Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (Delta Ins-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and Delta Ins-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than Delta Ins-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for Delta Ins-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, leishmanial ArgRS activates L-Arg in the absence of its cognate tRNA. Deletion of the unique insertion region within its active site led to a loss of tRNA binding capability and an alteration in its catalytic and structural features along with modification of inhibition modality by a benzothiazolo-coumarin derivative. Here, we present a detailed characterization of a new form of ArgRS that can be employed for developing novel anti-leishmanial agents. image