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. Author manuscript; available in PMC: 2012 Oct 27.
Published in final edited form as: J Med Chem. 2011 Sep 22;54(20):7150–7164. doi: 10.1021/jm200739e

Figure 7. Intracellular ATP levels and in situ GARFTase inhibition in R2/hPCFT4 cells treated with compound 3, compound 4, LMTX, and PMX.

Figure 7

Panel A: R2/hPCFT4 cells were treated with 1 µM of 3, 4, PMX, or LMTX, or solvent (0.5% DMSO for 3 and 4, H2O for PMX and LMTX) for 24 h. Cells were washed and nucleotides were extracted and analyzed by HPLC. Results are shown for percentage control ATP after the drug treatments. Panel B: GARFTase activity and inhibition were evaluated in situ with R2/hPCFT4 cells. Accumulation of [14C]formyl GAR from [14C]glycine was measured in R2/hPCFT4 cells treated with the antifolates. The production of [14C]formyl GAR was calculated as a percent of vehicle control over a range of antifolate concentrations. Results are presented as mean values ± standard errors from 3 experiments. Methodologic details are described in the Experimental Section. Results with PMX and LMTX were from Kugel Desmoulin et al.15 IC50s were as follows: 0.69 nM, 3; 1.96 nM, 4; 31.5 nM LMTX; and 7.3 nM, PMX.