Identification of tryparedoxin peroxidase as a chalcone target in Leishmania

Abstract

Leishmaniasis is a group of vector-borne neglected diseases caused by intracellular protozoan parasites of the Leishmania genus. Without an approved vaccine, current chemotherapy is based on drugs producing severe toxic side effects, and susceptible to resistance. To help the discovery of safer and more effective drugs, this work proposed to identify the parasite molecular target of chalcones, a promising new class of antileishmanial. The nitro chalcone ((2E)-3-(3-nitrophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one; compound 11) was employed as a model molecule for its high selective activity against Leishmania amazonensis (IC50= 0.34µM) and other parasite species. A NAT22 analogue containing an alkyne group ((2E)-1-[4-(hex-5-yn-1-yloxy)-2,6-dimethoxyphenyl]-3-(3-nitrophenyl)prop-2-en-1-one; 19), and a trifunctional probe accommodating biotin, rhodamine and an azide group (20) for chemical linkage to 19 were synthesized for protein target identification by Activity Based Protein Profiling (ABPP) approach. To validate the ABPP method, it was demonstrated that 19 retained anti-promastigote activity (IC50= 0.29 µM). Moreover, 19 was shown to permeate live promastigotes and bind to compartmentalized intracellular sites, as revealed by conjugation with the fluorescent probe (20). After SDS-PAGE, a single 22 kDa protein band was labelled by the probe. These were identified by mass spectrometry, and subsequently confirmed by Western blot, as the cytosolic tryparedoxin peroxidase (cTXNPx), a critical parasite detoxifying enzyme. Biophysical and molecular docking studies using recombinant cTXNPx, with chalcone 19 and a dihydrochalcone analogue (18) support a model in which chalcone 11 inhibits cTXNPx by covalent interaction of Cys52 with the α,β-unsaturated ketone. Lack of parasite survival after gene deletion using CRISPR-Cas9 approach suggested the essentiality of the identified enzyme. Overall, these results demonstrate that ABPP methods can be used for new drug target discovery in Leishmania, enabling the identification of cTXNPx as an important Leishmania drug target, providing detailed insight into the mechanism of action.