Figure 5. In situ.

GARFTase inhibition by compound 2 and Pmx in R1-11-PCFT4 and H2452 cells.

GARFTase activity and inhibition were evaluated in situ with R1-11-PCFT4 cells (panel A) and H2452 cells (panel B). For both cell lines, cells were treated with drug for 16 h at pH 6.8 in complete folate-free RPMI 1640 supplemented with 10% dialyzed fetal bovine serum and 25 mM HEPES/25 mM PIPES before incubating in the presence of 4 μM azaserine for 30 min, followed by [¹⁴C]glycine and L-glutamine treatment. After 8 h, radioactive metabolites were extracted and analyzed. Methods including those for quantifying [¹⁴C]formyl GAR are described under Materials and Methods. Results are presented for duplicate experiments (as average values plus/minus ranges) as percentages of vehicle controls over a series of drug concentrations. For H2452 cells, IC₅₀s for GARFTase inhibition were 44 nM ± 6 nM and 68.3 ± 0.8 nM for compound 2 and Pmx, respectively; for R1-11-PCFT4 cells, these values were 25.1 ± 11.8 nM and 33.6 ± 15.2 nM, respectively.