Gene Silencing of Histidyl-tRNA Synthetase in Leishmania Donovani Promastigotes Inhibits Parasite Growth and Reduces Virulence: A Comprehensive Computational and in Vitro Study

The majority of anti-leishmanial drugs used for treating trypanosomatid parasites help to reduce human morbidity and mortality. However, parasites have developed drug resistance, which has made it challenging to treat leishmaniasis. Therefore, new drugs and drug targets need to be identified. Protein synthesis is a crucial anabolic mechanism necessary for parasite survival. Histidyl-tRNA synthetase (HisRS) is an essential enzyme that is required for histidine incorporation into proteins. Recent studies on HisRS have shown differences between trypanosomatid HisRS and human HisRS, which could lead to the development of trypanosomatid HisRS structure-based inhibitors. This study aims to determine the role of L. donovani HisRS (LdHisRS) in parasite growth and virulence in vitro using RNAi. The silencing effect of LdHisRS expression was determined using qPCR. The results showed that after 24 and 48 h of incubation with 90 ng siRNAs, LdHisRS mRNA expression levels were significantly reduced by ∼3.14-fold and ∼3.90-fold, respectively. SiRNA-treated parasites also exhibited ∼46.6 % delayed growth and ∼47 % reduced virulence. Additionally, homology modeling, virtual screening, and molecular docking studies were performed with potential inhibitors that have significant suppressive activity in bacteria, fungi, and viruses. Halofuginone was found to have the best binding affinity of -9.09 kcal/mol as a potent inhibitor against LdHisRS. The molecular dynamics (MD) results showed that halofuginone could interact with the various active site segments, potentially blocking substrate access. The data on gene silencing through siRNA suggests that LdHisRS is essential for the parasite's growth and survival. The computational findings could lead to the development of a potent ligand (halofuginone) as a future anti-leishmanial drug, paving the way for an effective therapeutic treatment.