Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type


Degree Name



Cancer Biology

First Advisor

Larry H. Matherly


Folate is a B9 vitamin essential to DNA synthesis. The proton-coupled folate transporter (PCFT) is a newly discovered proton/folate symporter with an acidic pH optimum and broad expression across a variety of solid tumor types, with limited expression in normal tissues. Several antifolate molecules have been developed as cancer therapeutics, although these classical antifolates display numerous off-target effects due to transport by the ubiquitous reduced folate carrier (RFC). In this dissertation, we determine the roles of multiple PCFT structure/function domains, and develop PCFT-specific antifolates to target solid tumors. We utilize substituted cysteine accessibility methods (SCAM) to identify a novel reentrant loop structure between the second and third transmembrane domains (TMDs), which is critical to PCFT function, using membrane-impermeable thiol-reactive reagent MTSEA-biotin. We also utilize SCAM to identify and characterize the PCFT oligomerization interface at TMDs 3 and 6 via crosslinking reagent MTS-6-MTS. By the development of PCFT and ThTr1 chimera molecules, creation of PCFT half molecules, and cysteine- and alanine-scanning mutagenesis, we determined the purely structural role of the TMD6-7 connecting loop region. We studied novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to pemetrexed (PMX), a standard NS-NSCLC treatment. [AGF94 (2’,5’) and AGF154 (2’,4’)], which showed potencies comparable to PMX toward six NS-NSCLC cell lines. Uptake of [3H]AGF154 was comparable to that of [3H]PMX at acidic pH. De novo purine biosynthesis inhibition by AGF94/154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR. In vivo efficacy of AGF94 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Additonal antifolates were synthesized through the addition of fluorine to existing molecules and studied for their transport specifiticies correlating with anti-proliferative effects. In particular, AGF278 displayed increased specificity to PCFT, while also being very potent toward ovarian cancer cell lines. In conclusion, we established the role of three key structural domains of PCFT while also developing several novel PCFT-targeted antifolates for use as cancer therapeutics for NS-NSCLC and ovarian cancer. These novel antifolates are targeted to the acidic tumor microenvironment via selective transport by PCFT.