To evaluate the SNAP-based TR-FRET assay in a competition binding mode, we analyzed the binding of five known DHFR inhibitors to eDHFR (Figure 3B): the anti-cancer drugs methotrexate and trimetrexate targeting human DHFR, the anti-malaria drugs WR99210 and pyrimethamine targeting plasmodium DHFR, and the anti-bacterial trimethoprim. Here, DHFR is linked to malaria.