Antifolates as Tools for Chemical Biology
thesisposted on 10.12.2012 by Laura E. Pedro-Rosa
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Small molecule ligands and receptor protein pairs that associate to the exclusion of all other environmental components can be used as powerful biotechnology tools to analyze proteins not only in living cells but also in vitro. It has been shown that the antifolate trimethoprim (TMP) can be synthetically linked to fluorophores and used to selectively label genetically encoded fusions proteins of Escherichia coli dihydrofolate reductase (eDHFR) expressed in cultured mammalian cells. The high affinity (~1 nm Ki) of the TMP-eDHFR interaction, and the inertness of both TMP and eDHFR to other components present in mammalian cells makes this labeling technique possible. This dissertation presents the results of experiments that were designed to develop additional antifolate-DHFR pairs for live cell and in vitro protein labeling applications. Enzyme inhibition assays showed that triethyleneglycolamino derivatives of 2,4-diamino-5-(4-(3,4,5-trimethoxy)-3-ethoxybenzyl) pyrimidine (compound 2a) 2,4-diamino-5-(3,4,-dimethoxy)-5-carboxy-1-pentynylbenzyl) pyrimidine (compound 3a) were potent inhibitors (IC50 < 30 nM) of Plasmodium falciparum DHFR (pfDHFR) and Pneumoscystis carinii DHFR (pcDHFR), respectively. The antifolates were linked to fluorescein diacetate, and it was observed that the fluorescent analog of 2a labeled an overexpressed mutant of pfDHFR in live NIH3T3 cells. Additional experiments showed that conjugates of the antifolates TMP, methotrexate and 2a could be conjugated to a luminescent terbium complex and used for highly sensitive, time-resolved luminescence resonance energy transfer measurements of antifolate-DHFR affinity. The implications of these results for developing high throughput screening assays of ligand-protein and protein-protein interactions are explored herein.