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. 2023 May 22;8(10):e158798. doi: 10.1172/jci.insight.158798

Figure 6. Select compound 19-S series analogs show dual TS and DHFR inhibition.

Figure 6

(A) Tritium-based TS activity assay screen of compound 19-S series analogs. Compounds were initially screened at 250 μM to determine analogs showing TS inhibition. Mean ± SD of 4 data points from 2 independent experiments shown. DMSO, TS + 5,10-mTHF, and 5,10-mTHF controls represent 8 data points. P values are calculated using 2-tailed unpaired t test, ****P ≤ 0.0001. (B) Absorbance-based DHFR activity assay screen of compound 19-S series analogs; all compounds were initially screened at 1 μM concentration. Data are expressed as mean ± SD of n = 7 from 2 independent experiments. (C) Comparison showing TS activity utilizing the tritium-based activity assay in the presence of pemetrexed (PEM), methotrexate (MTX), and compounds 19-S, 19-S5, and 19-S7 at the indicated concentrations. Data are expressed as mean ± SD of n = 4 from 2 independent experiments. (D) Comparison showing DHFR activity utilizing the absorbance-based activity assay in the presence of PEM, MTX, and compounds 19-S, 19-S5, and 19-S7 at the indicated concentrations. Data are expressed as mean ± SD of n = 4. (E) MIA PaCa-2 cell line viability assays following 72-hour drug treatment with compound 19-S and 19-S series analogs 19-S5 and 19-S7 and known control antifolate inhibitors PEM and MTX. Data are expressed as mean ± SD (triplicates).