A gene-nutrient interaction between vitamin B6 and serine hydroxymethyltransferase (SHMT) affects genome integrity in Drosophila.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMTRNAi . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans.

Basic biochemical characterization of cytosolic enzymes in thymidine nucleotide synthesis in adult rat tissues: implications for tissue specific mitochondrial DNA depletion and deoxynucleoside-based therapy for TK2-deficiency.

BACKGROUND: Deficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues. RESULTS: In addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways. CONCLUSION: These results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders.

Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in Trypanosoma brucei: Intracellular Localization and Properties of the Enzyme.

Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2'-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism.IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5'-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.

Targeting Kinetoplastid and Apicomplexan Thymidylate Biosynthesis as an Antiprotozoal Strategy.

Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact on human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process, the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.

Toward a better understanding of folate metabolism in health and disease.

Folate metabolism is crucial for many biochemical processes, including purine and thymidine monophosphate (dTMP) biosynthesis, mitochondrial protein translation, and methionine regeneration. These biochemical processes in turn support critical cellular functions such as cell proliferation, mitochondrial respiration, and epigenetic regulation. Not surprisingly, abnormal folate metabolism has been causally linked with a myriad of diseases. In this review, we provide a historical perspective, delve into folate chemistry that is often overlooked, and point out various missing links and underdeveloped areas in folate metabolism for future exploration.

Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier.

Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N5-N10-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity.

Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability.

Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5-35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.

Arsenic trioxide targets MTHFD1 and SUMO-dependent nuclear de novo thymidylate biosynthesis.

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.

Alcohol Dehydrogenase 5 Is a Source of Formate for De Novo Purine Biosynthesis in HepG2 Cells.

Background: Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism.Objective: This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells.Methods:ADH5 knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [14C]-formate to [3H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [14C]-deoxyuridine to [3H]-thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis.Results: The [14C]-formate-to-[3H]-hypoxanthine ratio was greater in ADH5 knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%; P < 0.001) and folate sufficiency (+22%; P = 0.02). These data indicate that ADH5 deficiency increases the use of exogenous formate for de novo purine biosynthesis. The [14C]-deoxyuridine-to-[3H]-thymidine ratio did not differ between ADH5 knockout and wild-type cells, indicating that ADH5 deficiency does not affect de novo dTMP synthesis capacity relative to salvage synthesis. Under folate deficiency, ALDH2 knockdown cells exhibited a 37% lower ratio of [14C]-formate to [3H]-hypoxanthine (P < 0.001) compared with wild-type HepG2 cells, indicating decreased use of exogenous formate, or increased endogenous formate synthesis, for de novo purine biosynthesis.Conclusions: In HepG2 cells, ADH5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Formate is also shown to be limiting in the growth of HepG2 cells.

Targeting nuclear thymidylate biosynthesis.

Thymidylate (dTMP) biosynthesis plays an essential and exclusive function in DNA synthesis and proper cell division, and therefore has been an attractive therapeutic target. Folate analogs, known as antifolates, and nucleotide analogs that inhibit the enzymatic action of the de novo thymidylate biosynthesis pathway and are commonly used in cancer treatment. In this review, we examine the mechanisms by which the antifolate 5-fluorouracil, as well as other dTMP synthesis inhibitors, function in cancer treatment in light of emerging evidence that dTMP synthesis occurs in the nucleus. Nuclear localization of the de novo dTMP synthesis pathway requires modification of the pathway enzymes by the small ubiquitin-like modifier (SUMO) protein. SUMOylation is required for nuclear localization of the de novo dTMP biosynthesis pathway, and disruption in the SUMO pathway inhibits cell proliferation in several cancer models. We summarize evidence that the nuclear localization of the dTMP biosynthesis pathway is a critical factor in the efficacy of antifolate-based therapies that target dTMP synthesis.

Formate: The Neglected Member of One-Carbon Metabolism.

Formate, the only non-tetrahydrofolate (THF)-linked intermediate in one-carbon metabolism, is produced in mammals from a variety of metabolic sources. It occurs in serum of adults at a concentration of approximately 30 µM. Its principal function lies as a source of one-carbon groups for the synthesis of 10-formyl-THF and other one-carbon intermediates; these are primarily used for purine synthesis, thymidylate synthesis, and the provision of methyl groups for synthetic, regulatory, and epigenetic methylation reactions. Although formate is largely produced in mitochondria, these functions mostly occur in the cytoplasm and nucleus. Formate plays a significant role in embryonic development, as evidenced by the effectiveness of formate in the pregnant dam's drinking water on the incidence of neural tube defects in some genetic models. High formate concentrations in fetal lambs may indicate a role in fetal development and suggest that extracellular formate may play a role in the interorgan distribution of one-carbon groups.

Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

MTHFD1 regulates nuclear de novo thymidylate biosynthesis and genome stability.

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.

An unprecedented mechanism of nucleotide methylation in organisms containing thyX.

In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target; however, the lack of mechanistic understanding prohibits mechanism-based drug design. Here, we report trapping and characterization of two consecutive intermediates, which together with previous crystal structures indicate that the enzyme's reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. Furthermore, these results corroborate an unprecedented activation of the nucleotide that involves no covalent modification but only electrostatic polarization by the enzyme's active site. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target.

The 1,2-Diaminocyclohexane Carrier Ligand in Oxaliplatin Induces p53-Dependent Transcriptional Repression of Factors Involved in Thymidylate Biosynthesis.

Platinum-based chemotherapeutic drugs are widely used as components of combination chemotherapy in the treatment of cancer. One such drug, oxaliplatin, exerts a synergistic effect against advanced colorectal cancer in combination with 5-fluorouracil (5-FU) and leucovorin. In the p53-proficient colorectal cancer cell line HCT116, oxaliplatin represses the expression of deoxyuridine triphosphatase (dUTPase), a ubiquitous pyrophosphatase that catalyzes the hydrolysis of dUTP to dUMP and inhibits dUTP-mediated cytotoxicity. However, the underlying mechanism of this activity has not been completely elucidated, and it remains unclear whether factors other than downregulation of dUTPase contribute to the synergistic effect of 5-FU and oxaliplatin. In this study, we found that oxaliplatin and dachplatin, platinum-based drugs containing the 1,2-diaminocyclohexane (DACH) carrier ligand, repressed the expression of nuclear isoform of dUTPase (DUT-N), whereas cisplatin and carboplatin did not. Oxaliplatin induced early p53 accumulation, upregulation of primary miR-34a transcript expression, and subsequent downregulation of E2F3 and E2F1. Nutlin-3a, which activates p53 nongenotoxically, had similar effects. Introduction of miR-34a mimic also repressed E2F1 and DUT-N expression, indicating that this miRNA plays a causative role. In addition to DUT-N, oxaliplatin repressed, in a p53-dependent manner, the expression of genes encoding enzymes involved in thymidylate biosynthesis. Consequently, oxaliplatin significantly decreased the level of dTTP in the dNTP pool in a p53-dependent manner. These data indicate that the DACH carrier ligand in oxaliplatin triggers signaling via the p53-miR-34a-E2F axis, leading to transcriptional regulation that ultimately results in accumulation of dUTP and reduced dTTP biosynthesis, potentially enhancing 5-FU cytotoxicity.

Targeting the de novo biosynthesis of thymidylate for the development of a PET probe for pancreatic cancer imaging.

The development of cancer-specific probes for imaging by positron emission tomography (PET) is gaining impetus in cancer research and clinical oncology. One of the hallmarks of most cancer cells is incessant DNA replication, which requires the continuous synthesis of nucleotides. Thymidylate synthase (TSase) is unique in this context because it is the only enzyme in humans that is responsible for the de novo biosynthesis of the DNA building block 2'-deoxy-thymidylate (dTMP). TSase catalyzes the reductive methylation of 2'-deoxy-uridylate (dUMP) to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. Not surprisingly, several human cancers overexpress TSase, which makes it a common target for chemotherapy (e.g., 5-fluorouracil). We envisioned that [(11)C]-MTHF might be a PET probe that could specifically label cancerous cells. Using stable radiotracer [(14)C]-MTHF, we had initially found increased uptake by breast and colon cancer cell lines. In the current study, we examined the uptake of this radiotracer in human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 and found predominant radiolabeling of cancerous versus normal pancreatic cells. Furthermore, uptake of the radiotracer is dependent on the intracellular level of the folate pool, cell cycle phase, expression of folate receptors on the cell membrane, and cotreatment with the common chemotherapeutic drug methotrexate (MTX, which blocks the biosynthesis of endogenous MTHF). These results point toward [(11)C]-MTHF being used as PET probe with broad specificity and being able to control its signal through MTX co-administration.

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis.

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.

Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency.

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.

Standing the test of time: targeting thymidylate biosynthesis in cancer therapy.

Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.