Chemical and genetic validation of dihydrofolate reductase–thymidylate synthase as a drug target in African trypanosomes

Abstract

SummaryThe phenotypes of single-(SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR-TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR-TS is essential for parasite survival and represents a promising target for drug discovery.

Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

“…Animals were inspected daily for clinical signs of infection, and wet smears of tail blood were examined microscopically as appropriate. Parasitemia was determined using a Neubauer hemocytometer, as previously described, and infected mice were monitored for 30 days (34). Mice with parasitemia that exceeded 10 8 cells ml Ϫ1 were humanely killed, since prior experience indicated that animals would succumb to an overwhelming infection by the following day.…”

Interwoven Biology of the Tsetse Holobiont

“…has resulted in a highly restricted genomic repertoire, compensating for the absence of biosynthetic pathways by encoding transporters to sequester metabolites (including folate) from the environment (120,121). This enhanced biosynthetic capability may contribute to the higher reported vector competency of the G. morsitans host relative to G. brevipalpis (96)(97)(98)100), as trypanosomes necessitate exogenous folate for growth (122). Deeper investigation of this hypothesis is required.…”

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

“…This issue is not limited to drug-induced perturbations, as studies of genetic manipulation for target validation may also be complicated by excess nutrient availability (17). Indeed, this "metabolite rescue" approach is often used intentionally to generate conditional gene knockouts or to confirm chemical or genetic enzyme inhibition (17,18). However, the use of a rich culture medium may unintentionally hinder drug discovery efforts that utilize phenotypic screening approaches, as demonstrated for dihydrofolate reductase (DHFR)-thymidylate synthase inhibitors, which demonstrated significant trypanocidal activity only when the medium was depleted of folic acid (17).…”

“…Also, drug-induced metabolic perturbations can be easily masked by compensatory mechanisms if there are high metabolite concentrations available in the culture medium. This issue is not limited to drug-induced perturbations, as studies of genetic manipulation for target validation may also be complicated by excess nutrient availability (17). Indeed, this "metabolite rescue" approach is often used intentionally to generate conditional gene knockouts or to confirm chemical or genetic enzyme inhibition (17,18).…”

“…Indeed, this "metabolite rescue" approach is often used intentionally to generate conditional gene knockouts or to confirm chemical or genetic enzyme inhibition (17,18). However, the use of a rich culture medium may unintentionally hinder drug discovery efforts that utilize phenotypic screening approaches, as demonstrated for dihydrofolate reductase (DHFR)-thymidylate synthase inhibitors, which demonstrated significant trypanocidal activity only when the medium was depleted of folic acid (17). The same type of masking where highly abundant culture medium components compete with drugs for targets or transporters will also influence screening for other antimetabolites.…”