Abstract
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.
Highlights
W e and others are interested in gaining a better understanding of folate metabolism within kinetoplastids and using this information to identify potential drug targets for disease areas such as human African Trypanosomiasis (HAT), Leishmaniasis, and Chagas’ disease
In our (Cellzome) laboratories, we have developed powerful methods to quantitatively analyze binding of compounds to composed of nonselective inhibitors immobilized on beads that could effectively “pull down” a large number of different proteins in cell extracts
Article aFPGS, folypolyglutamate synthase; GCS, glycine cleavage system; SHMT, serine hydroxyl methyltransferase; dehydrogenase/methenyltetrahydrofolate cyclohydrolase (DHCH) MTFT, methionyl-tRNAformyltransferase; MTX, methotrexate; RTX, raltiterxed; PTX, pemetrexed; LEU, leucovorin; NTX, nolatrexed; THF, tetrahydrofolate. bCompounds with a folate pharmacophore possessing a diglutamate moiety that are proposed to bind to FPGS.[18] proteins, bound proteins were eluted from the beads, digested by trypsin, and analyzed by mass spectrometry
Summary
W e and others are interested in gaining a better understanding of folate metabolism within kinetoplastids and using this information to identify potential drug targets for disease areas such as human African Trypanosomiasis (HAT), Leishmaniasis, and Chagas’ disease. In our (Cellzome) laboratories, we have developed powerful methods to quantitatively analyze binding of compounds to composed of nonselective inhibitors immobilized on beads that could effectively “pull down” a large number of different proteins in cell extracts. As previously reported, this approach kinases or dioxygenases. Quantification of the kinases binding to inhibitors was performed by isobaric tagging with isotope-containing reagents (TMT, tandem mass tags) In this current study, we aimed to produce and develop an affinity matrix for enzymes involved in the folate metabolism of kinetoplastids, effectively the folateome of these parasites. Inhibitors were selected from the literature, where possible where there is literature data for inhibition of the kinetoplastid enzymes (see the references in Table 1 and further information in the Supporting Information)
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