.G.
      Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate.
      ).
      In the first major study to develop improved inhibitors of T. brucei MetRS, urea-based scaffolds held promise, firstly due to increased bioavailability and secondly, their likely permeability through the blood-brain barrier (Table 1) (
      • Shibata S.
      • Gillespie J.R.
      • Ranade R.M.
      • Koh C.Y.
      • Kim J.E.
      • Laydbak J.U.
      • Zucker F.H.
      • Hol W.G.
      • Verlinde C.L.
      • Buckner F.S.
      • Fan E.
      Urea-based inhibitors of Trypanosoma brucei methionyl-tRNA synthetase: selectivity and in vivo characterization.
      ,
      • Koh C.Y.
      • Kim J.E.
      • Wetzel A.B.
      • de van der Schueren W.J.
      • Shibata S.
      • Ranade R.M.
      • Liu J.
      • Zhang Z.
      • Gillespie J.R.
      • Buckner F.S.
      • Verlinde C.L.
      • Fan E.
      • Hol W.G.
      Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.
      ). Urea-based compounds inhibited parasite growth with low EC50 values (0.15 μM) and low toxicity to host cells. Compounds 2 and 26 showed superior membrane permeation in the in vitro MDR1-MDCKII model (which predicts and classifies compounds with blood barrier permeability), and improved oral pharmacokinetic properties in mice. Compound 26 also showed good suppressive activity against Trypanosoma brucei rhodesiense in the mouse model and compound 2 was seen to have entered the central nervous system in mice (
      • Shibata S.
      • Gillespie J.R.
      • Ranade R.M.
      • Koh C.Y.
      • Kim J.E.
      • Laydbak J.U.
      • Zucker F.H.
      • Hol W.G.
      • Verlinde C.L.
      • Buckner F.S.
      • Fan E.
      Urea-based inhibitors of Trypanosoma brucei methionyl-tRNA synthetase: selectivity and in vivo characterization.
      ). Subsequently, several urea-based inhibitors (UBIs) were designed against TbMetRS, having IC50 of 19nM (Table 1) (
      • Koh C.Y.
      • Kim J.E.
      • Wetzel A.B.
      • de van der Schueren W.J.
      • Shibata S.
      • Ranade R.M.
      • Liu J.
      • Zhang Z.
      • Gillespie J.R.
      • Buckner F.S.
      • Verlinde C.L.
      • Fan E.
      • Hol W.G.
      Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.
      ). UBIs bind to TbMetRS through conformational selection and very optimal binding in two pockets – the L-Met pocket and another conserved auxiliary pocket which is likely to be involved in tRNA binding. The UBIs do not compete with ATP for binding but rather interact with it via an h-bond. Thus, the omnipresent ATP-binding mode of MetRSs can be employed to design inhibitors for other disease-causing pathogens (
      • Koh C.Y.
      • Kim J.E.
      • Wetzel A.B.
      • de van der Schueren W.J.
      • Shibata S.
      • Ranade R.M.
      • Liu J.
      • Zhang Z.
      • Gillespie J.R.
      • Buckner F.S.
      • Verlinde C.L.
      • Fan E.
      • Hol W.G.
      Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.
      ). A beneficial fluorination spot for inhibitors targeting T. brucei MetRS was identified after the structural evaluation of TbMetRS complexes (Table 1) (
      • Zhang Z.
      • Barros-Álvarez X.
      • Gillespie J.R.
      • Ranade R.M.
      • Huang W.
      • Shibata S.
      • Molasky N.M.R.
      • Faghih O.
      • Mushtaq A.
      • Choy R.K.M.
      • de Hostos E.
      • Hol W.G.J.
      • Verlinde C.L.M.J.
      • Buckner F.S.
      • Fan E.
      Structure-guided discovery of selective methionyl-tRNA synthetase inhibitors with potent activity against Trypanosoma brucei.
      ). One series of compounds has a 1,3-dihydro-imidazol-2-one containing linker, while a second series includes a rigid fused aromatic ring. These distinct series inhibit parasite growth with high potency and EC50< 19nM with low general toxicity in mammalian cells. Selectivity was achieved in the range of 20-200-fold (
      • Larson E.T.
      • Kim J.E.
      • Zucker F.H.
      • Kelley A.
      • Mueller N.
      • Napuli A.J.
      • Verlinde C.L.
      • Fan E.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Merritt E.A.
      • Hol W.G.
      Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate.
      ). 5-fluoroimidazo[4,5-b]pyridine, when incorporated into compounds, imparts bioavailability and improved efficacy (
      • Zhang Z.
      • Barros-Álvarez X.
      • Gillespie J.R.
      • Ranade R.M.
      • Huang W.
      • Shibata S.
      • Molasky N.M.R.
      • Faghih O.
      • Mushtaq A.
      • Choy R.K.M.
      • de Hostos E.
      • Hol W.G.J.
      • Verlinde C.L.M.J.
      • Buckner F.S.
      • Fan E.
      Structure-guided discovery of selective methionyl-tRNA synthetase inhibitors with potent activity against Trypanosoma brucei.
      ). In another study, a large number of confirmed hits as inhibitors of TbMetRS was identified (1270) from MLSMR library by BioFocus DPI (San Francisco, USA) containing small molecules having molecular weight 350-410g/mol and containing both natural and synthetic products (Table 1) (
      • Pedró-Rosa L.
      • Buckner F.S.
      • Ranade R.M.
      • Eberhart C.
      • Madoux F.
      • Gillespie J.R.
      • Koh C.Y.
      • Brown S.
      • Lohse J.
      • Verlinde C.L.
      • Fan E.
      • Bannister T.
      • Scampavia L.
      • Hol W.G.
      • Spicer T.
      • Hodder P.
      Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.
      ). 52 of 54 compounds chosen for low-throughput screening were active in T. brucei aminoacylation activity assay. 12 of the 54 hit compounds inhibit the growth of T. brucei in culture, most likely via inhibition of TbMetRS (
      • Pedró-Rosa L.
      • Buckner F.S.
      • Ranade R.M.
      • Eberhart C.
      • Madoux F.
      • Gillespie J.R.
      • Koh C.Y.
      • Brown S.
      • Lohse J.
      • Verlinde C.L.
      • Fan E.
      • Bannister T.
      • Scampavia L.
      • Hol W.G.
      • Spicer T.
      • Hodder P.
      Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.
      ).
      Ursolic acid, a natural derivative taken from a reliable source of fresh leaves of Ochrosia elliptica Labill., family Apocyanaceae, displays potent antitrypanosomal and antileishmanial activities (Table 1) (
      • Labib R.M.
      • Ebada S.S.
      • Youssef F.S.
      • Ashour M.L.
      • Ross S.A.
      Ursolic Acid, a Natural Pentacylcic Triterpene from Ochrosia elliptica and Its Role in The Management of Certain Neglected Tropical Diseases.
      ). The IC50 values were encouraging, between 1.53 and 8.79 μg/ml and almost the same as that of pentamidine, an existing treatment for leishmaniasis though it has many side effects. Ursolic acid exhibited considerable affinity to MetRS with free binding energies from -42.54 to -63.93 kcal/mol (
      • Labib R.M.
      • Ebada S.S.
      • Youssef F.S.
      • Ashour M.L.
      • Ross S.A.
      Ursolic Acid, a Natural Pentacylcic Triterpene from Ochrosia elliptica and Its Role in The Management of Certain Neglected Tropical Diseases.
      ). Further, two new compounds containing the tetracyclic core of the Yohimbine and Corynanthe alkaloids showed potent inhibition TbMetRS aminoacylation activity and T. brucei parasite proliferation. Testing of multiple hydroxyalkyl δ-lactam, δ-lactam, and piperidine analogs, revealed one particular hydroxyalkyl δ-lactam derivative to be more effective against T. brucei. Still, they didn’t affect the aminoacylation activity of TbMetRS (Table 1) (
      • Lepovitz L.T.
      • Meis A.R.
      • Thomas S.M.
      • Wiedeman J.
      • Pham A.
      • Mensa-Wilmot K.
      • Martin S.F.
      Design, Synthesis, and Evaluation of Novel Anti-Trypanosomal Compounds.
      ).
      Two bacterial MetRS inhibitors, REP3123 and REP8839, affected P. falciparum pathogen survival at various stages, viz., ring stage, trophozoite, and schizont stage (Table 1) (
      • Hussain T.
      • Yogavel M.
      • Sharma A.
      Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.
      ) (Fig. 1). Three compounds, C1, C2, and C3, identified in silico were experimentally validated as they diminished protein translation by acting against PfMetRS as they stopped the progression of parasite growth from the ring to the trophozoite stage (
      • Hussain T.
      • Yogavel M.
      • Sharma A.
      Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.
      ) (Fig. 1). Class of imidazopyridine-containing compounds has shown promise against C. parvum and C. hominis infections in culture, likely via inhibition of Cryptosporidium MetRS (Table 1) (
      • Buckner F.S.
      • Ranade R.M.
      • Gillespie J.R.
      • Shibata S.
      • Hulverson M.A.
      • Zhang Z.
      • Huang W.
      • Choi R.
      • Verlinde C.L.M.J.
      • Hol W.G.J.
      • Ochida A.
      • Akao Y.
      • Choy R.K.M.
      • Van Voorhis W.C.
      • Arnold S.L.M.
      • Jumani R.S.
      • Huston C.D.
      • Fan E.
      Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.
      ). Compounds 2093, 2114, and 2259 showed the best in vivo activity; 2093 was not genotoxic. These compounds gradually stalled C. parvum infection in mouse models with no considerable side effects (
      • Buckner F.S.
      • Ranade R.M.
      • Gillespie J.R.
      • Shibata S.
      • Hulverson M.A.
      • Zhang Z.
      • Huang W.
      • Choi R.
      • Verlinde C.L.M.J.
      • Hol W.G.J.
      • Ochida A.
      • Akao Y.
      • Choy R.K.M.
      • Van Voorhis W.C.
      • Arnold S.L.M.
      • Jumani R.S.
      • Huston C.D.
      • Fan E.
      Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.
      ). This study points out that selectivity can be achieved over the human MetRS if treatment by these inhibitors is for short durations (e.g., < 1 week). A new class inhibitor, compound-1717, a fluro-imidazopyridine, targets Giardia intestinalis MetRS and has ‘cidal’ anti-Giardia activity as it inhibited trophozoites growth (Fig. 1) at 500nM with a therapeutic index of ∼100 (Table 1) (
      • Ranade R.M.
      • Zhang Z.
      • Gillespie J.R.
      • et al.
      Inhibitors of methionyl-tRNA synthetase have potent activity against Giardia intestinalis trophozoites.
      ). Compound-1717 satisfies Lipinski’s rule of 5 that determines the druggability of a molecule [Molecular mass less than 500 Da, high lipophilicity (expressed as partition coefficient LogP of less than 5), fewer than 5 hydrogen bond donors, less than 10 hydrogen bond acceptors, and molar refractivity between 40-130]. It was later seen to be highly effective in clearing Giardia infection within three days at variable doses in a mouse model of giardiasis (
      • Michaels S.A.
      • Shih H.W.
      • Zhang B.
      • Navaluna E.D.
      • Zhang Z.
      • Ranade R.M.
      • Gillespie J.R.
      • Merritt E.A.
      • Fan E.
      • Buckner F.S.
      • Paredez A.R.
      • Ojo K.K.
      Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model.
      ). Subsequently, another structurally novel class of inhibitors that contain a 4,6-diamino-substituted pyrazolopyrimidine core (the MetRS02 series) was identified (
      • Buckner F.S.
      • Ranade R.M.
      • Gillespie J.R.
      • Shibata S.
      • Hulverson M.A.
      • Zhang Z.
      • Huang W.
      • Choi R.
      • Verlinde C.L.M.J.
      • Hol W.G.J.
      • Ochida A.
      • Akao Y.
      • Choy R.K.M.
      • Van Voorhis W.C.
      • Arnold S.L.M.
      • Jumani R.S.
      • Huston C.D.
      • Fan E.
      Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.
      ). These compounds interestingly bind to an allosteric pocket in L. major MetRS. They also exhibit a non-competitive mode of inhibition in enzymatic studies (Table 1) (
      • Torrie L.S.
      • Robinson D.A.
      • Thomas M.G.
      • Hobrath J.V.
      • Shepherd S.M.
      • Post J.M.
      • Ko E.J.
      • Ferreira R.A.
      • Mackenzie C.J.
      • Wrobel K.
      • Edwards D.P.
      • Gilbert I.H.
      • Gray D.W.
      • Fairlamb A.H.
      • De Rycker M.
      Discovery of an Allosteric Binding Site in Kinetoplastid Methionyl-tRNA Synthetase.
      ). Compound DDD806905 worked against promastigotes (Fig. 1) but could not work in vivo. MetRS is the most widely studied aaRS in this review, as it has been investigated in all pathogens discussed here except Toxoplasma gondii.

      Phenylalanyl-tRNA synthetase (FRS)

      Bicyclic azetidines have been explored as inhibitors for parasitic PheRS. Cryptosporidium PheRS was validated as the molecular target of bicyclic azetidines. The most potent compound, BRD7929 eliminated parasites in vitro exponentially, with a half-life of ∼9.5 hours and ∼95 hours needed to kill 99.9% C. parvum parasites (Table 1) (
      • Vinayak S.
      • Jumani R.S.
      • Miller P.
      • Hasan M.M.
      • McLeod B.I.
      • Tandel J.
      • Stebbins E.E.
      • Teixeira J.E.
      • Borrel J.
      • Gonse A.
      • Zhang M.
      • Yu X.
      • Wernimont A.
      • Walpole C.
      • Eckley S.
      • Love M.S.
      • McNamara C.W.
      • Sharma M.
      • Sharma A.
      • Scherer C.A.
      • Kato N.
      • Schreiber S.L.
      • Melillo B.
      • Striepen B.
      • Huston C.D.
      • Comer E.
      Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase.
      ). Bicyclic azetidines have shown good selectivity as they eliminate parasites effectively in a mouse model with a once-daily dosing regimen. BRD7929 once-daily cured cryptosporidiosis in highly immunosuppressed mice and is thus promising for use in malnourished children and immunocompromised patients (
      • Vinayak S.
      • Jumani R.S.
      • Miller P.
      • Hasan M.M.
      • McLeod B.I.
      • Tandel J.
      • Stebbins E.E.
      • Teixeira J.E.
      • Borrel J.
      • Gonse A.
      • Zhang M.
      • Yu X.
      • Wernimont A.
      • Walpole C.
      • Eckley S.
      • Love M.S.
      • McNamara C.W.
      • Sharma M.
      • Sharma A.
      • Scherer C.A.
      • Kato N.
      • Schreiber S.L.
      • Melillo B.
      • Striepen B.
      • Huston C.D.
      • Comer E.
      Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase.
      ). Further, two compounds, bicyclic azetidines BRD7929 and BRD8494, were most potent across multiple stages of C. parvum growth in vitro across multiple stages, likely via inhibition of PheRS (Table 1) (
      • Funkhouser-Jones L.J.
      • Ravindran S.
      • Sibley L.D.
      Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro.
      ). BRD7929 was the most potent, possibly due to greater hydrophobicity. Series of bicyclic azetidines inhibit T. gondii growth in vitro and provide protection in a mouse model against acute and chronic toxoplasmosis (Table 1) (
      • Radke J.B.
      • Melillo B.
      • Mittal P.
      • Sharma M.
      • Sharma A.
      • Fu Y.
      • Uddin T.
      • Gonse A.
      • Comer E.
      • Schreiber S.L.
      • Gupta A.K.
      • Chatterjee A.K.
      • Sibley L.D.
      Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase.
      ). They are potent against tachyzoites (Fig. 1) at low nanomolar levels, and treatment of bradyzoites in vitro at EC90 concentrations leads to the complete killing of parasites. These compounds also exhibit better selectivity towards inhibiting T. gondii PheRS, thereby inhibiting parasite growth in vitro and in vivo. In particular, BRD7929 has an overall good bioavailability, potency and desirable selective profile which has remained a major challenge for aaRSs inhibitors so far.
      P. falciparum genome encodes for three different phenylalanyl-tRNA synthetases (PheRS), wherein two PfPheRS are localised to the cytosol and the apicoplast and a third unique PfPheRS is localized in the mitochondria (
      • Sharma A.
      • Sharma A.
      Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.
      ). A recent study revealed that BRD7929 had higher affinity and potent selective inhibition against P. falciparum cytoplasmic PheRS compared to the human PheRS (Table 1) (
      • Sharma M.
      • Mutharasappan N.
      • Manickam Y.
      • Harlos K.
      • Melillo B.
      • Comer E.
      • Tabassum H.
      • Parvez S.
      • Schreiber S.L.
      • Sharma A.
      Inhibition of Plasmodium falciparum phenylalanine tRNA synthetase provides opportunity for antimalarial drug development.
      ). BRD7929 inhibits P. falciparum growth at nanomolar concentrations (EC50 5 nM [Dd2 strain], 9 nM [3D7 strain]). It exhibited single-dose efficacy and promising pharmacokinetic properties in a mouse model. 3D structure of cytoplasmic PfPheRS with BRD7929 reveals binding of the inhibitor at the L-Phe pocket and an adjacent auxiliary pocket which is interesting as it is a departure from most aaRSs where inhibitors occupy one or the other or both two substrate binding sites (Table 1) (
      • Sharma M.
      • Mutharasappan N.
      • Manickam Y.
      • Harlos K.
      • Melillo B.
      • Comer E.
      • Tabassum H.
      • Parvez S.
      • Schreiber S.L.
      • Sharma A.
      Inhibition of Plasmodium falciparum phenylalanine tRNA synthetase provides opportunity for antimalarial drug development.
      ). These drugs are shown to kill parasites in vitro and in vivo in all stages of the parasite life cycle (Fig. 1). Bicyclic azetidines are also competitive inhibitors of L-Phe in P. vivax PheRS, as BRD1389 binds similarly to the L-Phe pocket and an adjacent auxiliary pocket (
      • Sharma M.
      • Malhotra N.
      • Yogavel M.
      • Harlos K.
      • Melillo B.
      • Comer E.
      • Gonse A.
      • Parvez S.
      • Mitasev B.
      • Fang F.G.
      • Schreiber S.L.
      • Sharma A.
      Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines.
      ). Thus, in both these studies, Pf and Pv cytoplasmic PheRS show a similar binding mode.
      PheRS has been explored as a druggable target only recently, and different bicyclic azetidines are now established as potent and promising inhibitors of PheRS from Plasmodium, Cryptosporidium, and Toxoplasma. PheRS holds a promise as an advanced target in parasites as it has shown the much-desired selectivity. Achieving selectivity remains a challenge in parasites aaRSs since most of them share high homology with the human homologs, and this is a major hindrance to successful progression from inhibitors to drugs. These inhibitors could be cross-tested for the other four pathogens discussed in this review.

      Prolyl-tRNA synthetase (PRS)

      Herman et al., 2015 showed the binding of halofuginone, a synthetic derivative of febrifugine, to the L-Pro and tRNA sites in Plasmodium prolyl-tRNA synthetase (ProRS), confirming the enzyme to be a functional target of both febrifugine and halofuginone (
      • Herman J.D.
      • Pepper L.R.
      • Cortese J.F.
      • Estiu G.
      • Galinsky K.
      • Zuzarte-Luis V.
      • Derbyshire E.R.
      • Ribacke U.
      • Lukens A.K.
      • Santos S.A.
      • Patel V.
      • Clish C.B.
      • Sullivan Jr., W.J.
      • Zhou H.
      • Bopp S.E.
      • Schimmel P.
      • Lindquist S.
      • Clardy J.
      • Mota M.M.
      • Keller T.L.
      • Whitman M.
      • Wiest O.
      • Wirth D.F.
      • Mazitschek R.
      The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.
      ). Febrifugine is well-established as a traditional Chinese herbal remedy for malaria fevers for a century (
      • Keller T.L.
      • Zocco D.
      • Sundrud M.S.
      • Hendrick M.
      • Edenius M.
      • Yum J.
      • et al.
      Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.
      ). Halofuginone also kills T. gondii parasites suggesting the broad efficacy of this compound (
      • Jain V.
      • Yogavel M.
      • Oshima Y.
      • Kikuchi H.
      • Touquet B.
      • Hakimi M.A.
      • Sharma A.
      Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis.
      ,
      • Mishra S.
      • Malhotra N.
      • Kumari S.
      • Sato M.
      • Kikuchi H.
      • Yogavel M.
      • Sharma A.
      Conformational heterogeneity in apo and drug-bound structures of Toxoplasma gondii prolyl-tRNA synthetase.
      ). Halofuginone though highly potent in killing Plasmodium parasites causes cytotoxicity to host cells (
      • Keller T.L.
      • Zocco D.
      • Sundrud M.S.
      • Hendrick M.
      • Edenius M.
      • Yum J.
      • et al.
      Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.
      ). Thus, many halofuginone and febrifugine derivatives with better safety profiles and improved therapeutic indices were designed for PfProRS (Table 1) (
      • Jain V.
      • Kikuchi H.
      • Oshima Y.
      • Sharma A.
      • Yogavel M.
      Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase.
      ,
      • Jain V.
      • Yogavel M.
      • Kikuchi H.
      • Oshima Y.
      • Hariguchi N.
      • Matsumoto M.
      • Goel P.
      • Touquet B.
      • Jumani R.S.
      • Tacchini-Cottier F.
      • Harlos K.
      • Huston C.D.
      • Hakimi M.A.
      • Sharma A.
      Targeting Prolyl-tRNA Synthetase to Accelerate Drug Discovery against Malaria, Leishmaniasis, Toxoplasmosis, Cryptosporidiosis, and Coccidiosis.
      ). Halofuginol, a new derivative of halofuginone was designed by modifying the linker region by replacing the ketone group with a secondary alcohol, is effective against the liver and blood stages (Fig. 1) of the parasite in a mouse model. Halofuginol demonstrated efficacy and tolerance in a P. berghei infected mouse model (administrated orally or intraperitoneally at a 25mg/kg dosage) (Table 1) (
      • Herman J.D.
      • Pepper L.R.
      • Cortese J.F.
      • Estiu G.
      • Galinsky K.
      • Zuzarte-Luis V.
      • Derbyshire E.R.
      • Ribacke U.
      • Lukens A.K.
      • Santos S.A.
      • Patel V.
      • Clish C.B.
      • Sullivan Jr., W.J.
      • Zhou H.
      • Bopp S.E.
      • Schimmel P.
      • Lindquist S.
      • Clardy J.
      • Mota M.M.
      • Keller T.L.
      • Whitman M.
      • Wiest O.
      • Wirth D.F.
      • Mazitschek R.
      The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.
      ).
      In a recent study, “double drugging” of Toxoplasma gondii ProRS by halofuginone and a novel ATP mimetic shows simultaneous binding at all three pockets in the active site since ATP mimetic L95 binds to the ATP site (
      • Manickam Y.
      • Malhotra N.
      • Mishra S.
      • Babbar P.
      • Dusane A.
      • Laleu B.
      • Bellini V.
      • Hakimi M.A.
      • Bougdour A.
      • Sharma A.
      Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.
      ). Both L95 and halofuginone are effective at nM concentrations when used individually (
      • Manickam Y.
      • Malhotra N.
      • Mishra S.
      • Babbar P.
      • Dusane A.
      • Laleu B.
      • Bellini V.
      • Hakimi M.A.
      • Bougdour A.
      • Sharma A.
      Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.
      ). Double drugging while managing dosage is a critical step towards likely achieving selectivity for ProRS and other aaRSs. Another study identified novel 1-(pyridin-4-yl)pyrrolidin-2-one derivatives as the cytoplasmic PfProRS inhibitors (
      • Okaniwa M.
      • Shibata A.
      • Ochida A.
      • Akao Y.
      • White K.L.
      • Shackleford D.M.
      • Duffy S.
      • Lucantoni L.
      • Dey S.
      • Striepen J.
      • Yeo T.
      • Mok S.
      • Aguiar A.C.C.
      • Sturm A.
      • Crespo B.
      • Sanz L.M.
      • Churchyard A.
      • Baum J.
      • Pereira D.B.
      • Guido R.V.C.
      • Dechering K.J.
      • Wittlin S.
      • Uhlemann A.C.
      • Fidock D.A.
      • Niles J.C.
      • Avery V.M.
      • Charman S.A.
      • Laleu B.
      Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Plasmodium Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.
      ). Compound 1 and its enantiomer 1-S, when tested against resistant Pf strains and the development of liver schizonts (Fig. 1), show potent low nanomolar activity. The slow killing and growth inhibition were seen in Pf and Pv field isolates. Thus, these derivatives show an encouraging off-target profile and oral efficacy in a Pf malaria murine model (
      • Okaniwa M.
      • Shibata A.
      • Ochida A.
      • Akao Y.
      • White K.L.
      • Shackleford D.M.
      • Duffy S.
      • Lucantoni L.
      • Dey S.
      • Striepen J.
      • Yeo T.
      • Mok S.
      • Aguiar A.C.C.
      • Sturm A.
      • Crespo B.
      • Sanz L.M.
      • Churchyard A.
      • Baum J.
      • Pereira D.B.
      • Guido R.V.C.
      • Dechering K.J.
      • Wittlin S.
      • Uhlemann A.C.
      • Fidock D.A.
      • Niles J.C.
      • Avery V.M.
      • Charman S.A.
      • Laleu B.
      Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Plasmodium Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.
      ). Halofuginone and its derivatives are well-established and promising inhibitors of ProRS from Plasmodium and Toxoplasma. ProRS can be similarly explored in the remaining five of the seven pathogens discussed here.

      Threonyl-tRNA synthetase (ThrRS)

      Borrelidin is a likely inhibitor for P. falciparum threonyl-tRNA synthetase (ThrRS), belonging to class II, as it successfully inhibits the proliferation of parasites in culture and the first asexual erythrocytic parasitic life-cycle indicating cytosolic inhibition (Fig. 1) (Table 1) (
      • Ishiyama A.
      • Iwatsuki M.
      • Namatame M.
      • Nishihara-Tsukashima A.
      • Sunazuka T.
      • Takahashi Y.
      • Ōmura S.
      • Otoguro K.
      Borrelidin, a potent antimalarial: stage-specific inhibition profile of synchronized cultures of Plasmodium falciparum.
      ,
      • Azcárate I.G.
      • Marín-García P.
      • Camacho N.
      • Pérez-Benavente S.
      • Puyet A.
      • Diez A.
      • Ribas de Pouplana L.
      • Bautista J.M.
      Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.
      ,
      • Otoguro K.
      • Ui H.
      • Ishiyama A.
      • Kobayashi M.
      • Togashi H.
      • Takahashi Y.
      • Masuma R.
      • Tanaka H.
      • Tomoda H.
      • Yamada H.
      • Omura S.
      In vitro and in vivo antimalarial activities of a non-glycosidic 18-membered macrolide antibiotic, borrelidin, against drug-resistant strains of Plasmodia.
      ). However, no effect is seen in the apicoplast despite PfThrRS exhibiting dual localization (
      • Ishiyama A.
      • Iwatsuki M.
      • Namatame M.
      • Nishihara-Tsukashima A.
      • Sunazuka T.
      • Takahashi Y.
      • Ōmura S.
      • Otoguro K.
      Borrelidin, a potent antimalarial: stage-specific inhibition profile of synchronized cultures of Plasmodium falciparum.
      ,
      • Azcárate I.G.
      • Marín-García P.
      • Camacho N.
      • Pérez-Benavente S.
      • Puyet A.
      • Diez A.
      • Ribas de Pouplana L.
      • Bautista J.M.
      Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.
      ). Increased concentrations of L-Thr in culture reduced parasite sensitivity indicating Thr utilization and PfThrRS as the targets for borrelidin. Subsequent studies showed in vivo effects of borrelidin as low doses cured mice of lethal rodent malaria infections caused by P. yoelii and possibly induced protective immune responses (
      • Azcárate I.G.
      • Marín-García P.
      • Camacho N.
      • Pérez-Benavente S.
      • Puyet A.
      • Diez A.
      • Ribas de Pouplana L.
      • Bautista J.M.
      Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.
      ,
      • Otoguro K.
      • Ui H.
      • Ishiyama A.
      • Kobayashi M.
      • Togashi H.
      • Takahashi Y.
      • Masuma R.
      • Tanaka H.
      • Tomoda H.
      • Yamada H.
      • Omura S.
      In vitro and in vivo antimalarial activities of a non-glycosidic 18-membered macrolide antibiotic, borrelidin, against drug-resistant strains of Plasmodia.
      ). Borrelidin though an excellent inhibitor of PfThrRS (it shows antimalarial activity against drug-resistant Pf parasites with IC50 of 0.93 ng/ml), faces the challenge of cytotoxicity. In this direction, borrelidin analogs and borrelidin-like series are promising and show reduced host cytotoxicity (
      • Sugawara A.
      • Tanaka T.
      • Hirose T.
      • Ishiyama A.
      • Iwatsuki M.
      • Takahashi Y.
      • Otoguro K.
      • Ōmura S.
      • Sunazuka T.
      Borrelidin analogues with antimalarial activity: design, synthesis and biological evaluation against Plasmodium falciparum parasites.
      ). Khan et al., 2011 discovered novel inhibitors of PfThrRS by in silico screening using structural models revealing compounds with moderate inhibition of P. falciparum growth (
      • Khan S.
      • Sharma A.
      • Jamwal A.
      • Sharma V.
      • Pole A.K.
      • Thakur K.K.
      • Sharma A.
      Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P.
      ).
      Eight potential Leishmania major ThrRS inhibitors were screened using a pre-validated MNP library (
      • Kelly P.
      • Hadi-Nezhad F.
      • Liu D.Y.
      • Lawrence T.J.
      • Linington R.G.
      • Ibba M.
      • Ardell D.H.
      Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens.
      ,
      • Schulze C.J.
      • Bray W.M.
      • Woerhmann M.H.
      • Stuart J.
      • Lokey R.S.
      • et al.
      Function-first" lead discovery: mode of action profiling of natural product libraries using image-based screening.
      ). Two compounds inhibited the aminoacylation activity at ∼50%, and four (1438C, 1758C, 2059D, and 2096B) inhibited the activity by greater than 75%, which continued to perturb aminoacylation throughout experiments. Of these four, 2059D and 2096B also inhibit L. major AlaRS (Table 1). Borrelidin, well-established as a natural product inhibitor of bacterial ThrRS, also inhibits T. brucei ThrRS by inhibiting parasite growth (
      • Kalidas S.
      • Cestari I.
      • Monnerat S.
      • Li Q.
      • Regmi S.
      • Hasle N.
      • Labaied M.
      • Parsons M.
      • Stuart K.
      • Phillips M.A.
      Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.
      ). Further, knockdown of T. brucei ThrRS studies results in rapid cell death. Borrelidin shows a strong affinity to the Leishmania donovani ThrRS (Kd: 0.04 μM), and it also inhibits the promastigotes stage of parasites (Fig. 1) (IC50: 21 μM) (Table 1) (
      • Chadha S.
      • Vijayan R.
      • Gupta S.
      • Munde M.
      • Gourinath S.
      • Madhubala R.
      Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target.
      ). Borrelidin and its analogs are well-established as inhibitors of ThrRS from Plasmodium, Trypanosoma and Leishmania and they can be explored using established approaches in four of the seven pathogens discussed here.

      Tryptophanyl-tRNA synthetase (TrpRS)

      Two genes encode two tryptophanyl-tRNA synthetases (TrpRS), belonging to class I, in T. brucei, wherein the first recognizes the tRNA in the cytosol, and the second recognizes the altered tRNA inside the mitochondria (
      • Charrière F.
      • Helgadóttir S.
      • Horn E.K.
      • Söll D.
      • Schneider A.
      Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.
      ). The second enzyme is needed for aminoacylation of the imported thiolated and the edited tRNATrp as it has a high substrate specificity (
      • Charrière F.
      • Helgadóttir S.
      • Horn E.K.
      • Söll D.
      • Schneider A.
      Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.
      ). Parasite-specific subdomains with structural differences are seen as TrpRS from Giardia Lamblia, C. parvum, T. brucei and B. histolytica, and P. falciparum which can guide selective drug design (
      • Arakaki T.L.
      • Carter M.
      • Napuli A.J.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      • Merritt E.A.
      The structure of tryptophanyl-tRNA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs.
      ,
      • Merritt E.A.
      • Arakaki T.L.
      • Gillespie R.
      • Napuli A.J.
      • Kim J.E.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Hol W.G.
      Crystal structures of three protozoan homologs of tryptophanyl-tRNA synthetase.
      ,
      • Khan S.
      • Garg A.
      • Sharma A.
      • Camacho N.
      • Picchioni D.
      • Saint-Léger A.
      • Ribas de Pouplana L.
      • Yogavel M.
      • Sharma A.
      An appended domain results in an unusual architecture for malaria parasite tryptophanyl-tRNA synthetase.
      ,
      • Koh C.Y.
      • Kim J.E.
      • Napoli A.J.
      • Verlinde C.L.
      • Fan E.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      Crystal structures of Plasmodium falciparum cytosolic tryptophanyl-tRNA synthetase and its potential as a target for structure-guided drug design.
      ). The activation reaction mechanism is different in basal eukaryote G. lamblia compared to human TrpRS as three critical residues stabilize interactions with a beta-hairpin are absent while retaining the overall dimer structure (
      • Arakaki T.L.
      • Carter M.
      • Napuli A.J.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      • Merritt E.A.
      The structure of tryptophanyl-tRNA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs.
      ). An inhibitor of the bacterial TrpRS called indolmycin, a natural tryptophan analog, was explored against P. falciparum TrpRS. Indolmycin, isolated from the bacteria Streptomyces griseus, affects parasite growth by specifically inhibiting the apicoplast PfTrpRS but not the cytosolic PfTrpRS (Table 1) (
      • Pasaje C.F.
      • Cheung V.
      • Kennedy K.
      • Lim E.E.
      • Baell J.B.
      • Griffin M.D.
      • Ralph S.A.
      Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum.
      ). The structure of the catalytic domain of cytoplasmic PfTrpRS is available in complex with L-tryptophan (
      • Khan S.
      • Garg A.
      • Sharma A.
      • Camacho N.
      • Picchioni D.
      • Saint-Léger A.
      • Ribas de Pouplana L.
      • Yogavel M.
      • Sharma A.
      An appended domain results in an unusual architecture for malaria parasite tryptophanyl-tRNA synthetase.
      ).

      Tyrosyl-tRNA synthetase (TyrRS)

      The first three-dimensional structure of the Leishmania major cytoplasmic tyrosyl-tRNA synthetase (TyrRS), belonging to class I, showed a pseudo-dimer with unique asymmetric domains and only a single functional, active site (near N-terminus) along with an anticodon site (near the C-terminus) (
      • Larson E.T.
      • Kim J.E.
      • Castaneda L.J.
      • Napuli A.J.
      • Zhang Z.
      • Fan E.
      • Zucker F.H.
      • Verlinde C.L.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      • Merritt E.A.
      The double-length tyrosyl-tRNA synthetase from the eukaryote Leishmania major forms an intrinsically asymmetric pseudo-dimer.
      ). Leishmania donovani TyrRS is characterized and validated as an essential enzyme (
      • Anand S.
      • Madhubala R.
      Twin Attributes of Tyrosyl-tRNA Synthetase of Leishmania donovani: A HOUSEKEEPING PROTEIN TRANSLATION ENZYME AND A MIMIC OF HOST CHEMOKINE.
      ). Fisetin (3,3′,4′,7-tetrahydroxyflavone), a flavonoid, inhibits parasite growth by inhibiting LdTyrRS aminoacylation activity, as seen earlier for trypanosomal TyrRS (
      • Larson E.T.
      • Kim J.E.
      • Castaneda L.J.
      • Napuli A.J.
      • Zhang Z.
      • Fan E.
      • Zucker F.H.
      • Verlinde C.L.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      • Merritt E.A.
      The double-length tyrosyl-tRNA synthetase from the eukaryote Leishmania major forms an intrinsically asymmetric pseudo-dimer.
      ,
      • Anand S.
      • Madhubala R.
      Twin Attributes of Tyrosyl-tRNA Synthetase of Leishmania donovani: A HOUSEKEEPING PROTEIN TRANSLATION ENZYME AND A MIMIC OF HOST CHEMOKINE.
      ). The structure of L. donovani TyrRS in complex with tyrosyl-adenylate, consists of two pseudo monomers (
      • Barros-Álvarez X.
      • Kerchner K.M.
      • Koh C.Y.
      • Turley S.
      • Pardon E.
      • Steyaert J.
      • Ranade R.M.
      • Gillespie J.R.
      • Zhang Z.
      • Verlinde C.L.M.J.
      • Fan E.
      • Buckner F.S.
      • Hol W.G.J.
      Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.
      ). P. falciparum TyrRS is localized in the cytosol and is present in the infected erythrocytes (
      • Bhatt T.K.
      • Kapil C.
      • Khan S.
      • Jairajpuri M.A.
      • Sharma V.
      • Santoni D.
      • Silvestrini F.
      • Pizzi E.
      • Sharma A.
      A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum.
      ). The extracellular activity of the PfYRS was detected via mimicking host cytokines to then induce immune system pro-inflammatory responses in the host. The apo structure of the PfTyrRS with bound tyrosyl-adenylate revealed an “extra pocket” near the adenine binding region, which is absent in human TyrRS.
      A recent study has identified an adenosine 5’-sulfamate (a close mimic of AMP) and ML901 from Takeda Pharmaceutical compound library, which showed potent activity against all strains of P. falciparum and also showed 800- to 5000-fold selectivity towards the parasite (
      • Xie S.C.
      • Metcalfe R.D.
      • Dunn E.
      • Morton C.J.
      • Huang S.C.
      • Puhalovich T.
      • Du Y.
      • Wittlin S.
      • Nie S.
      • Luth M.R.
      • Ma L.
      • Kim M.S.
      • Pasaje C.F.A.
      • Kumpornsin K.
      • Giannangelo C.
      • Houghton F.J.
      • Churchyard A.
      • Famodimu M.T.
      • Barry D.C.
      • Gillett D.L.
      • Dey S.
      • Kosasih C.C.
      • Newman W.
      • Niles J.C.
      • Lee M.C.S.
      • Baum J.
      • Ottilie S.
      • Winzeler E.A.
      • Creek D.J.
      • Williamson N.
      • Parker M.W.
      • Brand S.
      • Langston S.P.
      • Dick L.R.
      • Griffin M.D.W.
      • Gould A.E.
      • Tilley L.
      Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.
      ). In vivo study in mice engrafted with P. falciparum-infected RBCs showed a single dose [50mg/kg intraperitoneal injection] resulting in reduced parasitemia to baseline with no cytotoxicity. ML901 targets the PfTyrRS by a unique “reaction hijacking” mechanism. The PfYRS binds to ATP and tyrosine; the tRNATyr then binds to the tyrosine releasing AMP. This would normally make a “charged” tRNA but when inhibitor ML901 is present this drug binds to the tyrosine of Tyr-tRNATyr instead and the uncharged tRNATyr is released thereby inhibiting the PfTyrRS and stalling protein synthesis (
      • Xie S.C.
      • Metcalfe R.D.
      • Dunn E.
      • Morton C.J.
      • Huang S.C.
      • Puhalovich T.
      • Du Y.
      • Wittlin S.
      • Nie S.
      • Luth M.R.
      • Ma L.
      • Kim M.S.
      • Pasaje C.F.A.
      • Kumpornsin K.
      • Giannangelo C.
      • Houghton F.J.
      • Churchyard A.
      • Famodimu M.T.
      • Barry D.C.
      • Gillett D.L.
      • Dey S.
      • Kosasih C.C.
      • Newman W.
      • Niles J.C.
      • Lee M.C.S.
      • Baum J.
      • Ottilie S.
      • Winzeler E.A.
      • Creek D.J.
      • Williamson N.
      • Parker M.W.
      • Brand S.
      • Langston S.P.
      • Dick L.R.
      • Griffin M.D.W.
      • Gould A.E.
      • Tilley L.
      Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.
      ). This unique, promising approach can achieve the desired selectivity for aaRSs.

      Other structurally and functionally characterized aaRSs

      Aspartyl-tRNA synthetase (AspRS)

      The first apo structure of the aspartyl-tRNA synthetase (AspRS), belonging to class II, from Entamoeba histolytica, was determined early on (
      • Merritt E.A.
      • Arakaki T.L.
      • Larson E.T.
      • Kelley A.
      • Mueller N.
      • Napuli A.J.
      • Zhang L.
      • Deditta G.
      • Luft J.
      • Verlinde C.L.
      • Fan E.
      • Zucker F.
      • Buckner F.S.
      • Van Voorhis W.C.
      • Hol W.G.
      Crystal structure of the aspartyl-tRNA synthetase from Entamoeba histolytica.
      ). The T. brucei genome encodes two separate genes for AspRS, though the cell contains a single tRNAAsp isoacceptor. In vitro data shows that mitochondrial TbAspRS2 aminoacylates cytosolic and mitochondrial tRNAAsp, whereas the cytosolic TbAspRS1 only recognizes cytosolic tRNAAsp. Thus, cytosolic and mitochondrial tRNAAsp are derived from the same nuclear gene but are physically distinct, offering dual potential as drug targets (
      • Charrière F.
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      • Helgadóttir S.
      • Maréchal-Drouard L.
      • Cristodero M.
      • Horn E.K.
      • Söll D.
      • Schneider A.
      Dual targeting of a tRNAAsp requires two different aspartyl-tRNA synthetases in Trypanosoma brucei.
      ). Structural data reveals that the N-terminus of the P. falciparum AspRS contains a motif that may provide a strong RNA binding to plasmodial AspRS, and plasmodial insertion is required for AspRS dimerization, and thereby for its aminoacylation activity and other functions (
      • Bour T.
      • Akaddar A.
      • Lorber B.
      • Blais S.
      • Balg C.
      • Candolfi E.
      • Frugier M.
      Plasmodial aspartyl-tRNA synthetases and peculiarities in Plasmodium falciparum.
      ). AspRS has been reported in Entamoeba, Trypanosoma and Plasmodium but is yet to be explored in five of seven pathogens discussed. AspRS has been reported in Entamoeba, Trypanosoma and Plasmodium with 3D structures available from E. histolytica which can be utilized to develop first-ever inhibitors of AspRS.

      Glutamyl-tRNA synthetase (GluRS)

      The transcript of GltX, one of the glutamyl tRNA synthetases (GluRS), is expressed during the asexual blood stages of Babesia bovis, which confirms that the complete bipartite signal is in control of directing the reporter protein into the apicoplast, a compartment distinct from the nucleus and the mitochondrion (
      • Pedroni M.J.
      • Luu T.N.
      • Lau A.O.
      Babesia bovis: a bipartite signal directs the glutamyl-tRNA synthetase to the apicoplast.
      ). Further, the Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast is achieved by a vital indirect aminoacylation pathway where GluRS is first targeted in the apicoplast in the blood stages as it glutamylates tRNAGlu and tRNAGln (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.I.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in the plasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ). GluRS remains to be explored as no structure or inhibitor has been reported from the seven pathogens discussed here.

      Cysteinyl-tRNA synthetase (CysRS)

      The cysteinyl-tRNA synthetase (CysRS), belonging to class I, is encoded by a single gene in P. falciparum and localized in the cytosol and the apicoplast as the T. gondii CysRS (
      • Pham J.S.
      • Dawson K.L.
      • Jackson K.E.
      • Lim E.E.
      • Pasaje C.F.
      • Turner K.E.
      • Ralph S.A.
      Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites.
      ). Similar to the other well-studied dual location ThrRS, inhibition of CysRs will have dual effects, i.e., first killing the parasites via inhibition of cytosolic translation, and subsequently disrupting the apicoplast. CysRS has been partially studied in P. falciparum and remains to be explored in six of the seven pathogens discussed here, as no inhibitors have been discovered.

      Concluding remarks

      Aminoacyl-tRNA synthetases (aaRSs) are universally conserved enzymes essential for protein synthesis. Remarkable progress in the past two decades has thrust parasite-encoded aaRSs into focus as promising drug targets for many pathogens. Biochemical and structural studies have elucidated inhibition mechanisms that target various sites on these enzymes. Indeed, a remarkable example of double drugging of this enzyme family has also been validated wherein two different individually potent drugs co-bind to the prolyl-tRNA synthetase, occupying all the substrate binding sites on the enzyme. There is a wealth of knowledge on inhibitor identification, design, and development against numerous eukaryotic parasite aaRSs. These studies are based on various technologies that include in silico docking, high-throughput screening, enzyme inhibition assays, chemical modifications of lead compounds, and animal models. The libraries of hit compounds generated so far for individual pathogens can be tested across many more eukaryotic pathogens allowing cross-usage of existing and new inhibitors. These parasite aaRSs tend to have conserved 3D structures and conserved catalytic sites in their aminoacylation domains. Evaluating sequence conservation in the inhibitor-binding residues in complexes can be utilized make predictions and validate new hit compounds which could be used to target more than one pathogen. We have, in an earlier study, proposed “STOPP” i.e. structure-based targeting of orthologous pathogen proteins” (
      • Jain V.
      • Sharma A.
      Repurposing of Potent Drug Candidates for Multiparasite Targeting.
      ). Essential enzymes that are conserved (between hosts and pathogens and within different parasites) have the potential to be excellent drug targets if potential new compounds and chemical entities can differentiate subtle sequence/structure contrasts at the binding region. This knowledge can be leveraged when synthesizing novel entities. Such structure-based targeting of orthologous proteins will allow to jump-start inhibitor discovery across pathogen-encoded aaRSs.
      Resistance to antiparasitic drugs remains a significant issue and warrants special focus during the preclinical stages of inhibitor development. Early evaluations of inhibitors can put them on a robust path and make them promising scaffolds. Further, due to the universal nature of aaRSs enzymes, selectivity remains a challenge for developing successful drugs against parasite aaRSs even though these parasites, in some cases, are evolutionarily distinct and cause different diseases. The selectivity of an inhibitor towards parasite aaRS and not the human host aaRS is crucial as this results in host cytotoxicity. This issue of selectivity warrants attention and requires a thorough understanding of the structural underpinnings for specific drug design. The data consolidated in this work will pave the way for further dissection of aaRSs from eukaryotic pathogens and will steer the modification of promising inhibitors and scaffolds into selective drug-like compounds. Parasite-encoded aaRSs are undoubtedly exciting and promising druggable targets that warrant continued attention for the development of anti-infective drugs.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      AS is recipient of JC Bose National fellowship from Department of Science and Technology, Govt. of India.

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