Fig 1: DHFR-binding modes of C1 and methotrexate are predicted to be partially overlapping.(A, B, C, D, E, F) Surface representation of (A) crystal structure of hDHFR in complex with NADPH, (B) crystal structure of hDHFR in complex with NADP+ and folic acid (FOL), (C) crystal structure of hDHFR in complex NADP+ and 5,10-dideaza-THF (5,10THF), (D) crystal structure of hDHFR in complex with NADPH and methotrexate (MTX), (E) docking pose of C1 on hDHFR (PDB accession 4M6J), and (F) docking pose of C1 on hDHFR (PDB accession 5HPB). Residues involved in opening and closing of the active site are highlighted in green and were adopted from Bhabha et al (2013). (G, H, I, J) Active site crystal structures of the (G, H) hDHFR–NADPH (cyan), hDHFR–NADPH–folic acid (yellow), hDHFR–NADP+–5,10-dideaza–THF (magenta), and (I, J) hDHFR–NADPH–methotrexate complexes. (K, L) Docking poses of the hDHFR–NADPH–C1 complexes (orange is PDB accession 4KBN, purple is PDB accession 5HPB).
Fig 2: C1 potently forms a complex with DHFR in cells.Drug-induced HA–DHFR stabilization in cellular thermal shift assays on intact HEK293T cells overexpressing HA–DHFR and mTurquoise2 (negative control) or FPGS–FLAG. Cells were incubated with a drug concentration range for 4 h followed by a cellular thermal shift assay at 52°C. Data are average Z-scores and were collected in n = 2 biological replicates. Original Western blots are shown in Fig S6C.
Fig 3: Thermograms of isothermal titration calorimetry experiments, related to Table 1.(A) Representative thermograms demonstrating binding of DHFR to C1 and methotrexate. (B) Representative thermograms demonstrating that binding of DHFR to C1 and methotrexate is not altered in the presence of 125 μM NADPH.
Fig 4: Identification of compound C1 as a novel DHFR ligand.(A) Structures of methotrexate, pyrimethamine, and C1, a novel 2,4-diaminopyrimidine antifolate. (B) Comparison of C1 and methotrexate IC50 values for a panel of cancer cell lines. Methotrexate IC50 values were retrieved from the Genomics of Drug Sensitivity in Cancer database and determined using a Syto60 viability assay after 72 h treatment. C1 IC50 values were determined using a sulforhodamine B viability assay after 72 h treatment. (C) CellTiter-Glo viability assay on C1- or vehicle-treated LS 174T cells after 48 h treatment. Data were collected for n = 2 biological replicates. (D) Top panel: thermal proteome profile of intact LS 174T cells treated with 10 μM C1 for 1 h, including stability score and false discovery rate of all hits and candidates. Negative stability scores represent proteins that are destabilized by C1 treatment, and positive stability scores represent proteins that are stabilized by C1 treatment. Bottom panel: melting curve for DHFR in 10 μM C1- or DMSO-treated intact LS 174T cells. (E) Western blot analysis of a representative cellular thermal shift assay on intact HEK293T cells overexpressing HA–DHFR treated with 10 μM methotrexate or C1 for 1 h.
Fig 5: C1 and methotrexate inhibit DHFR and folate-mediated one-carbon metabolism.(A) In vitro inhibition of hDHFR activity by C1 and methotrexate, at 50 μM dihydrofolate and 60 μM NADPH. Data are shown as mean ± s.d. and were collected in n = 2 technical replicates. Results are representative of three independent experiments. (B) Thermal proteome profiling analysis of one-carbon metabolism–related proteins in cells treated with C1 or methotrexate, including Z-score transformed stability scores. Positive Z-scores represent stabilized proteins, negative Z-scores represent destabilized proteins. The thermal proteome profile for C1 was determined at 10 μM in LS 174T cells (Fig 1D). The thermal proteome profile for methotrexate was retrieved from Huber et al (2015) and determined at 10 μM in K562 cells. (C) Simplified schematic representation of tetrahydrofolate (THF)-mediated one-carbon transfer reactions involved in purine and thymidine synthesis. Rescue interventions are indicated with dashed lines. Abbreviations: 5,10-methylene-tetrahydrofolate (5,10-CH2-THF), formate (CHO), 10-formyl-tetrahydrofolate (10-CHO-THF), 5-formyl-tetrahydrofolate (5-CHO-THF), glycine cleavage system (GCS). (D) CellTiter-Glo viability analysis of C1- or methotrexate-treated A549 cells after 48 h treatment, in the absence or presence of 25 μM 5-formyl-tetrahydrofolate. Data were collected in n = 2 biological replicates. (E) CellTiter-Glo viability analysis of C1- or methotrexate-treated A549 or HCT-116 cells after 24 h treatment, including rescue treatments with one-carbon cycle metabolites. Data were collected in n = 2 biological replicates. (F) CellTiter-Glo viability analysis of C1-, methotrexate-, pyrimethamine-, or trimethoprim-treated A549 cells after 48 h treatment. Data were collected in n = 2 biological replicates.
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