Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target

Abstract

BackgroundAminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis.MethodologyRecombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human.ConclusionsRecombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.

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 .…”

pH effect on strain-specific transcriptomes of the take-all fungus

“…The gene coding for a threonyl-tRNA synthetase was overexpressed in acidic condition ( S6B Table ). This enzyme family, involved in the protein translation machinery, is often used as a target in a wide range of antibiotics [ 30 ], and its inactivation led to a reduced growth and attenuated virulence in Leishmania donovani [ 31 ]. In the same way, the regulation of its expression in G .…”

Structure-based inhibitor screening of natural products against NSP15 of SARS-CoV-2 revealed thymopentin and oleuropein as potent inhibitors

“…Experimental studies on NSP15 have revealed that His-235, His-250, and Lys-290 (Ricagno et al 2006) are the critical residues for catalysis that are located in the C-terminal domain of the enzyme. Docking studies were performed using GOLD (Jones et al 1997), and the protein was kept as a rigid molecule, and the number of Genetic Algorithm (GA) runs was set to 10 runs per ligand with the default search algorithm parameters similar to our earlier reports (Vijayan and Mallick 2007;Eniyan et al 2018;Chadha et al 2018; Vijayan and Manohayan 2015; Mahendran and Vijayan 2018). GOLD score was then used as the final scoring method.…”