A phase I open-label, dose-escalation study of NUC-3373, a targeted thymidylate synthase inhibitor, in patients with advanced cancer (NuTide:301).

PURPOSE: 5-fluorouracil (5-FU) is inefficiently converted to the active anti-cancer metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), is associated with dose-limiting toxicities and challenging administration schedules. NUC-3373 is a phosphoramidate nucleotide analog of fluorodeoxyuridine (FUDR) designed to overcome these limitations and replace fluoropyrimidines such as 5-FU. PATIENTS AND METHODS: NUC-3373 was administered as monotherapy to patients with advanced solid tumors refractory to standard therapy via intravenous infusion either on Days 1, 8, 15 and 22 (Part 1) or on Days 1 and 15 (Part 2) of 28-day cycles until disease progression or unacceptable toxicity. Primary objectives were maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) and schedule of NUC-3373. Secondary objectives included pharmacokinetics (PK), and anti-tumor activity. RESULTS: Fifty-nine patients received weekly NUC-3373 in 9 cohorts in Part 1 (n = 43) and 3 alternate-weekly dosing cohorts in Part 2 (n = 16). They had received a median of 3 prior lines of treatment (range: 0-11) and 74% were exposed to prior fluoropyrimidines. Four experienced dose-limiting toxicities: two Grade (G) 3 transaminitis; one G2 headache; and one G3 transient hypotension. Commonest treatment-related G3 adverse event of raised transaminases occurred in < 10% of patients. NUC-3373 showed a favorable PK profile, with dose-proportionality and a prolonged half-life compared to 5-FU. A best overall response of stable disease was observed, with prolonged progression-free survival. CONCLUSION: NUC-3373 was well-tolerated in a heavily pre-treated solid tumor patient population, including those who had relapsed on prior 5-FU. The MTD and RP2D was defined as 2500 mg/m2 NUC-3373 weekly. NUC-3373 is currently in combination treatment studies. TRIAL REGISTRATION: Clinicaltrials.gov registry number NCT02723240. Trial registered on 8th December 2015. https://clinicaltrials.gov/study/NCT02723240 .

The genetic landscape of a metabolic interaction.

While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focus on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We use deep mutational scanning to quantify the growth rate effect of 2696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

ARDS and aging: TYMS emerges as a promising biomarker and therapeutic target.

Background: Acute Respiratory Distress Syndrome (ARDS) is a common condition in the intensive care unit (ICU) with a high mortality rate, yet the diagnosis rate remains low. Recent studies have increasingly highlighted the role of aging in the occurrence and progression of ARDS. This study is committed to investigating the pathogenic mechanisms of cellular and genetic changes in elderly ARDS patients, providing theoretical support for the precise treatment of ARDS. Methods: Gene expression profiles for control and ARDS samples were obtained from the Gene Expression Omnibus (GEO) database, while aging-related genes (ARGs) were sourced from the Human Aging Genomic Resources (HAGR) database. Differentially expressed genes (DEGs) were subjected to functional enrichment analysis to understand their roles in ARDS and aging. The Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning pinpointed key modules and marker genes, with ROC curves illustrating their significance. The expression of four ARDS-ARDEGs was validated in lung samples from aged mice with ARDS using qRT-PCR. Gene set enrichment analysis (GSEA) investigated the signaling pathways and immune cell infiltration associated with TYMS expression. Single-nucleus RNA sequencing (snRNA-Seq) explored gene-level differences among cells to investigate intercellular communication during ARDS onset and progression. Results: ARDEGs are involved in cellular responses to DNA damage stimuli, inflammatory reactions, and cellular senescence pathways. The MEmagenta module exhibited a significant correlation with elderly ARDS patients. The LASSO, RRF, and XGBoost algorithms were employed to screen for signature genes, including CKAP2, P2RY14, RBP2, and TYMS. Further validation emphasized the potential role of TYMS in the onset and progression of ARDS. Immune cell infiltration indicated differential proportion and correlations with TYMS expression. SnRNA-Seq and cell-cell communication analysis revealed that TYMS is highly expressed in endothelial cells, and the SEMA3 signaling pathway primarily mediates cell communication between endothelial cells and other cells. Conclusion: Endothelial cell damage associated with aging could contribute to ARDS progression by triggering inflammation. TYMS emerges as a promising diagnostic biomarker and potential therapeutic target for ARDS.

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Sodium Butyrate Inhibits the Expression of Thymidylate Synthase and Induces Cell Death in Colorectal Cancer Cells.

The most commonly used chemotherapy for colorectal cancer (CRC) is the application of 5-fluorouracil (5-FU). Inhibition of thymidylate synthase (TYMS) expression appears to be a promising strategy to overcome the decreased sensitivity to 5-FU caused by high expression of TYMS, which can be induced by 5-FU treatment. Several compounds have been shown to potentially inhibit the expression of TYMS, but it is unclear whether short-chain fatty acids (SCFAs), which are naturally produced by bacteria in the human intestine, can regulate the expression of TYMS. Sodium butyrate (NaB) is the most widely known SCFA for its beneficial effects. Therefore, we investigated the enhancing effects on inhibition of cell viability and induction of apoptosis after co-treatment of NaB with 5-FU in two CRC cell lines, HCT116 and LoVo. This study suggests that the effect of NaB in improving therapeutic sensitivity to 5-FU in CRC cells may result from a mechanism that strongly inhibits the expression of TYMS. This study also shows that NaB inhibits the migration of CRC cells and can cause cell cycle arrest in the G2/M phase. These results suggest that NaB could be developed as a potential therapeutic adjuvant to improve the therapeutic effect of 5-FU in CRC.

The Association Between Thymidylate Synthase Gene Polymorphisms and the Risk of Ischemic Stroke in Chinese Han Population.

Family history of hypertension, smoking, diabetes and alcohol consumption and atherosclerotic plaque were identified as common risk factors in IS. We aimed at investigating the relationship between Thymidylate Synthase (TS) gene polymorphisms and ischemic stroke (IS).This case-control research selected and genotyped three single nucleotide polymorphisms (SNPs)of TS( rs699517, rs2790, and rs151264360) with Sanger sequencing in Chinese Han population. We also adopted logistic regression analysis in genetic models for calculating odds ratios and 95% confidence intervals. Genotype-Tissue Expression(GTEx) database analyzed the tissue-specific expression and TS polymorphisms. The ischemic stroke patients showed higher low-density lipoprotein cholesterol and total homocysteine (tHcy). It was found that patients with the TT genotype of rs699517 and GG genotype of rs2790 had larger degrees of tHcy than those with CC + CT genotypes and AA + AG genotypes, respectively. The genotype distribution of the three SNPs did not deviate from Hardy-Weinberg equilibrium (HWE). Haplotype analysis showed that T-G-del was the major haplotype in IS, and C-A-ins was the major haplotype in controls. GTEx database indicated that the rs699517 and rs2790 increased the expression of TS in healthy human and associated with TS expression level in a single tissue. In conclusion: This study has shown that TS rs699517 and rs2790 were significantly related to ischemic stroke patients.

Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?

The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.

Development and validation a radiomics nomogram for predicting thymidylate synthase status in hepatocellular carcinoma based on Gd-DTPA contrast enhanced MRI.

OBJECTIVES: The purpose of this study was to develop and validate a radiomics nomogram for predicting thymidylate synthase (TYMS) status in hepatocellular carcinoma (HCC) by using Gd-DTPA contrast enhanced MRI. METHODS: We retrospectively enrolled 147 consecutive patients with surgically confirmed HCC and randomly allocated to training and validation set (7:3). The TYMS status was immunohistochemical determined and classified into low TYMS (positive cells ≤ 25%) and high TYMS (positive cells > 25%) groups. Radiomics features were extracted from the arterial phases and portal venous phase of Gd-DTPA contrast enhanced MRI. Least absolute shrinkage and selection operator (LASSO) were applied for generating the Rad score. Clinical data and MRI findings were assessed to build a clinical model. Rad score combined with clinical features was used to construct radiomics nomogram. RESULTS: A total of 2260 features were extracted and reduced to 7 features as the most important discriminators to build the Rad score. InAFP was identified as the only independent clinical factors for TYMS status. The radiomics nomogram showed good discrimination in training (AUC, 0.759; 95% CI 0.665-0.838) and validation set (AUC, 0.739; 95% CI 0.585-0.860), and showed better discrimination capability (P < 0.05) compared with clinical model in training (AUC, 0.656; 95% CI 0.555-0.746) and validation set (AUC, 0.622; 95% CI 0.463-0.764). CONCLUSIONS: The radiomics nomogram shows favorable predictive efficacy for TYMS status in HCC, which might be helpful for the personalized treatment of HCC.

Thymidylate synthase promotes esophageal squamous cell carcinoma growth by relieving oxidative stress through activating nuclear factor erythroid 2-related factor 2 expression.

BACKGROUND: Thymidylate synthase (TYMS) is involved in the malignant process of multiple cancers, and has gained much attention as a cancer treatment target. However, the mechanism in carcinogenesis of esophageal squamous cell cancer (ESCC) is little reported. The present study was to clear the biological roles and carcinogenic mechanism of TYMS in ESCC, and explored the possibility to use TYMS as a tumor marker in diagnosis and a drug target for the treatment of ESCC. METHODS: Stably TYMS-overexpression cells established by lentivirus transduction were used for the analysis of cell proliferation. RNA sequencing was performed to explore the possible carcinogenic mechanisms. RESULTS: GEPIA databases analysis showed that TYMS expression in esophageal cancer tissues was higher than that in normal tissues. The MTT assay, colony formation assay, and nude mouse subcutaneous tumor model found that the overexpression of TYMS increased cell proliferation. Transcriptome sequencing analysis revealed that the promoted cell proliferation in TYMS-overexpression ESCC cells were mediated through activating genes expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 dependent antioxidant enzymes to relieve oxidative stress, which was confirmed by increased glutathione (GSH), glutathione peroxidase (GPX) activities, and reduced reactive oxygen species. Nrf2 active inhibitors (ML385) used in TYMS-overexpression cells inhibited the expression of Nrf2-dependent antioxidant enzyme genes, thereby increasing oxidative stress and blocking cell proliferation. CONCLUSION: Our study indicated a novel and effective regulatory capacity of TYMS in the cell proliferation of ESCC by relieving oxidative stress through activating expression of Nrf2 and Nrf2-dependent antioxidant enzymes genes. These properties make TYMS and Nrf2 as appealing targets for ESCC clinical chemotherapy.

Association of Thymidylate Synthase (TS) Gene Polymorphisms with Incidence and Prognosis of Coronary Artery Disease.

Coronary artery disease (CAD) is a prevalent cardiovascular condition characterized by the accumulation of plaque within coronary arteries. While distinct features of CAD have been reported, the association between genetic factors and CAD in terms of biomarkers was insufficient. This study aimed to investigate the connection between genetic factors and CAD, focusing on the thymidylate synthase (TS) gene, a gene involved in DNA synthesis and one-carbon metabolism. TS plays a critical role in maintaining the deoxythymidine monophosphate (dTMP) pool, which is essential for DNA replication and repair. Therefore, our research targeted single nucleotide polymorphisms that could potentially impact TS gene expression and lead to dysfunction. Our findings strongly associate the TS 1100T>C and 1170A>G genotypes with CAD susceptibility. We observed that TS 1100T>C polymorphisms increased disease susceptibility in several groups, while the TS 1170A>G polymorphism displayed a decreasing trend for disease risk when interacting with clinical factors. Furthermore, our results demonstrate the potential contribution of the TS 1100/1170 haplotypes to disease susceptibility, indicating a synergistic interaction with clinical factors in disease occurrence. Based on these findings, we propose that polymorphisms in the TS gene had the possibility of clinically useful biomarkers for the prevention, prognosis, and management of CAD in the Korean population.

Ensemble-based virtual screening of African natural products to target human thymidylate synthase.

Human thymidylate synthase (hTS) is a validated drug target for chemotherapy. A virtual screening experiment was used to prioritize a list of compounds from African Natural Products Databases docked against the orthosteric binding pocket of hTS. Consensus scores of binding affinities from ensemble-based virtual screening, hydrated docking and MM-PBSA calculations ranked compounds NEA4433 and NEA4434 as the best candidates owing to binding affinity scores in the picomolar order, their excellent ADMET profiles and the good stability of the protein-ligand complexes formed. The current study demonstrates the role of water in small molecule binding to hTS in mediating protein-ligand interactions. Similarly, the robust ensemble docking (relaxed scheme complex) ranked NEA4433 and NEA4434 as the best candidates. Furthermore, the best candidates prioritized were shown to strongly interact with the same residues that interacted with hTS substrate and cofactor.

Discovery of novel thymidylate synthase (TS) inhibitors that influence cancer angiogenesis and metabolic reprogramming in NSCLC cells.

Based on previous work, further search for more effective and less damaging thymidylate synthase (TS) inhibitors was the focus of this study. After further optimization of the structure, in this study, a series of (E)-N-(2-benzyl hydrazine-1-carbonyl) phenyl-2,4-deoxy-1,2,3,4-tetrahydro pyrimidine-5-sulfonamide derivatives were synthesized and reported for the first time. All target compounds were screened by enzyme activity assay and cell viability inhibition assay. On the one hand, the hit compound DG1 could bind directly to TS proteins intracellularly and promote apoptosis in A549 and H1975 cells. Simultaneously, DG1 could inhibit cancer tissue proliferation more effectively than Pemetrexed (PTX) in the A549 xenograft mouse model. On the other hand, the inhibitory effect of DG1 on NSCLC angiogenesis was verified both in vivo and in vitro. In parallel, DG1 was further uncovered to inhibit the expression of CD26, ET-1, FGF-1, and EGF by angiogenic factor antibody microarray. Moreover, RNA-seq and PCR-array assays revealed that DG1 could inhibit NSCLC proliferation by affecting metabolic reprogramming. Collectively, these data demonstrated that DG1as a TS inhibitor could be promising in treating NSCLC angiogenesis, deserving further investigation.

Delving in folate metabolism in the parasite Leishmania major through a chemogenomic screen and methotrexate selection.

Most of our understanding of folate metabolism in the parasite Leishmania is derived from studies of resistance to the antifolate methotrexate (MTX). A chemical mutagenesis screen of L. major Friedlin and selection for resistance to MTX led to twenty mutants with a 2- to 400-fold decrease in MTX susceptibility in comparison to wild-type cells. The genome sequence of the twenty mutants highlighted recurrent mutations (SNPs, gene deletion) in genes known to be involved in folate metabolism but also in novel genes. The most frequent events occurred at the level of the locus coding for the folate transporter FT1 and included gene deletion and gene conversion events, as well as single nucleotide changes. The role of some of these FT1 point mutations in MTX resistance was validated by gene editing. The gene DHFR-TS coding for the dihydrofolate reductase-thymidylate synthase was the second locus with the most mutations and gene editing confirmed a role in resistance for some of these. The pteridine reductase gene PTR1 was mutated in two mutants. The episomal overexpression of the mutated versions of this gene, but also of DHFR-TS, led to parasites several fold more resistant to MTX than those overexpressing the wild-type versions. Genes with no known link with folate metabolism and coding for a L-galactolactone oxidase or for a methyltransferase were mutated in specific mutants. Overexpression of the wild-type versions of these genes in the appropriate mutants reverted their resistance. Our Mut-seq approach provided a holistic view and a long list of candidate genes potentially involved in folate and antifolate metabolism in Leishmania.

Mitochondria-Targeted Thymidylate Synthase Inhibitor Based on Fluorescent Molecularly Imprinted Polymers for Tumor Antimetabolic Therapy.

Antimetabolites targeting thymidylate synthase (TS), such as 5-fluorouracil and capecitabine, have been widely used in tumor therapy in the past decades. Here, we present a strategy to construct mitochondria-targeted antimetabolic therapeutic nanomedicines based on fluorescent molecularly imprinted polymers (FMIP), and the nanomedicine was denoted as Mito-FMIP. Mito-FMIP, synthesized using fluorescent dye-doped silica as the carrier and amino acid sequence containing the active center of TS as the template peptide, could specifically recognize and bind to the active site of TS, thus inhibiting the catalytic activity of TS, and therefore hindering subsequent DNA biosynthesis, ultimately inhibiting tumor growth. The imprinting factor of FMIP reached 2.9, and the modification of CTPB endowed Mito-FMIP with the ability to target mitochondria. In vitro experiments demonstrated that Mito-FMIP was able to efficiently aggregate in mitochondria and inhibit CT26 cell proliferation by 59.9%. The results of flow cytometric analysis showed that the relative mean fluorescence intensity of Mito-FMIP accumulated in the mitochondria was 3.4-fold that of FMIP. In vivo experiments showed that the tumor volume of the Mito-FMIP-treated group was only one third of that of the untreated group. In addition, Mito-FMIP exibited the maximum emission wavelength at 682 nm, which allowed it to be used for fluorescence imaging of tumors. Taken together, this study provides a new strategy for the construction of nanomedicines with antimetabolic functions based on molecularly imprinted polymers.

Activity of pemetrexed in pre-clinical chordoma models and humans.

Chordomas are rare slow growing tumors, arising from embryonic remnants of notochord with a close predilection for the axial skeleton. Recurrence is common and no effective standard medical therapy exists. Thymidylate synthase (TS), an intracellular enzyme, is a key rate-limiting enzyme of DNA biosynthesis and repair which is primarily active in proliferating and metabolically active cells. Eighty-four percent of chordoma samples had loss of TS expression which may predict response to anti-folates. Pemetrexed suppresses tumor growth by inhibiting enzymes involved in folate metabolism, resulting in decreased availability of thymidine which is necessary for DNA synthesis. Pemetrexed inhibited growth in a preclinical mouse xenograft model of human chordoma. We report three cases of metastatic chordoma that had been heavily treated previously with a variety of standard therapies with poor response. In two cases, pemetrexed was added and objective responses were observed on imaging with one patient on continuous treatment for > 2 years with continued shrinkage. One case demonstrated tumor growth after treatment with pemetrexed. The two cases which had a favorable response had a loss of TS expression, whereas the one case with progressive disease had TS present. These results demonstrate the activity of pemetrexed in recurrent chordoma and warrant a prospective clinical trial which is ongoing (NCT03955042).

Virtual screening, structure based pharmacophore mapping, and molecular simulation studies of pyrido[2,3-d]pyrimidines as selective thymidylate synthase inhibitors.

Human thymidylate synthase is the rate-limiting enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate. dUMP (pyrimidine) and folate binding site hTS inhibitors showed resistance in colorectal cancer (CRC). In the present study, we have performed virtual screening of the pyrido[2,3-d]pyrimidine database, followed by binding free energy calculations, and pharmacophore mapping to design novel pyrido[2,3-d]pyrimidine derivatives to stabilize inactive confirmation of hTS. A library of 42 molecules was designed. Based on the molecular docking studies, four ligands (T36, T39, T40, and T13) were identified to have better interactions and docking scores with the catalytic sites [dUMP (pyrimidine) and folate binding sites] of hTS protein than standard drug, raltitrexed. To validate efficacy of the designed molecules, we performed molecular dynamics simulation studies at 1000 ns with principal component analysis and binding free energy calculations on the hTS protein, also drug likeness properties of all hits were in acceptable range. Compounds T36, T39, T40, and T13 interacted with the catalytic amino acid (Cys195), an essential amino acid for anticancer activity. The designed molecules stabilized the inactive conformation of hTS, resulting in the inhibition of hTS. The designed compounds will undergo synthesis and biological evaluation, which may yield selective, less toxic, and highly potent hTS inhibitors.Communicated by Ramaswamy H. Sarma.

TYMS promotes genomic instability and tumor progression in Ink4a/Arf null background.

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf, one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf -/- genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf -/- mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival.

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent.

INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Bacterial origins of thymidylate metabolism in Asgard archaea and Eukarya.

Asgard archaea include the closest known archaeal relatives of eukaryotes. Here, we investigate the evolution and function of Asgard thymidylate synthases and other folate-dependent enzymes required for the biosynthesis of DNA, RNA, amino acids and vitamins, as well as syntrophic amino acid utilization. Phylogenies of Asgard folate-dependent enzymes are consistent with their horizontal transmission from various bacterial groups. We experimentally validate the functionality of thymidylate synthase ThyX of the cultured 'Candidatus Prometheoarchaeum syntrophicum'. The enzyme efficiently uses bacterial-like folates and is inhibited by mycobacterial ThyX inhibitors, even though the majority of experimentally tested archaea are known to use carbon carriers distinct from bacterial folates. Our phylogenetic analyses suggest that the eukaryotic thymidylate synthase, required for de novo DNA synthesis, is not closely related to archaeal enzymes and might have been transferred from bacteria to protoeukaryotes during eukaryogenesis. Altogether, our study suggests that the capacity of eukaryotic cells to duplicate their genetic material is a sum of archaeal (replisome) and bacterial (thymidylate synthase) characteristics. We also propose that recent prevalent lateral gene transfer from bacteria has markedly shaped the metabolism of Asgard archaea.