Abstract 3822: A tumor-targeted 5-pyrrolo[2,3-d]pyrimidine antifolate is a selective substrate for folate receptor ≤ and potent inhibitor of 5-amino-4-carboxamide formyltransferase in de novo purine nucleotide biosynthesis
Authors: Mitchell-Ryan Shermaine K. , Wang Yiqiang , Cherian Christina , Etnyre Erika , Hou Zhanjun , Gangjee Aleem , Matherly Larry H. 
Published: August 2012

Abstract
Clinically used antifolates are transported into cells by three major systems including the reduced folate carrier (RFC), folate receptor (FR)α, and proton-coupled folate transporter (PCFT). RFC is ubiquitiously expressed in normal tissues, however, expression of FRα and PCFT is more limited. FRα and PCFT are expressed in human tumors, suggesting their potentials for tumor targeting with cytotoxic antifolates selective for these systems. We previously identified 6-substituted pyrrolo[2,3-d]pyrimidine benozyl antifolates characterized by 3 (AG17) and 4 (AG23) carbons in the bridge region with potent antiproliferative activities toward human tumor cells. AG17 and AG23 were selectively transported by FRα over RFC and inhibited de novo purine biosynthesis at α-glycinamide ribonucleotide formyltransferase (GARFTase). We synthesized the corresponding 5-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolates with 2 to 6 bridge carbons and tested growth inhibition of Chinese hamster ovary sublines that express human FRα (RT16), PCFT (R2/PCFT4), or RFC (PC43-10). Studies were extended to include KB human tumor cells expressing all three transporters. The 5-substituted analog (AG127) with a 4 carbon bridge was the most active of the series and inhibited proliferation of FRα-expressing RT16 (IC50 = 3.1 nM) and KB (IC50 = 1.9 nM) cells. Inhibitory effects of AG127 toward RFC-expressing PC43-10 and PCFT-expressing R2/PCFT4 cells were substantially reduced (IC50s of 54 and 733 nM, respectively), establishing FRα selectivity. In KB cells, AG17, AG23 and AG127 bound to FRα with high affinity. The antiproliferative effects of both 5- and 6-substituted analogs (AG127 and AG17/AG23, respectively) were protected by excess adenosine but not thymidine at drug concentrations up to 1 μM. Whereas 5-amino-4-imidazole carboxamide (AICA) completely protected KB cells from the effects of AG17/AG23, establishing GARFTase inhibition, AICA protection was incomplete for AG127. This suggests that AG127 inhibits AICA ribonucleotide formyltransferase (AICARFTase), the 2nd folate-dependent step in de novo purine biosynthesis, as its primary target. Consistent with targeting AICARFTase, using an in situ GARFTase assay in intact KB cells, inhibition by AG127 was modest and far less than for AG17 and AG23. Treatment of KB cells with AG127 resulted in time- and dose-dependent accumulation of the AICARFTase substrate ZMP (AICA ribonucleotide), confirming AICARFTase inhibition. ZMP accumulation was accompanied by activation of AMPK and inhibition of mTOR downstream targets. AG127 is a prototype of an entirely new generation of tumor-targeted antifolates with solid tumor selectivity over chemotherapy drugs currently available.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3822. doi:1538-7445.AM2012-3822