Elucidation of DNA Primase as a Drug Target in Leishmania donovani: Structure-Guided Inhibitor Identification and Biochemical Validation

Abstract

Replication is started by DNA primase, which synthesizes an oligoribonucleotide primer that DNA polymerases extend. Effective chemotherapy is still needed to manage and treat leishmaniasis, which continues to pose a threat to public health around the world. Repurposing drugs offers alternative uses for drugs with established pharmacological effects, saving money and increasing the pool of human resources available to create novel anti-leishmanials. Here, we used a non-radioactive primase-pyrophosphatase assay to biochemically characterize the Leishmania donovani nuclear DNA primase (LdPri) subunits, followed by in silico evaluation of inhibitors targeting LdPri. The best-evaluated inhibitors showed LdPri inhibition in vitro under conditions similar to those of the primase-pyrophosphatase experiment. The MTT assay also confirmed the inhibitors' anti-leishmanial properties. Parasite growth and morphological analysis were performed by culturing cells in the presence of inhibitors. Biochemical characterization revealed that LdPriS activity was unstable, but was highly stabilized upon association with LdPriL in LdPri. LdPri was found to be thermostable and possesses 3′-terminal nucleotidyltransferase activity. Additionally, in silico and in vitro studies evaluated Pritelivir (BAY 57-1293) as the most efficient LdPri complex inhibitor, followed by Epigallocatechin Gallate (EGCG). Moreover, kinetic studies showed that Pritelivir and EGCG exhibit competitive and uncompetitive inhibition, respectively, for both NTP and DNA substrates. Pritelivir effectively disrupted the L. donovani cell cycle, and the replication mechanism in treated promastigotes showed irregular shapes and short flagella.