Webster L, Thomas M, Urbaniak M, Wyllie S, Ong H, Tinti M, Fairlamb A, Boesche M, Ghidelli-Disse S, Drewes G, Gilbert I.
The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited by a number of drugs. However, there has been little success in developing drugs that target folate metabolism in the kinetoplastids. Despite compounds being identified which show significant inhibition of the parasite enzymes, this activity does not translate well into cellular and animal models of disease. Understanding to which enzymes antifolates bind under physiological conditions and how this corresponds to the phenotypic response could provide insight on how to target the folate pathway in these organisms. To facilitate this, we have adopted a chemical proteomics approach to study binding of compounds to enzymes of folate metabolism. Clinical and literature antifolate compounds were immobilized onto resins to allow for “pull down” of the proteins in the “folateome”. Using competition studies, proteins, which bind the beads specifically and nonspecifically, were identified in parasite lysate (Trypanosoma brucei and Leishmania major) for each antifolate compound. Proteins were identified through tryptic digest, tandem mass tag (TMT) labeling of peptides followed by LC-MS/MS. This approach was further exploited by creating a combined folate resin (folate beads). The resin could pull down up to 9 proteins from the folateome. This information could be exploited in gaining a better understanding of folate metabolism in kinetoplastids and other organisms.