Supplementary Data from Novel Pyrrolo[3,2-<i>d</i>]pyrimidine Compounds Target Mitochondrial and Cytosolic One-carbon Metabolism with Broad-spectrum Antitumor Efficacy

Abstract

&lt;p&gt;Supplementary Figure S1 shows in vitro antiproliferative activity and targeted pathways of previously reported AICARFTase and GARFTase inhibitors. Supplementary Figure S2 shows docking of AGF291, AGF320, and AGF347 (along with 5-formyl-THF) in human SHMT2 and rabbit SHMT1. Supplementary Figure S3 shows plasma membrane folate transporter expression levels in H460, HCT116, and MIA PaCa-2 human tumor cell lines as compared to those in the IGROV-1 epithelial ovarian cancer cell line. Supplementary Figure S4 shows in vitro antiproliferative activity and targeted pathways of AGF291, AGF320, and AGF347 along with previously reported GARFTase inhibitor AGF94 in H460, HCT116, and MIA PaCa-2 cell lines. Supplementary Figure S5 shows targeted metabolomics data on total serine, serine isotope labeling patterns, total GAR, total AICAR, total adenine nucleotides, and total dTTP along with isotope labeling patterns of H460, HCT116, and MIA PaCa-2 cell lines treated with AGF291, AGF320, and AGF347 not shown in main text Figure 4. Supplementary Figure S6 shows a Western blot confirming knockdown of SHMT2 in H460 SHMT2 KD cell line and knockout of SHMT2 in HCT116 SHMT2 KO cell line. Supplementary Figure S7 shows in vivo efficacies of AGF347 and gemcitabine towards MIA PaCa-2 early and late stage tumor xenograft models where mouse serum folate levels were not depleted to approximate those found in humans. Supplementary Figure S8 shows a cytochrome c oxidase assay on tumors harvested from the metabolomics arm of the late stage in vivo AGF347 trial. Table S1 shows docking scores of novel compounds in human SHMT2 and rabbit SHMT1 (corresponding to Supplementary Figure S2). Table S2 shows quantitative data from in vivo early- and late-stage trials of AGF347 and gemcitabine against MIA PaCa-2 tumor xenografts in NCR SCID mice.&lt;/p&gt;