DNA replication inhibitor as a potential antimicrobial agent targeting neglected tropical pathogens

Tropical diseases are a significant economic burden for developing nations, including Leishmaniasis, African sleeping sickness, and cryptococcosis caused by Leishmania spp., Trypanosoma brucei, and Cryptococcus neoformans, respectively. These diseases can be fatal without treatment. Current medicines are restricted by toxicity and resistance. Therefore, new treatments targeting these organisms are essential. Flap endonucleases (FENs) form a class of enzymes presented in all living beings and are involved in maintenance of the genome, assuming a significant part in DNA replication and DNA repair. Thus, it is likely to be vital for parasite and fungal survival, and subsequently potential drugs can be designed to target FENs. This project was developed using experimental and in silico techniques to specifically target microbial FENs by understanding the molecular mechanism of the enzyme and its biological structure. In silico studies were carried out using small molecule structures retrieved from publicly available databases and structures of the microbial FENs experimentally determined or predicted through AI algorithms. The in silico screening studies using Autodock and Autodock Vina identified potential inhibitors according to a binding energy score and further analysis was carried out. Additionally, over 1500 molecules were tested in vitro. Selective inhibitors were identified with IC50 as low as 23 μM. Crystallisation screens for recombinant FEN proteins were executed with the aim of elucidating experimental structures of microbial FENs. Protein crystals of Trypanosoma, Leishmania and Cryptococcus FEN were produced under different screening conditions. One of the conditions formed a Leishmania FEN crystal with a diffracting resolution of 1.4 Å, in which the data was used to model the protein structure. This work has potential impact in the future development of antimicrobial drugs and further studies can be performed (fragment structure optimization, in vivo assays and co-crystallisation of microbial FENs with inhibitors) to enrich structure-based drug design campaigns.