A High-Throughput Turbidometric Assay for Screening Inhibitors of Leishmania major Protein Disulfide Isomerase

Abstract

The use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity.

Leishmania major protein disulfide isomerase as a drug target

“…Indeed, preliminary experimental results led us to the identification of new molecules that have a strong effect on LmPDI activities and parasites growth in vitro and in infected macrophages (Ben Khalaf et al 2011). Moreover, as suggested by Krauth-Siegel and Inhoff (2003), as other enzymes involved in thiol metabolism, trypanthione, that is a PDI cofactor in Leishmania, could also constitute potential drug target candidate.…”

Kinases as Druggable Targets in Trypanosomatid Protozoan Parasites

“…A wide range of approaches have been pursued in the search for new drugs for kinetoplastid diseases, including target-based 35 or cell-based 36 high-throughput small molecule screens and identification of putative essential targets by detailed cell biology. Following identification of chemical matter in this way, further optimization is required in order to ensure high potency, cellular selectivity (low host toxicity), and the ability of new drugs to meet the target-product profiles for HAT, 37 leishmaniasis, 38 and Chagas disease.…”

Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

“…Given that PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of chemotherapy in some cancers [35,36]; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduced the infectivity of HIV [37]. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been reported [38,39], we described here a novel and simple micro-assay to test the chaperone activity of PDI enzymes that is amenable for HTS of PDI inhibitors.…”