Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Abstract

bHuman African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyltRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (␣ and ␤) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites

“…The development of target-based inhibitors is an attractive approach for addressing some of these concerns; however, few targets have been validated in T. cruzi or Leishmania due to their limited genetic tractability. Advances in T. brucei genetics have allowed genome-wide gene essentiality screens and the validation of new targets and chemotypes for the development of novel drugs (Alsford, et al, 2011; Cestari and Stuart, 2013; Kalidas, et al, 2014). Since the genomes of these parasites are highly conserved (~94% synteny and ~60% overall identity of orthologous genes) (El-Sayed, et al, 2005), the identification of new targets and discovery of T. brucei -specific inhibitors may be useful for chemical validation of orthologous enzymes in T. cruzi and Leishmania sp .…”

New Borrelidins from Onchidium sp. Associated Streptomyces olivaceus SCSIO LO13

“…The borrelidins have attracted significant attention from the natural products community, and their noteworthy anti‐infective and cytotoxic activities have especially drawn the interest and efforts of biosynthetic and chemical synthesis researchers . In this study, three new borrelidins ( 1 – 3 ), along with four previously reported family members ( 4 – 7 ), were isolated from a likely associated symbiont S. olivaceus SCSIO LO13 from the marine pulmonated mollusk Onchidium sp.…”

“…Borrelidin A ( 4 ) was the first member of the family discovered and has been shown to have antibiotic and antimalarial activities as well as the ability to inhibit tumorigenic and angiogenic processes . Borrelidin A ( 4 ) has been particularly important in demonstrating the importance of threonyl tRNA synthetase (ThrRS) inhibition as a viable means of targeting the causative agent for malaria . Although borrelidin A ( 4 ) is toxic to non‐malignant cell types, its antitumor potential, as reflected in part by its ability to inhibit the growth of hepatocellular carcinoma cells, has garnered a significant amount of clinical interest .…”

Novel Effects of Lapatinib Revealed in the African Trypanosome by Using Hypothesis-Generating Proteomics and Chemical Biology Strategies

“…2 and 3) after exposure to lapatinib. Trypanosome rounding has been observed in knockdown of proteins that are also involved in endocytosis (39), protein synthesis (40), and cell cycle (41,42). Trypanosomes have a subpellicular microtubule array (corset) that would need to be reorganized during cell rounding.…”