Glycinamide Ribonucleotide Research Articles

Using Variable Data-Independent Acquisition for Capillary Electrophoresis-Based Untargeted Metabolomics.

Capillary electrophoresis coupled with tandem mass spectrometry (CE-MS/MS) offers advantages in peak capacity and sensitivity for metabolic profiling owing to the electroosmotic flow-based separation. However, the utilization of data-independent MS/MS acquisition (DIA) is restricted due to the absence of an optimal procedure for analytical chemistry and its related informatics framework. We assessed the mass spectral quality using two DIA techniques, namely, all-ion fragmentation (AIF) and variable DIA (vDIA), to isolate 60-800 Da precursor ions with respect to annotation rates. Our findings indicate that vDIA, coupled with the updated MS-DIAL chromatogram deconvolution algorithm, yields higher spectral matching scores and annotation rates compared to AIF. Additionally, we evaluated a linear migration time (MT) correction method using internal standards to accurately align chromatographic peaks in a data set. Postcorrection, the data set exhibited less than 0.1 min MT drifts, a difference mostly equivalent to that of conventional reverse-phase liquid chromatography techniques. Moreover, we conducted MT prediction for metabolites recorded in mass spectral libraries and metabolite structure databases containing a total of 469,870 compounds, achieving an accuracy of less than 1.5 min root mean squares. Our platform provides a peak annotation platform utilizing MT information, accurate precursor m/z, and the MS/MS spectrum recommended by the metabolomics standards initiative. Applying this procedure, we investigated metabolic alterations in lipopolysaccharide (LPS)-induced macrophages, characterizing 170 metabolites. Furthermore, we assigned metabolite information to unannotated peaks using an in silico structure elucidation tool, MS-FINDER. The results were integrated into the nodes in the molecular spectrum network based on the MS/MS similarity score. Consequently, we identified significantly altered metabolites in the LPS-administration group, where glycinamide ribonucleotide, not present in any spectral libraries, was newly characterized. Additionally, we retrieved metabolites of false-negative hits during the initial spectral annotation procedure. Overall, our study underscores the potential of CE-MS/MS with DIA and computational mass spectrometry techniques for metabolic profiling.

Dual Template Molecularly Imprinted Polymers Targeting Blockade of CD47 for Enhanced Macrophage Phagocytosis and Synergistic Antimetabolic Therapy.

Glycinamide ribonucleotide formyltransferase (GARFT) is an important enzyme in the folate metabolism pathway, and chemical drugs targeting GARFT have been used in tumor treatments over the past few decades. The development of novel antimetabolism drugs that target GARFT with improved performance and superior activity remains an attractive strategy. Herein, we proposed a targeted double-template molecularly imprinted polymer (MIP) for enhancing macrophage phagocytosis and synergistic antimetabolic therapy. The double-template MIP was prepared by imprinting the exposed peptide segment of the extracellular domain of CD47 and the active center of GARFT. Owing to the imprinted cavities on the surface of MIP, it can actively target cancer cells and mask the "do not eat me" signal upon binding to CD47 thereby blocking the CD47-SIRPα pathway and ultimately enhancing phagocytosis by macrophages. In addition, MIP can specifically bind to the active center of GARFT upon entry into the cells, thereby inhibiting its catalytic activity and ultimately interfering with the normal expression of DNA. A series of cell experiments demonstrated that MIP can effectively target CD47 overexpressed 4T1 cancer cells and inhibit the growth of 4T1 cells. The enhanced phagocytosis ability of macrophages-RAW264.7 cells was also clearly observed by confocal imaging experiments. In vivo experiments also showed that the MIP exhibited a satisfactory tumor inhibition effect. Therefore, this study provides a new idea for the application of molecular imprinting technology to antimetabolic therapy in conjunction with macrophage-mediated immunotherapy.

Competing Endogenous RNAs Crosstalk in Hippocampus: A Potential Mechanism for Neuronal Developing Defects in Down Syndrome.

Down syndrome (DS) is the most example of aneuploidy, resulting from an additional copy of all or part of chromosome 21. Competing endogenous RNAs (ceRNAs) play important roles in neuronal development and neurological defects. This study aimed to identify hub genes and synergistic crosstalk among ceRNAs in the DS fetal hippocampus as potential targets for the treatment of DS-related neurodegenerative diseases. We profiled differentially expressed long non-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), differentially expressed microRNAs (DEmiRNAs), and differentially expressed messenger RNAs (DEmRNAs) in hippocampal samples from patients with or without DS. Functional enrichment analysis and gene set enrichment analysis were performed, and chromosome 21-related ceRNA and protein-protein interaction networks were constructed. Additionally, the correlations between lncRNA-mRNA and miRNA-mRNA expression in the samples and HEK293T cells were validated. Our finding of changes in the expression of some key genes and ncRNAs on chromosome 21 in DS might not fully conform to the gene dosage hypothesis. Moreover, we found that four lncRNAs (MIR99AHG, PLCB4, SNHG14, GIGYF2) and one circRNA (hsa_circ_0061697) may competitively bind with three miRNAs (hsa-miR-548b-5p, miR-730-5p, and hsa-miR-548i) and subsequently regulate five mRNAs (beta-1,3-galactosyltransferase 5 [B3GALT5], helicase lymphoid-specific [HELLS], thrombospondin-2 [THBS2], glycinamide ribonucleotide transformylase [GART], clathrin heavy chain like 1 [CLTCL1]). These RNAs, whether located on chromosome 21 or not, interact with each other and might activate the PI3K/Akt/mTOR and Wnt signaling pathways, which are involved in autophagosome formation and tau hyperphosphorylation, possibly leading to adverse consequences of trisomy 21. These findings provide researchers with a better understanding of the fundamental molecular mechanisms underlying DS-related progressive defects in neuronal development.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

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 with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-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, which 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.

GART Functions as a Novel Methyltransferase in the RUVBL1/β-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer.

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/β-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/β-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.

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.

GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

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.

Stereoselective Synthesis of β-Glycinamide Ribonucleotide.

A diastereoselective synthesis of the β-anomer of glycinamide ribonucleotide (β-GAR) has been developed. The synthesis was accomplished in nine steps from D-ribose and occurred in 5% overall yield. The route provided material on the multi-milligram scale. The synthetic β-GAR formed was remarkably resistant to anomerization both in solution and as a solid.

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.

Free-Energy Profile Analysis of the Catalytic Reaction of Glycinamide Ribonucleotide Synthetase.

The second step in the de novo biosynthetic pathway of purine is catalyzed by PurD, which consumes an ATP molecule to produce glycinamide ribonucleotide (GAR) from glycine and phosphoribosylamine (PRA). PurD initially reacts with ATP to produce an intermediate, glycyl-phosphate, which then reacts with PRA to produce GAR. The structure of the glycyl-phosphate intermediate bound to PurD has not been determined. Therefore, the detailed reaction mechanism at the molecular level is unclear. Here, we developed a computational protocol to analyze the free-energy profile for the glycine phosphorylation process catalyzed by PurD, which examines the free-energy change along a minimum energy path based on a perturbation method combined with the quantum mechanics and molecular mechanics hybrid model. Further analysis revealed that during the formation of glycyl-phosphate, the partial atomic charge distribution within the substrate molecules was not localized according to the formal charges, but was delocalized overall, which contributed significantly to the interaction with the charged amino acid residues in the ATP-grasp domain of PurD.

PH Effects and Cooperativity among Key Titratable Residues for Escherichia coli Glycinamide Ribonucleotide Transformylase.

Human glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis pathway that has been extensively studied as an anticancer target. To some extent, inhibition of GAR Tfase selectively targets cancer cells over normal cells and inhibits purine formation and DNA replication. In this study, we investigated E. coli GAR Tfase, which shares high sequence similarity with the human GAR Tfase, and most functional residues are conserved. Herein, we aim to predict the pH-activity curve through a computational approach. We carried out pH-replica exchange molecular dynamics (pH-REMD) simulations to investigate pH-dependent functions such as structural changes, ligand binding, and catalytic activity. To compute the pH-activity curve, we identified the catalytic residues in specific protonation states, referred to as the catalytic competent protonation states (CCPS), which maintain the structure, keep ligands bound, and facilitate catalysis. Our computed population of CCPS with respect to pH matches well with the experimental pH-activity curve. To compute the microscopic pKa values in the catalytically active conformation, we devised a thermodynamic model that considers the coupling between protonation states of CCPS residues and conformational states. These results allow us to correctly identify the general acid and base catalysts and interpret the pH-activity curve at an atomistic level.

Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity.

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

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.

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.