Abstract 4791: Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter
Authors: Wilson Mike R. , Ravindraa Manasa P. , Wallace-Povirk Adrianne , Polin Lisa , Hou Zhanjun , Gangjee Aleem , Matherly Larry H. 
Published: July 2016

The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) á delivers folate substrates into the cell via endocytosis. FRá and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRá and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRá (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRá without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 ìM) and 5-aminoimidazole-4-carboxamide (320 ìM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRá and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors.

Citation Format: Mike R. Wilson, Manasa P. Ravindraa, Adrianne Wallace-Povirk, Lisa Polin, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4791.