Ribonucleotide Formyltransferase Research Articles

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.

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.

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 β 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.

18F]Fluorothymidine(FLT)-PET imaging of thymidine kinase 1 pharmacodynamics in non-small cell lung cancer treated with pemetrexed.

3070 Background: Imaging of tumor proliferation has been studied with FLT-PET in various tumor types including NSCLC. Pemetrexed inhibits thymidylate synthase(TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). TS inhibition upregulates the thymidine salvage pathway including relocalisation of ENT1 to membrane and TK1 activation as a transient “flare” response. We hypothesise that this can be detected as an increase in FLT tumoral uptake that subsequently decreases with reduced proliferation. This study was conducted to assess FLT uptake as an early pharmacodynamics(PD) marker of pemetrexed response. Methods: This was an open-label imaging study in 21 patients with Stage 3/4 NSCLC treated with pemetrexed and platinum-based chemotherapy. Patients underwent FLT PET/CT scan at baseline and 4h after administration of pemetrexed. Platinum component of treatment was administered on the day after second FLT scan for cycle 1. Plasma for TK1 activity expression were collected before each scan time point and analysed by ELISA. Percentage change in standardized uptake value (%ΔSUV) was calculated as [SUV(PET2) – SUV(PET1)]/ SUV(PET1)*100. Treatment response calculated by RECIST 1. 1 and survival data were collected. Results: 17 patients had evaluable PET/CT scans for pemetrexed response. Median percentage difference for SUVmean and SUVmax in tumour lesions increased by 3% and 10.3% respectively. 5 patients showed homogeneous FLT flare at 4h after pemetrexed, 2 patients had decrease, 10 patients had heterogeneous FLT response (regardless of platinum doublet). There was no significant correlation between plasma TK1 activity and FLT flare. At 9 weeks, 4 patients had partial response, 9 stable disease and 4 progressive disease. Baseline and weighted average ΔSUVmax were not associated with survival. The 5 patients with FLT flare in all lesions showed a median OS of 31 months, unlike the group with heterogenous or decrease uptake(15 months). FLT uptake in bone marrow and small bowel significantly increased at 4h (t test p = 0.004, p = 0.004, respectively) indicating increased thymidine salvage activity. Early FLT uptake was not predictive for tumour RECIST response or OS. In multivariable cox regression analysis, pre-treatment TK1 activity, adjusted for performance status, smoking history and age, independently affected survival in this group (p = 0.011). Conclusions: Early FLT flare at 4h was seen in NSCLC post pemetrexed administration indicating activation of thymidine salvage pathway. Median overall survival of patients with an FLT flare response was more than twice longer than patients with mixed or no response. However, the small sample size lacked power to show statistical significance in the OS comparison. Further studies should evaluate this and the relationship to other prognostic variables in a larger cohort of patients. Clinical trial information: NCRI UK badge 9249.

VGLL3 increases the dependency of cancer cells on de novo nucleotide synthesis through GART expression.

Vestigial-like family member 3 (VGLL3) is a member of the VGLL family that serves as cofactors for TEA-domain transcription factors. Although VGLL3 is involved in the proliferation of cancer cells, the molecular mechanisms underlying VGLL3-mediated cell proliferation remain largely unknown. In this study, we found that stable expression of VGLL3 in human lung cancer A549 cells affects glutamine metabolism and increases their dependency on de novo nucleotide synthesis for proliferation. Mechanistically, VGLL3 was found to induce the expression of GART, which encodes a trifunctional enzyme that catalyzes de novo purine synthesis from glutamine. GART knockdown and the glycinamide ribonucleotide synthase, aminoimidazole ribonucleotide synthase, and glycinamide ribonucleotide formyltransferase trifunctional protein (GART) inhibitor lometrexol repressed the proliferation and survival of A549 cells stably expressing VGLL3. Mesenchymal breast cancer BT549 cells and MDA-MB-231 cells showed high expression of VGLL3, and VGLL3 knockdown was found to reduce GART expression. Lometrexol also repressed the proliferation of these breast cancer cells, whereas addition of inosine monophosphate, an important metabolite downstream of GART, rescued this repression. Taken together, these results suggest that VGLL3 induces GART expression and thereby confers de novo nucleotide-dependent cell proliferation in cancer cells.

Identification of monomorphic and polymorphic genes associated with recessive fertility defects in Holstein cows reared in Kazakhstan

Haplotypes of candidate genes namely: apoptotic protease activating factor 1 (APAF1 p.Q579X or HH1), glycinamide ribonucleotide formyltransferase (GART or HH4), structural maintenance of chromosomes 2 (SMC2 or HH3), and haplotype cholesterol deficiency (HCD) genes associated with recessive fertility defects (loss of fertility) were investigated in imported Canadian Holstein cows reared at “Medeu Commerce” LLP breeding farm in Kazakhstan. The genotypic profiling of the APAF1/HH1, GART/HH4 fertility haplotype carriers was carried out by PCR-RFLP methods using BstC8I and Tru9I and MseI, while the genotypic profiling of the SMC2/HH3, and HCD fertility haplotype carriers was carried out using our own primer designed by internal primer marker methods. The study revealed that the PCR-RFLP diagnostic markers APAF1/HH1 and GART/HH4 for recessive fertility defects were monomorphic in the Canadian Holstein cows investigated. However, the diagnostic markers SMC2/HH3 and HCD fertility haplotype carriers (our own design diagnostic markers) were polymorphic, with frequencies of 3% and 11%, respectively, in the investigated Canadian Holstein cows. The study concluded that genetic monitoring of recessive fertility defects enables the timely identification of carriers of harmful lethal mutations, and control of the fertility haplotype elimination process.

Synthesis, characterization, in vitro cytotoxic activity and molecular docking of dinuclear gold(I) complexes with terephthalaldehyde bis(thiosemicarbazones)

Four new bis(thiosemicarbazones) were synthesized and, by reaction with AuI, four dinuclear gold(I) complexes were obtained. Cytotoxic activity of both the ligands and the complexes against NCI-H460 (lung), A2780 and A2780cisR (ovarian) cancer cell lines and normal renal LLC-PK1 cells has been evaluated. The results indicate that the ligands do not show antiproliferative activity in the cell lines evaluated and the NCI-H460 line is only sensitive to the complex bearing cyclohexyl substituents. On the other hand, the four complexes display high cytotoxic activity towards A2780 and A2780 cisR and the complex with thiosemicarbazide could circumvent resistance to cisplatin. By contrast, none of the complexes show significant inhibition against the normal renal cell line LLC-PK1. Docking studies with carboxamide ribonucleotide formyltransferase (AICARFT) and cyclin-dependent kinases (CDK2) for lung and ovarian cancer, respectively, show that the best binding affinity corresponds to complex 4, which is in agreement with the in vitro cytotoxicity.

P25.01 Metabolic Characterization of Drug Resistance to Antifolate in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy that develops due to asbestos exposure. MPM is a diffuse disease with a poor prognosis. MPM develops in the pleural cavity and is highly resistant to a number of therapeutics. A combination of pemetrexed (PMX) and cisplatin (CDDP) is the first-line chemotherapy that remains useful today. PMX inhibits the enzymes dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART), which are involved in purine and pyrimidine synthesis. However, the median survival is approximately 12 months after treatment with PMX and CDDP, and the response rate is 41.3%. Therefore, patients with MPM show an approximately 60% incidence of resistance to PMX and CDDP. The mechanism of CDDP resistance has been investigated in several studies; however, the mechanism of PMX resistance is poorly understood. There are reports of TYMS overexpression being involved in PMX resistance; on the other hand, there are also reports of a lack of link to clinical results. In a previous study, I reported that the levels of glycine and IMP may be potential biomarkers of chemotherapy with PMX. For this reason, I hypothesized that MPM cells might acquire PMX resistance via changes in metabolic pathways. I established an MPM cell line of PMX resistance and examined the expression levels of PMX-targeted enzymes and intracellular metabolites of acquired PMX-resistant cell lines. In the first study, I changed medium the concentration of PMX was added to increase by 10% so that two PMX-resistant cell lines. Viable cells by WST-8 assay and the dose response curve. In a second study, I investigated the mRNA expression levels of parental cell lines and PMX-resistant cell lines to investigate whether PMX-resistant cell lines might change the expression levels of PMX-targeted enzymes and other enzymes. In the third study, I examined whether differences in intracellular metabolites between PMX-resistant and parent cell lines in the presence of PMX by metabolome analysis. MPM cells in the presence of PMX for a long time, and then established two PMX-resistant cell lines. These cell lines were extremely resistant to PMX. Next, I confirmed that increased TYMS expression levels by RT-qPCR when MPM cells exposed to PMX for a long time; however, DHFR and GART expression levels not change . Furthermore, expression levels of RFC and multidrug resistance protein (MRP) did not change . Next, I analyzed PMX-resistance and parent cell lines of intracellular metabolites in the presence of PMX, and found that glycine and adenosine changed between parental cell lines and PMX-resistant cell lines in the presence of PMX. In this study, I showed that TYMS expression levels, glycine and adenosine were changed by acquired PMX-resistance. Therefore, we think that finding regulatory genes of TYMS expression and glycine, adenosine production might elucidate the mechanism of drug resistance . I report these results, I would like to discuss.

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.

The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model of ATIC deficiency and ZMP accumulation

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFβ and other cellular processes.

Abstract 511: Targeted one-carbon (1C) metabolism inhibitors: Design, synthesis and biological evaluation of novel 5-substituted pyrrolo[3,2-d]pyrimidines with pyridyl- and ortho-fluoropyridyl glutamate side chains as selective folate receptors substrates

Abstract One-carbon (1C) metabolism, in tumor cells, provides basic metabolites needed for proliferation and is compartmentalized into the cytosol and mitochondria. The key enzymes in 1C metabolism are de novo purine biosynthesis catalyzing enzymes, glycinamide ribonucleotide formyl transferase (GARFTase) and 5-​aminoimidazole-​4-​carboxamide ribonucleotide formyltransferase (AICARFTase) and mitochondrial serine hydroxymethyltransferase 2 (SHMT2) involved in glycine synthesis and maintaining 1C pools. We have previously designed and evaluated a series of multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates with a phenyl glutamate side-chain (AGF291, AGF300 and AGF299), which inhibited KB tumor cell growth. AGF291 inhibited the enzymes crucial for both cytosolic and mitochondrial 1C metabolism. However, these compounds also were transported via the ubiquitously expressed reduced folate carrier (RFC) which can lead to dose-limiting toxicities as seen for classical antifolates such as pemetrexed (PMX). In this study, we designed conformationally restricted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates with pyridyl glutamate side-chains (AGF363, AGF369 and AGF370) and ortho-fluoropyridyl glutamate side-chains (AGF377 and AGF379) to decrease normal cell uptake by the ubiquitously present RFC and maintain or increase transport via tumor-selective folate receptor α (FRα) and folate receptor β (FRβ). Replacing the phenyl with the pyridyl side-chain abrogated the transport via RFC, thereby improved (AGF363 vs AGF291) or maintained (AGF369 vs AGF300 and AGF370 vs AGF299) selective uptake by FRα and FRβ over RFC and showed potent KB tumor cell inhibition (AGF363, IC50 = 10.78 nM and AGF369, IC50 = 6.97 nM). The ortho-fluorinated pyridyl analogs were also selective for FRα and FRβ over RFC but showed moderate potency towards KB tumor cells. Overall, this study identified two key pharmacophores, i.e. the pyridyl and the o-fluoropyridyl side-chains, to obtain absolute selectivity for FRα and FRβ over RFC in the 5-substituted pyrrolo[3,2-d]pyrimidine series of 1Cmetabolism inhibitors. Further preclinical studies are underway to afford viable candidates for future clinical application of selective compounds without toxicities as an improvement of currently used agents. Citation Format: Arpit Doshi, Adrianne Wallace-Povirk, Carrie O'Connor, Zhanjun Hou, Larry Matherly, Aleem Gangjee. Targeted one-carbon (1C) metabolism inhibitors: Design, synthesis and biological evaluation of novel 5-substituted pyrrolo[3,2-d]pyrimidines with pyridyl- and ortho-fluoropyridyl glutamate side chains as selective folate receptors substrates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 511.

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 -β over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRβ-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 -β over RFC. The N-substituted analogs 7–9 also preserved transport selectivity for FRα and -β over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRβ-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.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

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.

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.

Experimental characterization of two archaeal inosine 5'-monophosphate cyclohydrolases.

There is variability as to how archaea catalyze the final step of de novo purine biosynthesis to form inosine 5’-monophosphate (IMP) from 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR). Although non-archaea almost uniformly use the bifunctional PurH protein, which has an N-terminal IMP cyclohydrolase (PurH2) fused to a C-terminal folate-dependent aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (PurH1) domain, a survey of the genomes of archaea reveals use of PurH2 (with or without fusion to PurH1), the “euryarchaeal signature protein” PurO, or an unidentified crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of two representatives of the known IMP cyclohydrolase families. The locus TK0430 in Thermococcus kodakarensis encodes a PurO-type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes, the first functional characterization of a PurO from a non-methanogen. Kinetic characterization reveals a Km for FAICAR of 1.56 ± 0.39 μM and a kcat of 0.48 ± 0.04 s-1. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2-type IMP cyclohydrolase. This Archaeoglobus fulgidus PurH2 has a Km of 7.8 ± 1.8 μM and kcat of 1.32 ± 0.14 s-1, representing the first characterization of an archaeal PurH2 and the first characterization of PurH2 that naturally occurs unfused to an AICAR formyltransferase domain. Each of these two characterized IMP cyclohydrolases converts FAICAR to IMP in vitro, and each cloned gene allows the growth on purine-deficient media of an E. coli purine auxotroph lacking the purH2 gene.

Novel Pyrrolo[3,2-d]pyrimidine Compounds Target Mitochondrial and Cytosolic One-carbon Metabolism with Broad-spectrum Antitumor Efficacy.

Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, β-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

Abstract 811: Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic de novo purine synthesis enzymes as anti-tumor agents

Abstract Targeting cellular one-carbon (1C) metabolism in cancer cells with pyrrolo[3,2-d]pyrimidine analogs provides in vivo antitumor efficacy. We discovered a first-in-class series of 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347) with inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial 1C metabolism and a reported oncodriver. In addition, AGF291, AGF320 and AGF347 inhibited de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), and serine hydroxymethyltransferase 1 (SHMT1). In vivo studies demonstrated excellent antitumor efficacy for AGF347 against MIA PaCa-2 pancreatic adenocarcinoma xenografts in SCID mice with a median tumor growth delay (T-C) of >38 days for 4 mice, and 1 of 5 mice tumor-free 122 days post-treatment. AGF359, a new analog in this series, was a potent inhibitor of KB human tumor cell proliferation in vitro. AGF359 inhibition of KB human tumor cells was reversed with glycine and adenosine, establishing mitochondrial 1C metabolism and de novo purine biosynthesis as the targeted pathways; AICA plus glycine was incompletely protective, implicating AICARFTase as a direct cellular target. AGF359, like AGF291, AGF320, and AGF347, inhibited purified human SHMT2 (Ki = 0.399 ± 0.174 µM), SHMT1 (Ki = 0.70 ± 0.088 µM) and AICARFTase (Ki = 8.86 ± 2.83 µM). There was no inhibition of 5,10-methylene tetrahydrofolate dehydrogenase 2 (MTHFD2). Further structural modifications of these multi-targeted agents afforded pyrrolo[3,2-d]pyrimidine analogs AGF307 and AGF312. Cell-based glycine/nucleotide rescue experiments in KB tumor cells established that, as with AGF359, AGF307 and AGF312 were dual inhibitors of the mitochondrial 1C metabolism (AGF307) and de novo purine biosynthesis (AGF307, AGF312). These compounds are currently in further preclinical evaluation as a prelude to possible clinical development as antitumor agents. Citation Format: Md Junayed Nayeen, Khushbu Shah, Aamod Dekhne, Changwon Ning, Carrie O'Connor, Jade M. Katinas, Jennifer Wong, Zhanjun Hou, Charles E. Dann III, Larry H. Matherly, Aleem Gangjee. Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic de novo purine synthesis enzymes as anti-tumor agents [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 811.

Abstract 4794: Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs

Abstract Pancreatic cancer (PaC) represents the 4th leading cause of cancer-related deaths in the US with a mortality rate of 99%. The 5-year overall survival rate for PaC is currently 8%. One-carbon (C1) metabolism is frequently altered in cancer. For PaC, TCGA data sets show that elevated expression of key enzymes involved in cytosolic [5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and serine hydroxymethyltransferase (SHMT)1] and mitochondrial [SHMT2 and methylene tetrahydrofolate dehydrogenase 2 (MTHFD2)] C1 metabolism is associated with poor survival. Antifolate therapeutics disrupt cytosolic C1 pathways required for syntheses of thymidylate, purines, and certain amino acids, and are a mainstay for therapy of several cancers. Antifolate uptake into tumors and tissues involves the reduced folate carrier, the major tissue folate transporter, and the proton-coupled folate transporter (PCFT), which shows a more limited tissue distribution but is widely expressed in human solid tumors and is active only at acidic pHs characterizing the tumor microenvironment. We discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359) with PCFT transport that potently inhibited proliferation of PaC cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIA PaCa-2 and PANC-1), of which HPAC (KRAS mutant) and BxPC-3 (KRAS wild-type) cells were most sensitive. The PaC cell lines all expressed PCFT transcripts and proteins that were active for PCFT transport with 3H-AGF347 at acid pH. When HPAC cells were incubated with 3H-AGF347 over 48 h, drug accumulated in both cytosol and mitochondria. 3H-AGF347 was extensively metabolized to polyglutamates. Treatment of PaC cells with AGF347 and AGF359 inhibited proliferation by inducing glycine and adenosine auxotrophy that was rescued by excess glycine and adenosine. This implied that both mitochondria and cytosolic C1 metabolism was inhibited. Inhibition of mitochondrial SHMT2 and cytosolic SHMT1, glycinamide ribonucleotide formyltransferase and/or AICARFTase was confirmed by in vitro targeted metabolomics and assays with purified enzymes. Tumor cell killing was confirmed (with HPAC and BxPC-3) by colony-forming assays with AGF347 and AGF359 and drug-induced apoptosis with AGF347 was demonstrated (with HPAC) by annexin V-PI staining and flow cytometry. AGF347 and AGF359 depleted purine nucleotides and inhibited mTOR signaling via S6K1 at least in part (for BxPC-3) via activation of AMPK, likely due to elevated ZMP accompanying suppression of AICARFTase. Collectively, our studies identify first-in-class inhibitors and establish the considerable therapeutic potential of dual-targeting mitochondrial and cytosolic C1 metabolism in PaC independent of KRAS mutation status and reflecting cellular uptake by PCFT. Citation Format: Changwen Ning, Aamod Dekhne, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Josephine Frühauf, Xun Bao, Carrie O’Connor, Adrianne Wallace-Povirk, Jing Li, Charles E. Dann, Aleem Gangjee, Larry H. Matherly, Zhanjun Hou. Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs [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 4794.

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.