Table 1.
IC50 values for anti-proliferative activities toward engineered CHO and human tumor cell lines, and cell-free enzyme inhibition by 5-substituted pyrrolo[3,2-d]pyrimidine inhibitors compared to AGF94 and PMX.
| Compound | Proliferation Inhibition [IC50 (nM)] | In vitro enzyme assays [Ki (μM)] | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| R2 (null) |
PC43–10 (RFC) | R2/PCFT4 (PCFT) | HCT116 | HCT116 SHMT2 KO | H460 | MIA PaCa-2 | GARFTase | AICARFTase | SHMT2 | MTHFD2 | SHMT1 | |
| AGF94 | 136 (3) | 110 (15) | 3 (1) | 191 (62) | 295 (59)* | 166 (59) | 1924 (539) | 0.88 (0.71) | NI | 188.0 (91.8) | NI | 112 (57) |
| PMX | 395 (83) | 124 (39) | 24 (5) | 293 (81) | 333 (40) | 165 (59) | 281 (23) | 5.19 (1.63) | 0.88 (0.56) | NI | NI | NI |
| AGF291 | NI | 454 (87) | 282 (22) | 2266 (450) | 108 (66)** | 461 (163) | 3664 (721) | NI | 13.31 (3.16) | 0.63 (0.29) | NI | 0.90 (0.06) |
| AGF347 | NI | 224 (17) | 479 (63) | 437 (180) | 42(19)*** | 214 (88) | 1381 (182) | 3.13 (0.66) | 3.72 (1.61) | 2.19 (0.23) | NI | 2.91 (0.59) |
| AGF331 | NI | NI | 661 (62) | 2199 (817) | 969 (254) | 2367 (711) | 6681 (604) | ND | ND | ND | ND | ND |
| AGF320 | NI | NI | 694 (56) | 737 (195) | 117 (60)* | 573 (144) | 2703 (400) | 0.33 (0.22) | 2.36 (1.99) | 0.056 (0.020) | NI | 0.126 (0.030) |
Proliferation inhibition assays were performed using the engineered CHO cell lines R2 (folate transporter-null), PC43–10 (expresses RFC only), and R2/PCFT4 (expresses PCFT only), and human tumor cell lines, including HCT116 (colon cancer), H460 (lung cancer), and MIA PaCa-2 (pancreatic cancer). For HCT116, both wild-type (WT) and CRISPR/Cas9 SHMT2 KO cells (22,23) were tested. For SHMT2 KO cells, glycine was added to the media to circumvent the impact of loss of SHMT2, permitting cell growth. Results are shown as mean IC50 values (± standard deviations), corresponding to the concentrations that inhibit growth by 50% relative to vehicle-treated control cells, from at least four biological replicates. p < 0.10;
p < 0.05;
p < 0.01;
p < 0.001 in HCT116 SHMT2 KO relative to WT cells. For the in vitro enzyme assays, Ki values are presented as mean values (+/− standard deviations) from at least 3 replicate experiments. “ND” denotes assay not done. “NI” denotes “no inhibition” at 1000 nM inhibitor for the CHO cell line proliferation experiments and up to 200 μM inhibitor for the in vitro enzyme assays.