| Folates are important cofactors involved in biosynthesis of nucleotides and cellular metabolism amino acids. Mammalian cells cannot synthesize folates de novo, and rely on folates taken from diet sources. Since folates are anions at physiological pH, which cannot diffuse across cell membranes efficiently, cell membrane folate transporter systems are required for folate uptake. The reduced folate carrier (RFC) is a ubiquitously expressed, high capacity folate transporter system, recognized as the predominant membrane transport system for folates, as well as most antifolates.;To understand the molecular mechanism of substrate transport by human RFC, I used N-hydroxysuccinamide (NHS) activated substrates, including methotrexate, to probe the substrate binding domain on human RFC, which interacts with carboxyl groups on substrates during substrate transport. Site-directed mutagenesis was used to generate single amino acid mutants of RFC to evaluate the function of specific residues. Human RFC substrate analogs with two carboxyl groups replaced with hydrogen or methyl group were used to examine the roles of two carboxyl groups on substrate during substrate transport. The results indicated that the gamma carboxyl group on the substrate was covalently labeled onto lysine411 in transmembrane domain 11 during NHS-mediated affinity labeling, which indicated that lysine411 was in a substrate binding pocket on human RFC. However, the interaction between gamma carboxyl group on substrate and lysine411 on human RFC had an antagonistic effect on substrate transport by human RFC. The alpha rather than the gamma carboxyl group on the transport substrate was shown to be important for substrate transport by human RFC.;Folate receptors (FRs) are another type of membrane folate transport systems in mammalian cells. The alpha and beta isoforms of FRs can transport substrates across cell membrane by endocytosis. The expression of FRs in normal tissues is very limited and not accessible by circulating substrates. However, the alpha and beta isoforms of FRs are over-expressed in some malignant tissues, as ovarian carcinoma (for FRα) and acute myelogenous leukemia (for FRβ), which, in combination with their high affinity towards folic acid, renders them as important cell surface receptors for tumor-specific drug delivery. In this study, I characterized two series of folate analogs, 6-substituted pyrrolo[2,3-d]pyrimidines and 6-substituted thieno[2,3-d]pyrimidines, which were transported by FRs, but not RFC, and inhibited glycinamide ribonucleotide formyltransferase (GARFTase) in the de novo purine synthesis pathway. The cell membrane transport and cell growth inhibition properties of these compounds were first identified with a series of Chinese hamster ovary cell lines over-expressing different folate transporter systems including human RFC, FRα, or FRβ, and two human cancer cell lines over-expressing FRα, KB and IGROV1. The lead compounds in those series showed potent growth inhibition, at nanomolar levels, toward cells expressing FRs, but not those expressing RFC. The membrane transport specificity of the compounds was further characterized in direct cell membrane transport or binding assays. Cytotoxicity protection assays with nucleosides, adenosine and thymidine, and a precursor of an intermediate metabolite, aminoimidazole-4-carboxamide (AICA), identified GARFTase in de novo purine synthesis pathway as the folate-dependent enzyme inhibited by these compounds. This was further confirmed and compared with classical antifolates in vitro, with purified mouse recombinant enzyme inhibition, and in situ, by examining incorporation of 14C-labeled glycine into 14C-formyl-GAR in cultured KB cells. Colonyforming inhibition and reversibility assays with KB cells confirmed that colony formation was irreversibly inhibited by the pyrrolopyrimidine and thiophenepyrimidine antifolate compounds, as they were by classical antifolates. However, apoptosis is not the predominant form of cell death as measured by annexin V-FITC staining flow cytometry. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogs as antitumor agents. |
