Abstract #1684: Discovery of a glycinamide ribonucleotide formyltransferase inhibitor with solid tumor selectivity via its transport by the proton-coupled folate transporter
Authors: Sita Kugel Desmoulin, Yiqiang Wang, Jianmei Wu, Zhanjun Hou, Christina Cherian, Aleem Gangjee, Larry Matherly
Journal: Cancer Research (2009)

The human proton-coupled folate transporter (hPCFT) is a folate-proton symporter that functions optimally at low pH. hPCFT could be used as a means of selectively targeting antifolates, taking advantage of the acidic microenvironments of solid tumors. However, the therapeutic potential of hPCFT is unestablished and the ability to selectively target hPCFT over the ubiquitously expressed human reduced folate carrier (hRFC) has not been determined. Real-time RT-PCR was used to determine transcript levels of hPCFT in 42 solid tumor and 29 leukemia cell lines. hPCFT transcripts were significantly elevated in the majority of solid tumor cell lines but were generally low in the leukemia cell lines. To determine the functional properties of hPCFT and to create a model for screening novel hPCFT-targeted therapeutics, a transport-defective Chinese hamster ovary subline (R2) was stably transfected with Myc-His6-tagged hPCFT (R2-PCFT#4), and expression and functional transport at low pH were validated. Transport kinetics for  3 H-methotrexate ( 3 H-MTX) were established in R2-PCFT#4 cells and found to be pH dependent. Using a fluorescence-based cytotoxicity assay, with R2-PCFT#4 cells, we screened a series of 6-substituted pyrrolo[2,3- d ]pyrimidine antifolates with bridge length variations from 1-6 carbons. A novel analog was identified (compound 2) with an IC 50  value (~30 nM) comparable to the clinically relevant hPCFT substrate pemetrexed (PMX). However, unlike PMX, compound 2 had an IC 50  of greater than 1000 nM in hRFC-expressing pC43-10 cells. Transport inhibition of  3 H-MTX cellular uptake was measured for both compound 2 and PMX in R2-PCFT#4 and pC43-10 cells from pH 5.5-7.2. Compound 2 was equipotent to PMX in R2-PCFT#4 cells, with increased potency at low pHs. In hRFC-expressing pC43-10 cells, PMX competitively inhibited  3 H-MTX uptake at pH 7.2 and compound 2 was completely inert, once again demonstrating the selectivity of compound 2 for hPCFT over hRFC. To determine the mechanism of action for compound 2, cytotoxicity experiments were paired with adenosine (60 \#956;M), thymidine (10 \#956;M) or 5-amidazole-4-carboxamide (AICA) (320 \#956;M) treatments. Adenosine and AICA completely protected cells from compound 2, establishing glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) in the  de novo  purine biosynthetic pathway as the primary intracellular target. These results were substantiated by an  in situ  assay of GARFTase activity at pH 6.8 involving  14 C-glycine incorporation into  14 C-formyl GAR. In conclusion, we have discovered a novel GARFTase inhibitor that is specific for hPCFT over hRFC. This finding raises exciting new possibilities for tumor targeting, based on an inherent biological feature of solid tumors, namely their acidic microenvironments, for selective delivery of potent cytotoxic drugs.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1684.