Thymidylate Synthase Activity Research Articles

An insight into thymidylate synthase inhibitor as anticancer agents: an explicative review.

Cancer, a widespread challenge to global health, remains a puzzle of intricate molecular dynamics. This review article delves into the mystery of cancer, with a keen focus on understanding the contributory role of thymidylate synthase (TS) in cancer. TS, a vital enzyme in DNA synthesis and repair, emerges as a significant player in the narrative of cancer development. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is a major step in producing DNA. Numerous malignancies, including those of the breast, colon, lung, and ovary, have been linked to dysregulation of TS activity. Overexpression or mutations of TS lead to uncontrolled cell proliferation and tumorigenesis molecular interactions and signalling pathways involving TS come under scrutiny, revealing the nuanced connections that propel its involvement in cancer progression. Beyond overexpression and mutations, there emerges a subtle layer of regulation that involves microRNAs (miRNAs). These tiny particles attach to the TS messenger RNA, causing translational repression or its degradation, which in turn affects TS activity. Moving towards the therapeutic realm, thymidylate synthase inhibition acts as a promising anti-cancer strategy. Targeting TS with small-molecule inhibitors could provide a novel approach to treat various cancers. By reducing the number of available nucleotides, TS inhibition would slow down or halt cancer cell division, thus depriving the tumor of the building blocks required for its proliferation and growth. The aim is to assess the viability and effectiveness of targeting TS to halt or slow down cancer progression. There is growing evidence that, in comparison to traditional TS inhibitors, few novel antifolate TS inhibitors are effective against a wider variety of neoplasms, such as lung carcinomas. It has been discovered that TS inhibitors increase cancer tissues' sensitivity to chemotherapy and radiation, increasing their vulnerability to these treatments. This article aims to provide a comprehensive insight into TS, examining its cellular details, detailing the heterocyclic moieties and molecular foundations, and providing a promising future outlook.

Review of 5-FU resistance mechanisms in colorectal cancer: clinical significance of attenuated on-target effects.

The emergence of chemoresistant disease during chemotherapy with 5-Fluorouracil-based (5-FU-based) regimens is an important factor in the mortality of metastatic CRC (mCRC). The causes of 5-FU resistance are multi-factorial, and besides DNA mismatch repair deficiency (MMR-D), there are no widely accepted criteria for determining which CRC patients are not likely to be responsive to 5-FU-based therapy. Thus, there is a need to systematically understand the mechanistic basis for 5-FU treatment failure and an urgent need to develop new approaches for circumventing the major causes of 5-FU resistance. In this manuscript, we review mechanisms of 5-FU resistance with an emphasis on: (1) altered anabolic metabolism limiting the formation of the primary active metabolite Fluorodeoxyuridylate (5-Fluoro-2'-deoxyuridine-5'-O-monophosphate; FdUMP); (2) elevated expression or activity of the primary enzymatic target thymidylate synthase (TS); and (3) dysregulated programmed cell death as important causes of 5-FU resistance. Importantly, these causes of 5-FU resistance can potentially be overcome through the use of next-generation fluoropyrimidine (FP) polymers (e.g., CF10) that display reduced dependence on anabolic metabolism and more potent TS inhibitory activity.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

High Folate, Perturbed One-Carbon Metabolism and Gestational Diabetes Mellitus.

Folate is a dietary micronutrient essential to one-carbon metabolism. The World Health Organisation recommends folic acid (FA) supplementation pre-conception and in early pregnancy to reduce the risk of fetal neural tube defects (NTDs). Subsequently, many countries (~92) have mandatory FA fortification policies, as well as recommendations for periconceptional FA supplementation. Mandatory fortification initiatives have been largely successful in reducing the incidence of NTDs. However, humans have limited capacity to incorporate FA into the one-carbon metabolic pathway, resulting in the increasingly ubiquitous presence of circulating unmetabolised folic acid (uFA). Excess FA intake has emerged as a risk factor in gestational diabetes mellitus (GDM). Several other one-carbon metabolism components (vitamin B12, homocysteine and choline-derived betaine) are also closely entwined with GDM risk, suggesting a role for one-carbon metabolism in GDM pathogenesis. There is growing evidence from in vitro and animal studies suggesting a role for excess FA in dysregulation of one-carbon metabolism. Specifically, high levels of FA reduce methylenetetrahydrofolate reductase (MTHFR) activity, dysregulate the balance of thymidylate synthase (TS) and methionine synthase (MTR) activity, and elevate homocysteine. High homocysteine is associated with increased oxidative stress and trophoblast apoptosis and reduced human chorionic gonadotrophin (hCG) secretion and pancreatic β-cell function. While the relationship between high FA, perturbed one-carbon metabolism and GDM pathogenesis is not yet fully understood, here we summarise the current state of knowledge. Given rising rates of GDM, now estimated to be 14% globally, and widespread FA food fortification, further research is urgently needed to elucidate the mechanisms which underpin GDM pathogenesis.

Abstract 225: Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model

Abstract The purpose of this study is to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively analyze the treatment outcome of 5-fluorouracil (5-FU) in colon cancer. Predicting the outcome of chemotherapeutic agents plays an essential role in improving dose strategies. To that end, we have developed an integrated mechanistic PK/PD model to analyze the kinetics of tumor response to 5-FU treatment. Compared to the existing PK/PD models, our model is novel in the way it incorporates the 5-FU metabolism pathway and downstream antiproliferative events to provide a comprehensive model of treatment outcome. 5-FU has been widely used to treat cancers, including colon cancer. 5-FU metabolites can be incorporated into RNA, DNA and also interfere with thymidylate synthase (TS) catalytic activity, which is the main mechanism of 5-FU cytotoxicity. TS activity has been shown to be the predictor for treatment efficacy. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate catalyzed by TS is the sole intracellular de novo source of thymidylate. The formation of a ternary complex between fluorodeoxyuridylate (FdUMP) and TS obstructs the access of dUMP to the TS nucleotide-binding site. Resultant thymidine deprivation and dUMP accumulation give rise to the deoxynucleotide triphosphate (dNTP) pool imbalance, which could trigger S-phase specific DNA fragmentation, cell cycle arrest and apoptosis. In our PK model, compartmental analysis is developed to track the distribution of 5-FU from its entry into body to interstitial fluid outside the tumor cells and to its cellular uptake. In the cell model, the connection of TS inhibition and dNTP pool imbalance is implemented. In addition to the central plasma and peripheral tissue compartments, our model includes compartments for: 5-FU concentration in the extracellular fluids; intracellular 5-FU; 5-FU anabolites; genomic 5-FU in RNA, and in DNA; and, TS - FdUMP complex. Posttreatment free TS level is used to compute the perturbed TS catalytic activity. Using the model outcome on TS inhibition, we modeled the kinetics of dNTP pool perturbation. The dNTP pool imbalance leads to double strand break (DSB) formation that initiates DNA damage repair response, kinetics of which are also modeled. Our model uses the law of mass action and Michaelian kinetics and each molecular reaction is expressed as a nonlinear differential equation. Time course data from the literature are used in parameter estimation. Our simulation results successfully captured the kinetics of treatment outcome from the administration of the 5-FU to downstream effects in the tumor. The next step in our effort is to expand the model to include the tumor growth kinetics. Citation Format: Chenhui Ma, Alexandru Almasan, Evren Gurkan-Cavusoglu. Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 225.

Coptidis Rhizoma Extract Reverses 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells via Modulation of Thymidylate Synthase.

Colorectal cancer (CRC) is a malignancy of the colon or rectum. It is ranked as the third most common cancer in both men and women worldwide. Early resection permitted by early detection is the best treatment, and chemotherapy is another main treatment, particularly for patients with advanced CRC. A well-known thymidylate synthase (TS) inhibitor, 5-fluorouracil (5-FU), is frequently prescribed to CRC patients; however, drug resistance is a critical limitation of its clinical application. Based on the hypothesis that Coptidis Rhizoma extract (CRE) can abolish this 5-FU resistance, we explored the efficacy and underlying mechanisms of CRE in 5-FU-resistant (HCT116/R) and parental HCT116 (HCT116/WT) cells. Compared to treatment with 5-FU alone, combination treatment with CRE and 5-FU drastically reduced the viability of HCT116/R cells. The cell cycle distribution assay showed significant induction of the G0/G1 phase arrest by co-treatment with CRE and 5-FU. In addition, the combination of CRE and 5-FU notably suppressed the activity of TS, which was overexpressed in HCT116/R cells, as compared to HCT116/WT cells. Our findings support the potential of CRE as an adjuvant agent against 5-FU-resistant colorectal cancers and indicate that the underlying mechanisms might involve inhibition of TS expression.

Synthesis of New 1, 3, 4-Oxadiazole-Incorporated 1, 2, 3-Triazole Moieties as Potential Anticancer Agents Targeting Thymidylate Synthase and Their Docking Studies.

Thymidylate synthase (TS) has emerged as a hot spot in cancer treatment, as it is directly involved in DNA synthesis. In the present article, nine hybrids containing 1,2,3-triazole and 1,3,4-oxadiazole moieties (6–14) were synthesized and evaluated for anticancer and in vitro thymidylate synthase activities. According to in silico pharmacokinetic studies, the synthesized hybrids exhibited good drug likeness properties and bioavailability. The cytotoxicity results indicated that compounds 12 and 13 exhibited remarkable inhibition on the tested Michigan Cancer Foundation (MCF-7) and Human colorectal Carcinoma (HCT-116) cell lines. Compound 12 showed four-fold inhibition to a standard drug, 5-fluoruracil, and comparable inhibition to tamoxifen, whereas compound 13 exerted five-fold activity of tamoxifen and 24-fold activity of 5-fluorouracil for MCF-7 cells. Compounds 12 and 13 inhibited thymidylate synthase enzyme, with an half maximal inhibitory concentration, IC50 of 2.52 µM and 4.38 µM, while a standard drug, pemetrexed, showed IC50 = 6.75 µM. The molecular docking data of compounds 12 and 13 were found to be in support of biological activities data. In conclusion, hybrids (12 and 13) may inhibit thymidylate synthase enzyme, which could play a significant role as a chemotherapeutic agent.

Phase I pharmacological study of continuous chronomodulated capecitabine treatment

PurposeCapecitabine is an oral pre-pro-drug of the anti-cancer drug 5-fluorouracil (5-FU). The biological activity of the 5-FU degrading enzyme, dihydropyrimidine dehydrogenase (DPD), and the target enzyme thymidylate synthase (TS), are subject to circadian rhythmicity in healthy volunteers. The aim of this study was to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK) and pharmacodynamics (PD) of capecitabine therapy adapted to this circadian rhythm (chronomodulated therapy).MethodsPatients aged ≥18 years with advanced solid tumours potentially benefitting from capecitabine therapy were enrolled. A classical dose escalation 3 + 3 design was applied. Capecitabine was administered daily without interruptions. The daily dose was divided in morning and evening doses that were administered at 9:00 h and 24:00 h, respectively. The ratio of the morning to the evening dose was 3:5 (morning: evening). PK and PD were examined on treatment days 7 and 8.ResultsA total of 25 patients were enrolled. The MTD of continuous chronomodulated capecitabine therapy was established at 750/1250 mg/m2/day, and was generally well tolerated. Circadian rhythmicity in the plasma PK of capecitabine, dFCR, dFUR and 5-FU was not demonstrated. TS activity was induced and DPD activity demonstrated circadian rhythmicity during capecitabine treatment.ConclusionThe MTD of continuous chronomodulated capecitabine treatment allows for a 20% higher dose intensity compared to the approved regimen (1250 mg/m2 bi-daily on day 1–14 of every 21-day cycle). Chronomodulated treatment with capecitabine is promising and could lead to improved tolerability and efficacy of capecitabine.

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

BackgroundDeficiency 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.ResultsIn 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.ConclusionThese 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.

AGENT: An open-label phase III study of arfolitixorin versus leucovorin in modified FOLFOX-6 for first-line treatment of metastatic colorectal cancer.

TPS268 Background: 5-fluorouracil (5FU), in combination with folates, is an established cornerstone of metastatic colorectal cancer (mCRC) treatment. All folates currently approved for mCRC need to be metabolically activated to [6R]-5,10-methylenetetrahydrofolic acid ([6R]-MTHF), the active thymidylate synthase co-substrate that potentiates the effect of 5FU. Arfolitixorin does not require multi-step metabolic activation, and may produce higher, and less inter- and intraindividually variable, concentrations of [6R]-MTHF than leucovorin. Methods: The phase III AGENT trial (NCT03750786) is a randomized, multicenter, parallel-group study comparing the efficacy of arfolitixorin versus leucovorin in mCRC patients treated with first-line 5FU, oxaliplatin, and bevacizumab. Patients are randomized (1:1) to the investigational arm (arfolitixorin + 5FU + oxaliplatin [ARFOX] + bevacizumab) or the comparator arm (leucovorin + 5FU + oxaliplatin [modified FOLFOX-6] + bevacizumab), and treated until disease progression based on RECIST 1.1 criteria. Recruitment is ongoing, and aims to randomize 440 patients in 18 months. Eligibility criteria include non-resectable mCRC; eligibility for 5FU, oxaliplatin, and bevacizumab therapy; ECOG PS 0 or 1. The study will be conducted across approximately 100 sites in Australia, Austria, Canada, France, Germany, Greece, Japan, Spain, Sweden, and USA. The primary endpoint is objective response rate. Key secondary endpoints are progression-free survival and duration of response. Additional secondary endpoints include overall survival, quality of life, safety and tolerability, and number of patients undergoing curative metastasis resection. A translational program will evaluate expression levels of several folate metabolism- and transportation-related genes in mCRC tumor biopsies to determine their relationship to treatment outcome. A broad array of genes will analyzed, including ATP-binding cassette C3 (ABCC3) transporter, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), proton-coupled folate transporter (PCFT), and serine hydroxymethyltransferase 1 (SHMT1). Interim data are expected in mid 2020. Clinical trial information: NCT03750786.

Abstract C059: Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation

Abstract Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. NUC-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on NUC-3373 and the role of NUC-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating NUC-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and NUC-3373. Thymidine supplementation was used to indirectly assess the effect of NUC-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments NUC-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following NUC-3373 treatment. Supplementation with exogenous thymidine rescued cells from NUC-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for NUC-3373. In selected cell lines treated with NUC-3373, TS ternary complexes were detected for at least 72 hours. Treatment with NUC-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received NUC-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, NUC-3373 was not catabolised by DPD, highlighting the ability of NUC-3373 to bypass a key rate-limiting factor associated with 5-FU. NUC-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that NUC-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of NUC-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059

664TiP - Open label phase III study of arfolitixorin vs leucovorin in mFOLFOX-6 for first-line treatment of metastatic colorectal cancer: AGENT

Background5-fluorouracil (5-FU) in combination with the folate Leucovorin (LV) has been the cornerstone in the treatment of colorectal cancer (CRC) for decades. All folates currently approved for use in the clinical setting need to be metabolically activated to [6R] 5,10-methylenetetrahydrofolic acid ([6R]-MTHF), which is the active thymidylate synthase co-substrate that potentiates the effect of 5-FU. Arfolitixorin does not need multi step metabolic activation like currently available folates. It is therefore hypothesized that the administration of arfolitixorin will result in higher, and less individual variability, intracellular concentrations of the active thymidylate synthase co-substrate [6R]-MTHF in all patients compared to LV administration. Trial designPrimary endpoint ORR by Blinded Independent Central Review (BICR) Key secondary endpoints Progression Free Survival (PFS) Duration of Response (DoR) Secondary endpoints Overall Survival (OS) Quality of Life (QoL) Safety and Tolerability Patients undergoing curative metastasis resection Study Design This is a randomized, multicenter, parallel-group, Phase III study to compare the efficacy of arfolitixorin versus LV in patients with mCRC treated with 5-FU, oxaliplatin, and bevacizumab. Patients will be randomized in a 1:1 ratio to either the investigational arm or the comparator arm. The study target is to randomize 440 patients in 18 months. An adaptive study design includes the possibility to increase the sample size to 660 patients as determined by the DSMB during the interim analysis to be conducted after 330 patients have conducted their 16 weeks scan. Key inclusion criteria: Adults > 18 years of age Colorectal adenocarcinoma verified by biopsy Non-resectable mCRC No prior treatment for first line mCRC ECOG performance status 0 or 1Key exclusion criteria: Malignant tumors other than colorectal adenocarcinomas Less than 6 months since last anti-cancer treatment DPD deficiency Clinically significant cardiovascular disease Central nervous system metastases Study Countries Australia, Austria, Canada, France, Germany, Greece, Spain, Sweden and USA FPI: December 18, 2018. Clinical trial identificationNCT03750786; 2017-004154-41. Legal entity responsible for the studyIsofol Medical AB. FundingIsofol Medical AB. DisclosureJ. Tabernero: Advisory / Consultancy: Array Biopharma; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: BeiGene; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Chugai; Advisory / Consultancy: Genetech Inc; Advisory / Consultancy: Genemab A/S; Advisory / Consultancy: Halozyme; Advisory / Consultancy: Imugene Limited; Advisory / Consultancy: Inflection Biosciences Limited; Advisory / Consultancy: Ipsen; Advisory / Consultancy: Kura Oncology; Advisory / Consultancy: Lilly; Advisory / Consultancy: MSD; Advisory / Consultancy: Menarini; Advisory / Consultancy: Merck Serono; Advisory / Consultancy: Merrimack; Advisory / Consultancy: Merus; Advisory / Consultancy: Molecular Partners; Advisory / Consultancy: Novartis. G. Prager: Advisory / Consultancy: Merck Serono; Advisory / Consultancy: Roche; Advisory / Consultancy: Amgen; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Lilly; Advisory / Consultancy: Servier; Advisory / Consultancy: Taiho; Advisory / Consultancy: Bayer; Advisory / Consultancy: Halozyme; Advisory / Consultancy: BMS; Advisory / Consultancy: Cellgene; Advisory / Consultancy: Shire. S. Stintzing: Advisory / Consultancy: Amgen; Advisory / Consultancy: Bayer; Advisory / Consultancy: Roche; Advisory / Consultancy: Merck; Advisory / Consultancy: Lilly; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Taiho; Advisory / Consultancy: Takeda; Advisory / Consultancy: Servier. H.J. Lenz: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Merck; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Travel / Accommodation / Expenses: Boehringer Ingelheim. H.P. Nygren: Shareholder / Stockholder / Stock options: Oncopeptides; Research grant / Funding (institution), Licensing / Royalties: Respos Pharma. C. Papadimitriou: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Roche; Research grant / Funding (self): BMS; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Genesis; Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Merck.

Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP

In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused “uncoupling” of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Abstract 2079: Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells

Abstract Background: Mutant KRAS is a key stratification factor in determining colorectal cancer (CRC) treatment. Nearly 50% of CRC tumors have KRAS mutations and these patients are unlikely to respond to anti-EGFR therapy. 5-fluorouracil (5-FU)-based chemotherapy is the preferred treatment for CRC patients with KRAS mutations; however, response rates remain very low for patients with advanced disease. Further, a sub-set of patients treated with 5-FU-based regimens suffers from serious side effects resulting from RNA-directed effects that do not contribute to the anti-cancer activity. We are developing polymeric fluoropyrimidines, such as CF10, to treat CRC more effectively and with reduced side effects. CF10 is >300-fold more potent than 5-FU across the NCI 60 cell line screen with particularly strong potency to KRAS-mutant CRC cells. We investigate the role of KRAS mutation on CF10 and 5-FU response as part of an overall effort to determine if CF10-based regimens can more effectively treat CRC patients for whom 5-FU-based regimens are currently the preferred treatment option. Methods: HKe3-wtKRAS and HKe3-mtKRAS were generated from HCT-116 human CRC cells (ATCC) by transfection with the pMSCVpuro vector encoding HA-tagged mutant or wild-type KRAS. Cytotoxicity was determined using an Alamar blue assay. Thymidylate synthase (TS) inhibition was evaluated by detecting ternary complex formation by Western blot and using a TS activity assay. Topoisomerase 1 cleavage complex (Top1cc) was evaluated using a RADAR assay and by immunofluorescence. DNA double strand breaks were detected by IF for γH2AX. Results: CF10 is highly potent to CRC cells regardless of KRAS mutation status. In isogenic cell lines that differ only in KRAS, CF10 is more potent to KRAS-mutant relative to wild type cells. The improved potency of CF10 relative to 5-FU and increased sensitivity of KRAS-mutant cells is enhanced for cells grown in 3D culture. CF10 cytotoxicity is mediated by TS inhibition and Top1cc formation that induces DNA DSBs and activates apoptosis. In vivo, CF10 displays significantly improved anti-tumor activity relative to 5-FU in HCT-116 orthotopic tumors and causes minimal systemic toxicity. Conclusions. KRAS mutation confers increased sensitivity to the DNA-directed fluoropyrimidine CF10. The overall mechanism of CF10 cytotoxicity to CRC cells is similar in both KRAS-WT and KRAS-MT CRC cells and involves TS inhibition, and misincorporation of FdU into DNA followed by generation of Top1-mediated DNA DSBs. Our studies indicate FP polymers are likely to be effective in CRC regardless of KRAS-mutation. The increased sensitivity of KRAS-mutant CRC to CF10 together with the very low systemic toxicity indicates that CRC patients currently being treated with 5-FU-based therapy could more effectively be treated with CF10-based regimens. Citation Format: William H. Gmeiner, Mandira Manandhar, Amanda M. Hinds, Julija Holmes, Pierre Vidi, Toshiyuki Tsunoda, Senji Shirasawa. Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2079.

RESEASRCH POGRESS FOR THYMIDYLATE SYNTHASE AND INHIBITORS IN CANCERS COUPLE IN CHEMICAL STRUCTURES

Cancer new drug development and testing mostly rely on existing drug which plays a great role as templates, and the biomarkers. Thymidylate Synthase Inhibitors (TSI) tabulated below are available for the diseases conditions like cancer which encircle the thymidylate synthase (TS). Sparse data are available for TS involvement in cancer. TS is prudent in the synthesis of thymidylate which is useful for body functionalities, and its production pathways are observed to contain supportive proteins. The loss of thymine in cancerous cells couple in abnormal DNA synthesis influenced cell apoptosis. An increased TS protein expression seen in many cancers, serving as aggressive phenotype. Cancers is TS disease, still without a cure, taking away the lives of at least 8 million people every year irrespective of nationality, remained a burden that let world health organization to request for urgent treatment. The trouble encompassing cancer in general and that which generated from TS is the principal base of this review. Although, it has been used in other disease and have not yield total cure in cancer yet, TSI potentially utilized to offset the problem responsible for low circulated thymidine in human plasma, is useful to manage cancer disease. Members of TSI bears five or six cycle ring in their structure. The pterine and pyrimidine are compounds which are incorporation with another compound by utilizing the technique of nucleophilic displacement, multicomponent condensation and ultimate combination principle, molecular hybridization strategy and the structural activity relationship mechanism to develop new small TS inhibitors. Through the available English articles about TS related cancer and TSI in ScienceDirect database are utilized here. The search was made by key in the keywards singularly and in pairs to present this comprehensive knowledge about TSI inhibitors, and evaluate TS enzyme mechanisms which contributes to cancer and how the TSI can circumvent cancer disease. Conclusively, the series of TSI utilized the TS protein enzyme to ombat TS human cancer, are also in use as a fundamental molecular template for new drug. TS activity can produce the novel thymidylate. This work will be of prudent to researchers in drug design and synthesis and as well to hospital clinician to support the judgement inside cancer disease treatment.

Inhibition of thymidylate synthase affects neural tube development in mice

Thymidylate synthase (TYMS) is a key enzyme in the de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP) from 2′-deoxyuridine-5′-monophosphate (dUMP). Our aim was to investigate the role of dTMP dysmetabolism via inhibition of TYMS by an inhibitor, 5-fluorouracil (5-FU) in the occurrence of neural tube defects (NTDs). We found that a high incidence of NTDs occurred after treatment with 5-FU at 12.5 mg/kg body weight. TYMS activity was significantly inhibited with decreased dTMP and accumulation of dUMP after 5-FU injection. The proliferation of neuroepithelial cells were markedly inhibited in NTDs compared with control. Expressions of proliferating cell nuclear antigen and phosphohistone H3 were significantly decreased in NTDs, while phosphorylated replication protein A2, P53 and Caspase3 were significantly increased in NTDs compared with control. These results indicated that inhibition of TYMS affected the balance between proliferation and apoptosis in neuroepithelial cells, which might shed some lights on the mechanisms involved in NTDs.

Synthesis, characterization and molecular docking studies of thiouracil derivatives as potent thymidylate synthase inhibitors and potential anticancer agents.

Thymidylate synthase (TS), one of folate-dependent enzymes, is a key and well-recognized target for anticancer agents. In this study, a series of 6-aryl-5-cyano thiouracil derivatives were designed and synthesized in accordance with essential pharmacophoric features of known TS inhibitors. Nineteen compounds were screened in vitro for their anti-proliferative activities toward HePG-2, MCF-7, HCT-116, and PC-3 cell lines. Compounds [Formula: see text], [Formula: see text], and 24 exhibited high anti-proliferative activity, comparable to that of 5-fluorouracil. Additionally, ten compounds with potent anti-proliferative activities were further evaluated for their ability to inhibit TS enzyme. Six compounds ([Formula: see text], [Formula: see text], [Formula: see text], 22, 23 and 24) demonstrated potent dose-related TS inhibition with [Formula: see text] values ranging from 1.57 to [Formula: see text]. The in vitro TS activity results were consistent with those of the cytotoxicity assay where the most potent anti-proliferative compounds of the series showed good TS inhibitory activity comparable to that of 5-fluorouracil. Furthermore, molecular docking studies were carried out to investigate the binding pattern of the designed compounds with the prospective target, TS (PDB-code: 1JU6).

Abstract 1214: The polymeric fluoropyrimidine F10 is effective towards cell and organoid models of 5-FU-resistant colorectal cancer

Abstract Background: Fluoropyrimidine drugs (FPs, e.g. 5-fluorouracil (5-FU)) constitute the backbone of most chemotherapy regimens for treating locally-advanced and metastatic colon cancer (e.g. FOLFOX, FOLFIRI, FOLFOXIRI). While current FPs improve survival and are the preferred drugs for most colorectal (CRC) cases, drug resistance is a serious problem. One of the most prevalent causes of 5-FU resistance is mutation in TP53 which occur in more than one-third of stage III CRC tumors. 5-FU requires wtp53 for initiation of apoptosis and CRC cells with mtp53 are markedly less sensitive to 5-FU. Methods: To test if F10 induced p53-independent apoptosis in CRC cells, we obtained p53-/- and p53+/+ HCT-116 cells from Professor Vogelstein (Johns Hopkins) and evaluated drug-response in cell-based, organoid, and xenograft models. Cell viability and apoptosis were assessed using Cell-Titer Glo and Caspase Glo assays (Promega). Cell-cycle distribution was evaluated by flow cytometry analysis of propidium iodide stained cells. Thymidylate synthase (TS) activity was assessed using a 3H-release assay and Topoisomerase 1 cleavage complex (Top1cc) formation was assessed using an in vivo complex of enzyme (ICE) bioassay. Cell division was quantified by time-lapse video microscopy. Organoids were developed by bioprinting cells on a biogel matrix to produce a center comprised of tumor cells encompassed by stromal cells. Drug effects in each cell type were evaluated using a “Live-Dead” assay. Drug effects in vivo were evaluated using xenograft models. Results: p53-/- HCT-116 cells were resistant to 5-FU, but sensitive to F10 and underwent p53-independent apoptosis. While F10 is more efficient than 5-FU at inhibiting TS, it uniquely supported continued DNA synthesis, mitosis, and cell division in p53-null cells while 5-FU-treated cells underwent S-phase arrest under thymineless conditions. Following cell division, F10 induced high levels of Top1cc in daughter cells causing massive DNA damage. Using a DT40 knockout system we determined that Tdp1 and PARP-1 were important for F10-induced DNA damage. Organoid models demonstrated selective killing of malignant cells relative to stroma by F10, but not 5-FU. Xenograft models are in progress. Conclusions: F10 is much more potent than 5-FU and induces p53-independent apoptosis. F10 differs from 5-FU in supporting continued DNA synthesis under thymineless conditions leading to Top1-mediated DNA damage, Top1 depletion, genomic instability and activation of the extrinsic apoptotic pathway. Citation Format: William H. Gmeiner, Anthony Dominijanni, Olcay Boyacioglu, Steven Forsyth, Aleks Skardal, Boris Pasche. The polymeric fluoropyrimidine F10 is effective towards cell and organoid models of 5-FU-resistant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1214. doi:10.1158/1538-7445.AM2017-1214

Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana.

In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities.

Drug-Repositioning Screens Identify Triamterene as a Selective Drug for the Treatment of DNA Mismatch Repair Deficient Cells

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial.Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivoResults: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks.Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880-90. ©2016 AACR.