Applications of Mass spectrometry in natural product drug discovery for malaria: Targeting Plasmodium falciparum thioredoxin reductase

Abstract

Malaria is considered to be the dominant cause of death in low income countries especially in Africa. Malaria caused by Plasmodium falciparum is a most lethal form of the disease because of its rapid spread and the development of drug resistance. The main problem in the treatment of malaria is the emergence of drug resistant malaria parasites. Over the years/decades, natural products have been used for the treatment or prevention of number of diseases. They can serve as compounds of interest both in their natural form and as templates for synthetic modification. Nature has provided a wide variety of compounds that inspired the development of potential therapeutics such as quinine, artemisinin and lapachol as antimalarial agents. As the resistance to known antimalarials is increasing, there is a need to expand the antimalarial drug discovery efforts for new classes of molecules to combat malaria. This research work focuses on the applications of ultrafiltration, mass spectrometry and molecular modeling based approaches to identify inhibitors of Plasmodium falciparum thioredoxin reductase (PfTrxR), our main target and Plasmodium falciparum glutathione reductase (PfGR) as an alternative target for malaria drug discovery. In our first approach, we used an ultrafiltration and liquid chromatography mass spectrometry based approach to screen one hundred and thirty three structurally diverse natural compounds to determine their binding affinity towards PfTrxR. Along with the set of natural products, different plant extracts were also subject to binding experiments to identify ligands of PfTrxR followed by identification and structure elucidation of identified ligands using mass spectrometry. In our second approach, we had developed an LC-MS based functional assay to identify inhibitors of PfTrxR by quantifying the reduced thioredoxin (Trx–(SH)2), the product formed in the enzymatic reaction. Thioredoxin is a 11.7 kDa protein. To validate the developed functional assay we have screened reference compounds 2,4-dinitrophenyl sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT) and 3-(dimethylamino)-propiophenone (3-DAP) for their PfTrxR inhibitory activity and ten natural compounds (at 10 mM) which were earlier identified as ligands of PfTrxR by a UF-LC-MS based binding assay. In the third approach, our goal was to identify natural products which can selectively target Plasmodium falciparum thioredoxin reductase (PfTrxR), Plasmodium falciparum glutathione reductase (PfGR) enzymes of the parasite distinct from the host enzymes. In our study, the binding affinities of natural products towards PfTrxR, PfGR, human TrxR and human GR were determined using a mass spectrometry based ligand binding assay. The in vitro antimalarial activity (IC50) and cytotoxicity of these ligands were also determined. In silico molecular modeling was used to ascertain and further confirm the binding affinities and key interactions of these ligands towards PfTrxR, PfGR and human isoforms of these enzymes.