Many of the oldest and most efficient drugs have been part of anti-folate treatments. These drugs target different parts of the folate pathway. Folate, also known as Vitamin B9, is an essential nutrient needed to act as a one-carbon donor in purine biosynthesis. Folate also plays an important role as a cofactor which is necessary for the synthesis of Tetrahydrofolate (THF) via Dihydrofolate reductase or DHFR. THF is a precursor for nucleic acid synthesis, mRNA translation and DNA methylation. Due to these significant roles, DHFR is a strong target for Malaria and other types of cancers. However, DHFR has mutated to form resistance towards drugs that target folate metabolism. The increasing resistance of the Malaria parasite to commonly used anti-malarial drugs such as Sulfadoxine (SP) and Pyrimethamine (PYR) has increased research on the mutations. Recent research describes the way mutations are able to change the conformation of the enzyme and decrease its binding affinity for the inhibitors which results in the resistance of the enzyme to these structural analogs. However, once the drug pressure is taken off the field the parasite favors the expression only of the wild type protein. To understand the enzyme kinetics and the effect of the mutations, Plasmodium falciparum DHFR wild type protein and drug resistant mutants are cloned into a protein expression vector. The expression and purification of the protein is required to study the mutant and wild type protein by mass spectrometry hydrogen-deuterium exchange (HDX). This will allow the identification of solvent-exposed regions of the wild type protein and drug resistant mutants, which is significant in understanding how the mutations unfavorably affect the activity of the DHFR enzyme. We will present the data collected on the cloning of the different drug resistant mutants.

Characterization of Malaria DHFR Mutants

253rd National Meeting (2017)

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