Glycinamide Ribonucleotide Formyltransferase Inhibitor Research Articles

Structure-Based Design of Transport-Specific Multitargeted One-Carbon Metabolism Inhibitors in Cytosol and Mitochondria.

Multitargeted agents provide tumor selectivity with reduced drug resistance and dose-limiting toxicities. We previously described the multitargeted 6-substituted pyrrolo[3,2-d]pyrimidine antifolate 1 with activity against early- and late-stage pancreatic tumors with limited tumor selectivity. Structure-based design with our human serine hydroxymethyl transferase (SHMT) 2 and glycinamide ribonucleotide formyltransferase (GARFTase) structures, and published X-ray crystal structures of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC), SHMT1, and folate receptor (FR) α and β afforded 11 analogues. Multitargeted inhibition and selective tumor transport were designed by providing promiscuous conformational flexibility in the molecules. Metabolite rescue identified mitochondrial C1 metabolism along with de novo purine biosynthesis as the targeted pathways. We identified analogues with tumor-selective transport via FRs and increased SHMT2, SHMT1, and GARFTase inhibition (28-, 21-, and 11-fold, respectively) compared to 1. These multitargeted agents represent an exciting new structural motif for targeted cancer therapy with substantial advantages of selectivity and potency over clinically used antifolates.

Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors

We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3–9) with 3–4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRβ, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3–5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.

Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical antifolates with selective uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a “first-in-class” classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.

Abstract 2992: Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-d]pyrimidine antifolates

Abstract Folate-dependent one-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a number of metabolites critical to tumor propagation. Folates are taken up by the plasma membrane facilitative transporters, reduced folate carrier (RFC; major tissue transporter) and proton-coupled folate transporter (PCFT; narrow physiological niche, but commonly expressed in solid tumors), and then transported into mitochondria via the mitochondrial folate transporter (MFT; SLC25A32). Although drug-targeting of cytosolic 1CM remains a clinical mainstay for a variety of cancers, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in a host of different cancers and, overall, SHMT2 is the fifth-most differentially expressed metabolic enzyme in cancer versus normal tissues. Despite its unequivocal oncogenic importance and therapeutic potential, there are no clinically relevant inhibitors of SHMT2. In this study, we characterized cellular pharmacodynamics of novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320, and AGF347) which show in vitroantitumor efficacy toward H460 lung, HCT-116 colon, and MIA PaCa-2 pancreatic cancer cells. Inhibition of mitochondrial SHMT2 and cytosolic 1CM at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) by this series was established by in vitro targeted metabolomics in H460, HCT-116, and MIA PaCa-2 cells and in vitro cell-free assays with purified enzymes. By depleting SHMT2-derived formate, these compounds potentiate their own direct inhibition of GARFTase and AICARFTase. Depletion of adenine nucleotide pools in vitro by all compounds led to inhibition of mTOR signaling to S6K1 in HCT116 cells. Subcellular fractionation of MIA PaCa-2 and MFT-null and human MFT-transfected glyB Chinese hamster ovary cells confirmed synthesis of polyglutamyl forms of AGF347 in both cytosol and mitochondria with mitochondrial uptake of AGF347 in part mediated by MFT. Treatment by all compounds decreased the cellular GSH/GSSG ratio, indicating depleted ability to combat oxidative stress. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice (n=5) with median tumor growth delay (T-C) in 4 mice >38 days and 1 of 5 tumor-free survivors (cured). In vivo metabolomics on tumor xenografts confirmed inhibition of serine catabolism and purine biosynthesis. Collectively, our studies establish the exceptional therapeutic potential of inhibitors dual-targeting mitochondrial and cytosolic 1CM. Citation Format: Aamod Dekhne, Khushbu Shah, Gregory S. Ducker, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Arpit Doshi, Xun Bao, Hasini Kalpage, Jenney Liu, Seongho Kim, Adrianne Wallace-Povirk, Changwen Ning, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Jing Li, Maik Hüttemann, Joshua D. Rabinowitz, Charles E. Dann, Aleem Gangjee, Larry Matherly. Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2992.

Abstract 2798: Folate receptor-targeted cancer chemotherapy with novel 6-substituted thieno[2,3-d]pyrimidine compounds with nitrogen and fluorine containing aromatic side chains as de novo purine biosynthesis inhibitors

Abstract Targeted cancer chemotherapy inhibits the growth of cancer cells by interfering with critical pathways required for tumor progression. Three major transporters are involved in membrane transport of folates into mammalian cells and include the reduced folate carrier (RFC), folate receptors (FRs) α and β, and proton-coupled folate transporter (PCFT). The clinically used agents methotrexate, pemetrexed, raltitrexed and pralatrexate are all transported by the ubiquitously expressed RFC into both tumor cells and normal tissues, and hence are non-selective. The lack of tumor selectivity results in dose-limiting toxicity. We reported 6-substituted thieno[2,3-d]pyrimidine (6TP) compounds with a phenyl side-chain and bridge lengths of 2-8 carbons as potent inhibitors that are selectively transported by FRs over RFC and inhibit proliferation of FR-expressing KB tumor cells with low nanomolar IC50s. The most potent compound (AGF50), a thieno[2,3-d]pyrimidine with a 4-carbon bridge and a phenyl side chain, showed FR selectivity over RFC and potent inhibition of de novo purine biosynthesis at least in part due to inhibition of glycinamide ribonucleotide formyltransferase (GARFTase). In this report, fluorine was strategically placed on the aryl side chain which could induce conformational restriction due to possible intramolecular fluorine-hydrogen bonding interactions. We found that fluorine substitution on the phenyl side-chain, ortho (o-) to L-glutamate (AGF304) improved potency and selectivity for FRα (4.5-fold) and FRβ (20-fold) compared to AGF50, as reflected in inhibition of proliferation of Chinese hamster ovary cells expressing human FRs. The pyridine nitrogen can also form H-bonds with NH of the L- glutamate amide, which could improve the potency and selectivity of 6TPs with pyridine side-chains. Analogs with systematic pyridine side-chain modifications, as well as ortho fluorine substitution on the pyridine ring, were designed, modeled, and synthesized. The 2',5'-pyridine analog AGF132 was ~9-fold more active toward FRα-expressing cells but approximately equipotent toward FRβ. The fluorinated 2',5'-pyridine analog AGF305 was ~5-fold less potent for FRα and was >50-fold more potent for FRβ compared to AGF132. AGF132, AGF304 and AGF305 inhibited growth of KB human tumor cells approximately equivalent to AGF50. Whereas adenosine completely protected cells from growth inhibition by the 6TPs, protection with 5-aminoimidazole-4-carboxamide (AICA) was incomplete, suggesting inhibition at AICA ribonucleotide formyltransferase (AICARFTase), along with GARFTase. Our 6TPs with aryl side-chain modifications are slated for further preclinical studies to identify potential clinical candidates that target the de novo purine biosynthesis pathway. Citation Format: Nian Tong, Aleem Gangjee, Adrianne W. Povrik, Carrie O. Connor, Jianjun Hou, Larry Matherly. Folate receptor-targeted cancer chemotherapy with novel 6-substituted thieno[2,3-d]pyrimidine compounds with nitrogen and fluorine containing aromatic side chains as de novo purine biosynthesis inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2798.

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

Abstract 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.

Abstract 4393: Design and synthesis of novel conjugates for targeting the folate receptor: Exploiting cytotoxic GARFTase inhibitors for delivering additional chemotherapeutic payloads to cancer cells

Abstract Background: Antifolates targeting glycinamide ribonucleotide formyltransferase (GARFTase) disrupt cell division (mitosis) by inhibiting the de novo purine biosynthesis pathway. Recently, novel GARFTase inhibitors, exhibiting high folate receptor (FR) binding affinity and low affinity for the reduced folate carrier (RFC), have been explored as chemotherapeutic agents. Theoretically, these GARFTase inhibitors could also be used as FR-targeting ligands for delivering additional payloads to tumor types over-expressing the FR. In this poster, we will present our practical approach to explore the dual role of these new GARFTase inhibitors as cytotoxic agents and as targeting ligands for delivering tubulysins. Tubulysins are natural products isolated from myxobacterial species and are highly potent mitotic poisons that inhibit tubulin polymerization. Methods: We developed a general method for preparing dual drug/ligand conjugates possessing two disulfide-based self-immolative linker systems. In our synthetic protocol we relied on consecutive introduction of two heterosubstituted disulfide bonds. A GARFTase inhibitor was first connected to a hydrophilic spacer unit through the first disulfide-based linker system. The obtained conjugate contains also an S-9-fluorenylmethyl (Fm)-protected thiol in its molecular architecture. Treatment with a strong organic base resulted in spontaneous removal of the Fm moiety. The deprotected thiol reacted in situ with a propreatary thiophilic tubulysin derivative, present in the reaction mixture, and a second self-immolative disulfide-based linker system was formed. Results: Novel conjugates of GARFTase inhibitor with tubulysins were prepared using newly designed synthetic strategies. In a high-yielding chemoselective reaction we introduced a second disulfide bond into a molecular framework without destroying the pre-existing disulfide linker system. The versatility of this synthetic protocol was demonstrated by synthesizing several related drug conjugates. Conclusion: In the poster to be presented, we will disclose, for the first time, the structure and synthesis of novel conjugates of GARFTase inhibitors with tubulysins. The target compounds contain two self-immolative disulfide linker systems. The observed excellent in vivo activity in tumor xenografts demonstrates the dual role of GARFTase inhibitors as therapeutic agents and as FR-targeting ligands for simultaneously delivering two highly potent payloads. Citation Format: Iontcho R. Vlahov, Fei You, Hanna F. Klein, Paul J. Kleindl, Melissa Nelson, Marilynn Vetzel, Joseph A. Reddy, Christopher P. Leamon, Larry H. Matherly, Aleem Gangjee. Design and synthesis of novel conjugates for targeting the folate receptor: Exploiting cytotoxic GARFTase inhibitors for delivering additional chemotherapeutic payloads to cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2015-4393

Abstract 5403: Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT)

Abstract Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for first-line and maintenance therapy of NSCLC. Whereas correlations were reported between clinical responses in NSCLC and levels of folate receptor (FR) α, FRα is not a pharmacologically relevant PMX transporter. Rather, PMX is primarily transported by the reduced folate carrier (RFC) and PCFT. RFC is ubiquitously expressed, whereas PCFT shows more limited expression in normal tissues, although both are broadly expressed in tumor cells. Unlike RFC, PCFT is active at acidic pH, with significant levels at pH 6.8. Using patient cDNAs from normal lung (n = 8) and NSCLC samples (n = 37), we measured transcripts for RFC and PCFT, and for intracellular targets for PMX, including thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFTase), by real-time RT-PCR (qPCR). PCFT was detected at similar levels in normal lung and NSCLC specimens, while RFC was decreased in tumors compared to normal lung. Expression of TS and GARFTase were increased in NSCLC samples, compared to normal tissue. In NSCLC cell lines A549, CRL5872, H460, H1650 and H2030, PCFT and RFC, but not FRα, were detected by qPCR. We synthesized novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to PMX [AGF94 (2′,5′) and AGF154 (2′,4′)] as potential antitumor agents. Based on patterns of inhibition of proliferation of engineered Chinese hamster ovary cells expressing human RFC or PCFT, AGF94 and AGF154, unlike PMX, are not transported by RFC but are excellent substrates for PCFT. AGF94 and AGF154 showed potencies comparable to PMX toward all the NSCLC cell lines. We found that [3H]PMX and [3H]AGF154 were transported by PCFT into the NSCLC cell lines at pH 5.5 and pH 6.8 and uptake could be blocked by non-radioactive AGF94. AGF154 accumulation over sustained exposure at pH 5.5 was greater than that of PMX, whereas accumulations of AGF154 and PMX were nearly identical at pH 6.8. In H460 cells treated with PMX, thymidine (10 μM) partially protected cells from growth inhibition, whereas neither adenosine (10 μM) nor 5-aminoimidazole-4-carboxamide (AICA) (320 μM) were protective. In cells treated with AGF94 or AGF154, adenosine and AICA, but not thymidine, were completely protective, consistent with inhibition of de novo purine biosynthesis at GARFTase. Potent GARFTase inhibition by AGF94 and AGF154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR (FGAR), the GARFTase product. Our results suggest that novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating NSCLC, reflecting their selective membrane transport by PCFT over RFC and potent inhibition of de novo purine biosynthesis. Citation Format: Mike R. Wilson, Zhanjun Hou, Lei Wang, Si Wang, Manohar Ratnam, Aleem Gangjee, Larry Matherly. Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2015-5403

Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and as potential antitumor agents.

A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

Abstract 1620: Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates

Abstract Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) α and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates occurs in part, to their lack of selectivity for tumor cells over normal cells due to RFC transport. Antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. Our analog AG17F had shown selective FR and PCFT transport over RFC with excellent cell inhibitory activity in KB tumor cell line (IC50 = 1.7 nM). On the basis of AG17F, AG140F with sulfur in place of carbon at C10, AG182F with oxygen in place of carbon at C10 and AG183F with nitrogen in place of carbon at C10 were designed and synthesized. This isosteric replacement of carbon with heteroatoms in the bridge provides compounds with different chain lengths, angles, conformations and extra hydrogen bond donors and/or acceptors compared to the parent carbon chain analogues. AG17F had shown inhibitory activity toward the growth of a CHO cell line (hRFC) expressing human RFC (IC50 = 304 nM), the three heteroatom analogues AG140F, AG182F and AG183F were inactive in the CHO cell line expressing RFC transport at concentrations up to 1000 nM, but were highly active in inhibiting CHO cell lines (RT16) expressing human FRα (IC50s of 0.62, 1.10, and 2.3 nM respectively) and inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 267, 57.3, and 87.4 nM respectively). AG140F, AG182F, and AG183F were potently inhibitory toward KB tumor cells (IC50s of 2, 0.32 and 0.34 nM respectively) and IGROV1 tumor cells (IC50s of 0.51, 0.58 and 0.48 nM respectively). AG140F, AG182F, and AG183F showed binding affinities to FRα in RT16 cells exceeding that for AG17F, and comparable to folic acid. With R2/PCFT4 cells, all compounds showed high affinities with binding (reflected in Ki values) in the order of AG17F>AG182F∼AG183F>AG140F. In KB cells, adenosine and 5-aminoimidazole-4-carboxamide protected from growth inhibition suggesting de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase) and the likely targets. By in situ assays in KB cells, GARFTase inhibition was confirmed at concentrations similar to those which inhibit cell proliferation. AG183F was further evaluated for against early stage human IGROV tumor xenograft in female SCID mice models and was found to be remarkably efficacious (T/C = 12%) without any acute or long term toxicities. Citation Format: Lalit Kumar Golani, Aleem Gangjee, Christina Cherian, Steven Orr, Shermaine Mitchell-Ryan, Lisa Polin, Adrianne Wallace, Larry H. Matherly. Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2014-1620

Structure-activity profiles of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with modified amino acids for cellular uptake by folate receptors α and β and the proton-coupled folate transporter.

Structure–activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10–13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9–13 toward FRα- and FRβ-expressing CHO cells were only partly reflected in binding affinities to FRα and FRβ or in the docking scores with molecular models of FRα and FRβ. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.

Discovery of 5-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates as Dual-Acting Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase in De Novo Purine Nucleotide Biosynthesis: Implications of Inhibiting 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase to AMPK Activation and Antitumor Activity

We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.

Abstract 5494: Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis.

Abstract Epithelial ovarian cancers (EOC) represent a unique treatment challenge as over 50% of patients are diagnosed with advanced stage disease when surgical and chemotherapeutic interventions prove futile. Approximately 90% of EOC are folate receptor (FR) α positive and FR levels correlate with stage and grade. FRα-targeted therapies represent a rational treatment option for patients with advanced EOC whose treatments fail. There are three primary mechanisms of folate transport in mammalian cells, the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and FRs. RFC is the major transporter for folates and is expressed ubiquitously in normal tissues and tumors. Hence, developing novel antifolates that bypass cellular accumulation by RFC should reduce dose-limiting toxicities. PCFT is expressed in many solid tumors including ovarian cancer and is active at acidic pH. Previous studies established that a novel 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolate with an isosteric thienoyl substitution and a three carbon bridge (AG94) showed selective cellular uptake by FRα and PCFT over RFC. [1,3]Thienoyl regioisomers (AG150 and AG154) of AG94 showed subnanomolar potencies toward both KB nasopharyngeal carcinoma (IC50s = 0.1 and 0.09 nM, respectively) and IGROV1 ovarian carcinoma (IC50s of 0.64 and 0.1nM, respectively). While AG150 and AG154 were substrates for both PCFT and FRs, like AG94, their inhibitory effects at low drug concentrations were mediated primarily via FRα, as indicated by their capacities to bind with high affinities to FRα and protection by excess folic acid. Nucleoside protection assays with adenosine and 5-amino-4-imidazole carboxamide established that the first folate-dependent enzyme in the de novo purine biosynthesis pathway, glycinamide ribonucleotide (GAR) formyltransferase (GARFT), was the primary intracellular target for AG150 and AG154. GARFT targeting was confirmed by in situ metabolic labeling with [14C]glycine and measurement of accumulated [14C]formyl GAR in KB cells, for which IC50s were 1.35 and 1.46 nM, respectively. The purine salvage enzyme methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with CDKN2a in solid tumors including ovarian cancer. Growth inhibitory effects of AG150 and AG154 were reversed in the presence of methylthioadenosine in MTAP-deficient IGROV1 cells transfected with MTAP cDNA. Our results demonstrate potent anti-tumor activities of the 6-pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG94, AG150 and AG154, associated with their FRα-mediated cellular uptake and inhibition of GARFT in de novo purine biosynthesis. Preservation of purine salvage including MTAP is likely an important cellular determinant of antitumor efficacy for this class of novel tumor-targeted agents. Citation Format: Shermaine K. Mitchell-Ryan, Lei Wang, Steven Orr, Sita Kugel, Christina Cherian, Aleem Gangjee, Larry H. Matherly. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2013-5494

Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC). HeLa sublines expressing exclusively PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) and H2452 MPM cells were assayed for transport with [(3)H]compound 2. [(3)H]Polyglutamate metabolites of compound 2 were measured in R1-11-PCFT4 and H2452 cells. In vitro cell proliferation assays and colony formation assays were performed. Inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) was assayed by nucleoside protection assays and in situ GARFTase assays with [(14)C]glycine. In vivo efficacy was established with early- and advanced-stage H2452 xenografts in severe-combined immunodeficient (SCID) mice administered intravenous compound 2. [(3)H]Compound 2 was selectively transported by PCFT and was metabolized to polyglutamates. Compound 2 selectively inhibited proliferation of R1-11-PCFT4 cells over R1-11-RFC6 cells. H2452 human MPM cells were sensitive to the antiproliferative effects of compound 2. By colony-forming assays with H2452 cells, compound 2 was cytotoxic. Compound 2 inhibited GARFTase in de novo purine biosynthesis. In vivo efficacy was confirmed toward early- and advanced-stage H2452 xenografts in SCID mice administered compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.

Abstract 3826: Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

Abstract Pemetrexed (Pmx; Alimta) is among the most active agents for treating malignant pleural mesothelioma (MPM). Pmx is transported into cells by 3 systems, the reduced folate carrier (RFC), folate receptor (FR) β, and proton-coupled folate transporter (PCFT). RFC is present in all tissues and tumors, whereas PCFT and FRα are more narrowly expressed. By real-time RT-PCR, clinically-relevant MPM cell lines express both RFC and PCFT but not FRα. The reported clinical efficacy of Pmx toward MPM may partly reflect high levels of PCFT in this tumor. Novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with significant PCFT substrate activity but with greater specificities toward PCFT over RFC than Pmx, might offer significant advantages for selective therapeutic targeting MPM with reduced toxicity. We directly tested this notion for AG94, a 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate with a 3 carbon bridge. In HeLa sublines expressing moderate PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) levels, AG94 selectively inhibited (>35-fold) proliferation of PCFT-expressing R1-11-PCFT4 cells, in contrast to the nearly identical inhibitions with Pmx toward these sublines. H2452 human MPM cells showed similar sensitivities to the anti-proliferative effects of AG94 and Pmx (IC50s of 72 nM and 64 nM, respectively). Analogous results were obtained by colony-forming assays. Growth inhibition by AG94 was reversed with excess adenosine and 5-amino-4-imidazolecarboxamide but not thymidine. Potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase), the first folate-dependent step in de novo purine biosynthesis, was confirmed by an in situ GARFTase assay in both R-1-11-PCFT and H2452 cells. Assays with [3H]AG94 with engineered Chinese hamster ovary cells confirmed selective PCFT transport, characterized by an acidic pH optimum and transport superiority over Pmx at both pH 5.5 and pH 6.8. Analogous results were seen with R1-11-PCFT4 and H2452 cells. In both R1-11-PCFT4 and H2452 cells, [3H]Pmx and [3H]AG94 were metabolized to polyglutamates, including di- to heptaglutamyl forms. Efficacy trials with subcutaneous early and upstage H2452 tumors were performed in SCID mice administered AG94, compared to gemcitabine and cisplatin. AG94 showed antitumor efficacies superior to gemcitabine and cisplatin. All mice tolerated the treatment regimens well. Our results demonstrate potent antitumor efficacies of AG94 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamate conjugates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by potent antifolate drugs such as AG94 presents an exciting new opportunity for treating solid tumors including MPM over chemotherapy agents 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 3826. doi:1538-7445.AM2012-3826

Abstract 3823: 2-Methyl-6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates as selective folate receptor substrates, glycinamide ribonucleotide formyltransferase inhibitors and antitumor agents

Abstract The important role of reduced folates in one-carbon transfer reactions has rendered folate metabolism an attractive target in cancer chemotherapy for decades. Clinically used antifolates enter cells via folate uptake systems, such as the reduced folate carrier (RFC), folate receptors (FRs), and the proton-coupled folate transporter (PCFT). Toxicity of clinically used antifolates is due to their lack of selectivity for tumor cells, since these antifolates are transported by RFC which occurs in normal as well as tumor cells. FRs are not usually expressed in normal cells. However, the ≤ isoform of FR is abundantly expressed in certain tumor cells, such as ovarian, endometrial, etc. Thus, selective FR-targeted antifolates that also possess cytotoxic activity without transport by RFC would likely circumvent the major toxicities of currently used antifolates. 2-Methyl-6-substituted pyrrolo[2,3-d]pyrimidine antifolates were designed to evaluate the importance of the 2-amino group for transport by RFC, FR, and /or PCFT, as well as for inhibition of α-glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis, compared with the 2-amino analogs. The most active compound in our series, AG179, showed an IC50 of 1.6 nM in inhibiting proliferation of KB human tumor cells, and was ∼3-fold less potent than the corresponding 2-amino compound AG71 (0.55 nM). In Chinese hamster ovary (CHO) cells engineered to individually express the major folate transporters, AG179 also inhibited growth of FRα-expressing RT16 cells but was inactive toward RFC-expressing PC43-10 and PCFT-expressing R2/PCFT4 CHO cells. The 2-amino analog, AG71, is inert toward RFC but is avidly transported by PCFT and is cytotoxic toward PCFT-expressing cells. In KB cells, growth inhibition by AG179 like AG71 is protected by excess adenosine and 5-amino-4-imidazole carboxamide, establishing GARFTase and de novo purine biosynthesis as the likely intracellular targets. These results establish that replacement of the 2-amino group of AG71 with a methyl group has a limited impact on FRα uptake and anti-proliferative activity, however, this replacement has a profound impact on cellular uptake by PCFT. This report will present the details of the design, synthesis, biological evaluation and molecular modeling of the title compounds. 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 3823. doi:1538-7445.AM2012-3823

Abstract 1364: Design, synthesis and evaluation of potent antiproliferative and cytotoxic antifolates with selective FRs and PCFT transport over RFC and potent GARFTase inhibition

Abstract Lack of selectivity for folate transporters is one of the major causes of toxicity and chemotherapy failure with clinically used antifolates. Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT), account for most of the folate influx activity in mammalian cells. We recently reported potent inhibition of cell proliferation by a classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine with a thienoyl for benzoyl-substituted side chain and a 4-carbon bridge (AG71). AG71 was selective for transport by FRs and PCFT over RFC and inhibited glycinamideribonucleotide formyl transferase (GARFTase) in de novo purine biosynthesis. In SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. A series of five thienoyl regioisomers of AG71 with [1,3] (AG117 and AG118) and [1,2] (AG114, AG115, AG116) thienoyl substitutions by the 4-carbon bridge and the L-glutamate were synthesized. Analogs were evaluated for anti-proliferative activities in engineered Chinese hamster ovary (CHO) cells expressing RFC, FR, or PCFT to determine the optimum structures for selective transport by FRs and/or PCFT. Additional studies were performed in KB and IGROV1 tumor cells, using inhibition of cell proliferation or colony formation as metrics. Like AG71, none of the novel thienyol regioisomers were substrates for RFC. AG117 and AG118 were potently active toward FR- and PCFT-expressing CHO cells and toward KB and IGROV1 tumor cells, equivalent or exceeding levels for AG71. In colony-forming assays in KB cells, AG117 and AG118 were cytotoxic. AG114, AG115, and AG116 were substantially less active or completely inert. AG117 and AG118, like AG71, were high affinity ligands for FRα by competitive ligand binding with 3H-folic acid, and were potent substrates for PCFT by direct competition 3H-methotrexate transport. By protection assays, AG71 and AG117 preserved potent anti-proliferative activity even in the presence of 50 nM leucovorin; adenosine and 5-imidazole-4-carboxamide were completely protective, establishing the de novo purine biosynthetic pathway and GARFTase as the principal intracellular targets. GARFTase inhibition was confirmed by in situ GARFTase metabolic assays with 14C-glycine as a radiotracer. Our results identify 6-subsituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG71 as potent anti-proliferative and cytotoxic antifolates, reflecting selective transport by PCFT and FR over RFC and inhibition of GARFTase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1364. doi:10.1158/1538-7445.AM2011-1364

Abstract 3250: Design, synthesis and biological evaluation of side chain pemetrexed (PMX) analogues as potent inhibitors of tumors expressing folate receptors via GARFTase inhibition

Abstract Two major hurdles in cancer therapy are the inability of chemotherapy agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The most active of this series (AG71) included a 4 carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) (but not RFC) whereupon it inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 was highly active toward both KB and IGROV1 tumors; in SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine structure of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogs with 1 to 6 carbon atoms in the bridge (AG133-AG138) as hybrid molecules of PMX and AG71. The compounds were synthesized via a Heck coupling of the thiophene iodide with the alkene alcohols followed by α-bromonation and cyclization with the 2,4-diamino-6-oxo-pyrimidine to afford the 5-substituted pyrrolo[2,3-d]pyrimidine and elaborated to the L-glutamate. These analogs could be envisaged to afford the multi-targeted attributes of PMX, while preserving FRα and/or PCFT specificity of our 6-substituted analogs. Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). Analogs with 4 and 5-carbon atom chains (AG136 and AG137) showed IC50 values of 17.1 nM and 18.1 nM, respectively, against KB tumor cells in culture; other analogs of this series were 13- to >58-fold less active. In the CHO sublines, AG136 and AG137 were growth inhibitory in the order RT-16 = PC43-10 > R2/PCFT4 and RT-16 >> PC43-10 = R2/PCFT4, respectively. Thus, at least for AG136 unlike AG71, significant RFC activity is preserved. Whereas PMX is a multi-targeted agent with inhibitory effects on both de novo thymidylate and purine nucleotide biosynthetic pathways, the anti-proliferative effects of AG136 and AG137 were abolished by adenosine but not thymidine, establishing exclusive inhibition of purine biosynthesis. Our results suggest that 6-subtitution of the pyrrolo[2,3-d]pyrimidine scaffold is more effective in conferring selective transport of FRα over RFC, although inhibition of de novo purine biosynthesis is preserved in the 5-substituted compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3250. doi:10.1158/1538-7445.AM2011-3250

ERK Suppression in DU145 Cells Selected for AG2034 Resistance is Associated with Decreased ATP, GARFT Inactivation and Increased ERK Signaling

Background: Previously, we showed that AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) in the pathway for de novo purine synthesis, is cytotoxic to different prostate cancer cell lines in the absence of inosine and hypoxanthine in the culture media. Therefore, we examined DU145 androgen- independent prostate cancer cells selected for drug resistance over a prolonged period of time, in the absence of media components (inosine and hypoxanthine) required for salvage purine synthesis and in the continuous presence of AG2034. Methods and Results: DU145 cells were selected over time for 4-(2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H- pyrimidino(5,4,6)(1,4) thiazin-6-yl)-(S)-ethyl)-2,5-thienoylamino-L-glutamic acid (AG2034) resistance by gradually decreasing hypoxanthine concentrations from 1.7 μM to zero in the continuous presence of 10 nM 5-methyl tetrahydrofolate and 50 nM AG2034. Since the inhibition of GARFT blocks glycine incorporation into the purine ring, total cellular ATP was quantified by reverse-phase HPLC and ( 14 C)-glycine incorporation into ATP was determined by liquid scintillation counting of HPLC-isolated ATP fractions. Total and phosphorylated proteins were determined by western blotting. Quantitative RT-PCR was utilized to characterize mRNA expression levels before and after transient transfection studies with small interfering RNA (siRNA). We show that the exposure of parental DU145 cells to AG2034, an inhibitor of GARFT, leads to rapid enzyme inhibition and depletion of ATP. However, resistant DU145 cells are able to re-activate GARFT, restore ATP pools and increase mRNA expression and phosphorylation of ERK1/2 thus, stimulating drug-resistant growth. Moreover, siRNA knockdown of ERK1/2 mRNA in resistant DU145 cells inhibits ERK protein expression and phosphorylation and results in the depletion of ATP and the inhibition of GARFT activity in the absence of changes in GARFT mRNA and protein expression levels. Conclusions: The inhibition of ERK1/2 signaling by siRNA transfection affects AG2034 resistance by inhibiting GARFT activity and depleting ATP pools. Hence, we conclude that ERK plays an important role in the maintenance of the AG2034-resistant DU145 phenotype.

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

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.