Selection and properties of Leishmania tropica resistant to 10-propargyl-5,8-dideazafolate, an inhibitor of thymidylate synthetase

Abstract

Leishmania tropica promastigotes have been selected which are highly resistant to the thymidylate synthetase (TS) inhibitor, 10-propargyl-5,8-dideazafolate (CB3717). As reported for L. tropica resistant to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), CB3717-resistant organisms have high levels of the bifunctional TS-DHFR and amplified DNA sequences. TS-DHFR represents up to 2% of the protein in cell extracts and does not appear to have a structural alteration that contributes to drug resistance. The amplified unit of DNA has a uniform restriction-site map throughout the selection and is nearly identical to the 30 kb amplified unit of R-region DNA found in MTX-resistant cells, except for a small increase in size of the fragment that contains a junction believed to be the site of DNA rearrangements generated during amplification. CB3717-resistant cells do not possess the amplified H-region DNA found in MTX-resistant cells. The amplified DNA in cells resistant to low levels of CB3717 appears as a 30-kb extrachromosomal circle, similar to the amplified DNA of MTX-resistant organisms. In cells resistant to higher levels of drug, the amplified DNA appeared as higher molecular weight forms. When resistant cells were grown in the absence of drug, the amplified DNA and levels of TS-DHFR gradually fell to approximately 10% of the resistant levels. These findings support the proposal that the R-region DNA possesses the sequences that encode the bifunctional protein.