title: Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of de novo purine biosynthesis in the chemotherapy of solid tumors.
abstract: The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-L-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-L-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [(3)H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.
authors.affiliation: Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
authors.forename: Sita Kugel
authors.initials: SK
authors.lastname: Desmoulin
authors.forename: Yiqiang
authors.initials: Y
authors.lastname: Wang
authors.forename: Jianmei
authors.initials: J
authors.lastname: Wu
authors.forename: Mark
authors.initials: M
authors.lastname: Stout
authors.forename: Zhanjun
authors.initials: Z
authors.lastname: Hou
authors.forename: Andreas
authors.initials: A
authors.lastname: Fulterer
authors.forename: Min-Hwang
authors.initials: MH
authors.lastname: Chang
authors.forename: Michael F
authors.initials: MF
authors.lastname: Romero
authors.forename: Christina
authors.initials: C
authors.lastname: Cherian
authors.forename: Aleem
authors.initials: A
authors.lastname: Gangjee
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems / Q000379:methods*; D005260:Female; D005493:Folic Acid Antagonists / Q000378:metabolism*; D006131:Growth Inhibitors / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D058979:Proton-Coupled Folate Transporter; D011687:Purines / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D011743:Pyrimidines / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D011758:Pyrroles / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D006131:Growth Inhibitors; D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; D011687:Purines; D011743:Pyrimidines; D011758:Pyrroles; C450639:Pyrrolo(2,3-d)pyrimidine; C505058:SLC46A1 protein, human