Structural Characterization of 5-Substituted Pyrrolo[3,2-d]pyrimidine Antifolate Inhibitors in Complex with Human Serine Hydroxymethyl Transferase 2.

We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3-5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.

Mitochondrial and cytosolic one-carbon metabolism is a targetable metabolic vulnerability in cisplatin-resistant ovarian cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared to normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin sensitive (A2780, CaOV3, IGROV1) and resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359 and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism that offers a promising new platform for therapy.

Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport.

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.

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 ß 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.

Multitargeted 6-Substituted Thieno[2,3-d]pyrimidines as Folate Receptor-Selective Anticancer Agents that Inhibit Cytosolic and Mitochondrial One-Carbon Metabolism.

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3-9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or ß but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1',4'-phenyl ring with 2',5'-pyridyl, or 2',5'-pyridyl with a fluorine insertion ortho to l-glutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4-9 were highly active (IC50's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17- to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.

Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

Biology and therapeutic applications of the proton-coupled folate transporter.

INTRODUCTION: The proton-coupled folate transporter (PCFT; SLC46A1) was discovered in 2006 as the principal mechanism by which folates are absorbed in the intestine and the causal basis for hereditary folate malabsorption (HFM). In 2011, it was found that PCFT is highly expressed in many tumors. This stimulated interest in using PCFT for cytotoxic drug targeting, taking advantage of the substantial levels of PCFT transport and acidic pH conditions commonly associated with tumors. AREAS COVERED: We summarize the literature from 2006 to 2022 that explores the role of PCFT in the intestinal absorption of dietary folates and its role in HFM and as a transporter of folates and antifolates such as pemetrexed (Alimta) in relation to cancer. We provide the rationale for the discovery of a new generation of targeted pyrrolo[2,3-d]pyrimidine antifolates with selective PCFT transport and inhibitory activity toward de novo purine biosynthesis in solid tumors. We summarize the benefits of this approach to cancer therapy and exciting new developments in the structural biology of PCFT and its potential to foster refinement of active structures of PCFT-targeted anti-cancer drugs. EXPERT OPINION: We summarize the promising future and potential challenges of implementing PCFT-targeted therapeutics for HFM and a variety of cancers.

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment.

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRß is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRß-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism.

The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.

Design, Synthesis, and Biological Evaluation of 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines as Microtubule Targeting Agents.

A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and SNAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC50 values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC50 values of 53-125 nM). Additionally, compounds 4-8, 10 and 12-13 circumvented Pgp and ßIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI50 of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates.

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure-activity relationship, in vitro and in vivo evaluation as antitumor agents.

The design, synthesis, and biological evaluation of a series novel N1­methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11-13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 µM, respectively. Compounds 14-16 inhibited the polymerization of tubulin with IC50s near ∼1 µM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented ßIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9-17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (ßIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.

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 FRß, 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.

Potential of substituted quinazolines to interact with multiple targets in the treatment of cancer.

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-ß. Low nanomolar inhibition of EGFR was observed for 1-3 and 9-10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer.

New therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. To identify new approaches for targeting EOC, metabolic vulnerabilities must be discovered and strategies for the selective delivery of therapeutic agents must be established. Folate receptor (FR) α and the proton-coupled folate transporter (PCFT) are expressed in the majority of EOCs. FRß is expressed on tumor-associated macrophages, a major infiltrating immune population in EOC. One-carbon (C1) metabolism is partitioned between the cytosol and mitochondria and is important for the synthesis of nucleotides, amino acids, glutathione, and other critical metabolites. Novel inhibitors are being developed with the potential for therapeutic targeting of tumors via FRs and the PCFT, as well as for inhibiting C1 metabolism. In this review, we summarize these exciting new developments in targeted therapies for both tumors and the tumor microenvironment in EOC.

The 3-D conformational shape of N-naphthyl-cyclopenta[d]pyrimidines affects their potency as microtubule targeting agents and their antitumor activity.

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6'-methoxynaphthyl-1'-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5'-methoxynaphthyl-2'-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.

Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer.

One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria, and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy, and "antifolates" targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, its relationship with the oncogenic phenotype, and its therapeutic potential for cancer.

Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism.

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.