Thymidine Salvage Pathway Research Articles

Metabolic engineering of Escherichia coli for thymidine production

Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E. coli. Firstly, deoA, tdk, udp, rihA, rihB, and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E. coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5'-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Decarboxylation Mechanism of iso-Orotate Decarboxylase Revisited.

iso-Orotate decarboxylase (IDCase), which is involved in the thymidine salvage pathway, has attracted considerable interest owing to its chemical similarity to a hypothetical DNA decarboxylase in mammals. Although valuable insights into the active DNA demethylation of 5-methyl-cytosine can be obtained from the decarboxylation mechanism of 5-carboxyl-uracil (5caU) catalyzed by IDCase, this mechanism remains under debate. In this study, the catalytic mechanism of 5caU decarboxylation by IDCase was studied using hybrid quantum mechanics/molecular mechanics (QM/MM) methodologies and density functional theory (DFT) calculations with a truncated model. The calculations supported a mechanism involving three sequential stages: activation of the 5caU substrate via proton transfer from an arginine (R262') to the carboxyl group of 5caU, formation of a tetrahedral intermediate, and decarboxylation of the tetrahedral intermediate to generate uracil as the product. The reaction pathways and structures obtained using the QM/MM and DFT methods coincided with each other. These simulations provided detailed insights into the unique mechanism of IDCase, clarifying various unresolved issues, such as the critical role of R262'. In addition, aspartate D323 was found to act as a general base in the tetrahedral intermediate formation step and a general acid in the later C-C bond cleavage step.

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

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

Protease Responsive Nanogels for Transcytosis across the Blood−Brain Barrier and Intracellular Delivery of Radiopharmaceuticals to Brain Tumor Cells (Adv. Healthcare Mater. 20/2021)

Blood–Brain Barrier In article number 2100812 by Smriti Singh, Agnieszka Morgenroth, and co-workers, Matrix metalloprotease (MMP) sensitive nanogels are synthesized for effective delivery of a radiopharmaceutical ([125I]ITdU) across the blood-brain barrier (BBB). The nanogels show efficient transcytosis across the in vitro model of BBB co-cultured with glioblastoma cells. Once transcytosed, nanogels degrade in an up-regulated MMP2/9 environment of glioblastoma and release the radiopharmaceutical, which gets incorporated into the DNA of tumor cells by thymidine salvage pathway.

Protease Responsive Nanogels for Transcytosis across the Blood-Brain Barrier and Intracellular Delivery of Radiopharmaceuticals to Brain Tumor Cells.

Despite profound advances in treatment approaches, gliomas remain associated with very poor prognoses. The residual cells after incomplete resection often migrate and proliferate giving a seed for highly resistant gliomas. The efficacy of chemotherapeutic drugs is often strongly limited by their poor selectivity and the blood brain barrier (BBB). Therefore, the development of therapeutic carrier systems for efficient transport across the BBB and selective delivery to tumor cells remains one of the most complex problems facing molecular medicine and nano-biotechnology. To address this challenge, a stimuli sensitive nanogel is synthesized using pre-polymer approach for the effective delivery of nano-irradiation. The nanogels are cross-linked via matrix metalloproteinase (MMP-2,9) substrate and armed with Auger electron emitting drug 5-[125 I]Iodo-4"-thio-2"-deoxyuridine ([125 I]ITdU) which after release can be incorporated into the DNA of tumor cells. Functionalization with diphtheria toxin receptor ligand allows nanogel transcytosis across the BBB at tumor site. Functionalized nanogels efficiently and increasingly explore transcytosis via BBB co-cultured with glioblastoma cells. The subsequent nanogel degradation correlates with up-regulated MMP2/9. Released [125 I]ITdU follows the thymidine salvage pathway ending in its incorporation into the DNA of tumor cells. With this concept, a highly efficient strategy for intracellular delivery of radiopharmaceuticals across the challenging BBB is presented.

Sequence-dependent Combination of 5-Fluorouracil and Targeted Agents in RAF-mutant Colorectal Cancer Cell Lines

V-raf murine sarcoma viral oncogene homolog B (BRAF)-mutant colorectal cancer has limited therapeutic options for targeted agent and showed poor response to standard chemotherapy containing 5-fluorouracil (5-FU). We previously reported that 5-FU induced the expression of thymidine kinase 1 (TK1), a rate-limiting enzyme for thymidine salvage pathway, which is related with acquired resistance for 5-FU. As TK1 expression was dependent on growth-stimulatory signal such as Braf-MEK and PI3K-AKT pathway, we investigated whether the sequential treatment of 5-FU followed by targeted agent could induce synergy for cell viability and repress TK1 expression in compared with the treatment in reverse sequence. Treatment with 5-FU for 2 days followed by vemurafenib, a mutant BRAF inhibitor, for another 2 days induced significant increase of cell death in compared with the single treatment in colo205 and RKO cell lines. However, the combined treatment in reverse sequence showed the similar response to single treatment. Treatment of dactolisib (a PI3K/mTOR inhibitor) or MK-2206 (a AKT inhibitor) preceded by 5-FU induced cell death more than single agent. Induction of TK1 by 5-FU was abrogated by subsequent treatment with vemurafenib, dactolisib, or MK-2206, whereas 5-FU still induced TK1 expression in the cells pretreated with these targeted agents. In summary, the sequence-dependent treatment of 5-FU and targeted agents is effective through the extinction of 5-FU-induced TK1 activation, which could give a promising strategy for patients having BRAF-mutant colon cancers.

Reaction Mechanism and Substrate Specificity of Iso-orotate Decarboxylase: A Combined Theoretical and Experimental Study.

The C-C bond cleavage catalyzed by metal-dependent iso-orotate decarboxylase (IDCase) from the thymidine salvage pathway is of interest for the elucidation of a (hypothetical) DNA demethylation pathway. IDCase appears also as a promising candidate for the synthetic regioselective carboxylation of N-heteroaromatics. Herein, we report a joint experimental-theoretical study to gain insights into the metal identity, reaction mechanism, and substrate specificity of IDCase. In contrast to previous assumptions, the enzyme is demonstrated by ICPMS/MS measurements to contain a catalytically relevant Mn2+ rather than Zn2+. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds via a (reverse) electrophilic aromatic substitution with formation of CO2. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of could be ruled out on the basis of prohibitively high energy barriers. In contrast to related o-benzoic acid decarboxylases, such as γ-resorcylate decarboxylase and 5-carboxyvanillate decarboxylase, which exhibit a relaxed substrate tolerance for phenolic acids, IDCase shows high substrate fidelity. Structural and energy comparisons suggest that this is caused by a unique hydrogen bonding of the heterocyclic natural substrate (5-carboxyuracil) to the surrounding residues. Analysis of calculated energies also shows that the reverse carboxylation of uracil is impeded by a strongly disfavored uphill reaction.

Dexamethasone pretreatment impairs the thymidylate synthase inhibition mediated flare in thymidine salvage pathway activity in non-small cell lung cancer.

IntroductionSuccessful inhibition of thymidylate synthase (TS) by pemetrexed, a TS inhibitor, results in a reproducible transient burst or “flare” in thymidine salvage pathway activity at 2 hrs. of therapy which can be measurable with FLT-PET ([18F]fluorothymidine-positron emission tomography) in non-small cell lung cancer (NSCLC). Routine administration of dexamethasone with pemetrexed-based therapy could potentially confound this imaging approach since dexamethasone is known to inhibit expression of thymidine kinase 1, a key enzyme in the thymidine salvage pathway. Here we examine the potential impact of dexamethasone on the TS inhibition-mediated thymidine salvage pathway “flare” in NSCLC.Materials and methodsIn order to determine NSCLC cell line sensitivity to dexamethasone and pemetrexed, IC50 studies were performed on NSCLC cell lines H23, H1975, H460, H1299. TS inhibition-mediated “flare” in thymidine salvage pathway activity was then measured at 2hrs. of exposure to pemetrexed and cisplatin in NSCLC cells lines following using 3H-thymidine incorporation assays under the following conditions: control (no chemotherapy or dexamethasone), or treated with pemetrexed and cisplatin without dexamethasone, with 24 hrs. pre-treatment of dexamethasone or with dexamethasone administered together with chemotherapy. These conditions were chosen to model the delivery of pemetrexed-based therapy in the clinic.ResultsThe IC50 of H23, H1975, H460, H1299 for dexamethasone and pemetrexed were 40, 5.9, 718, 362 μM and 0.22, 0.73, 0.14 and 0.66 μM respectively. Significant blunting of the thymidine salvage pathway “flare” is observed at 2hrs. of pemetrexed-based therapy when dexamethasone sensitive cell lines H23 and H1975 were pretreated with dexamethasone but not when dexamethasone was given together with pemetrexed therapy or in the setting of dexamethasone resistance (H460 and H1299).Conclusion24 hr. pretreatment with dexamethasone, but not same day co-administration of dexamethasone with therapy, impairs the TS inhibition-mediated “flare” in thymidine salvage pathway activity in NSCLC.

Abstract 3049: Dexamethasone pretreatment impairs the TS inhibition-mediated flare in thymidine salvage pathway activity

Abstract Introduction: Inhibition of thymidylate synthase (TS) by pemetrexed, an inhibitor of thymidylate synthase, results in an early transient burst or “flare” in thymidine salvage pathway activity at 2 hrs of therapy, which, we have demonstrated in prior publications, can be measurable with FLT ([18F]thymidine)-positron emission tomography (PET) in non-small cell lung cancer (NSCLC). Administration of dexamethasone with pemetrexed-based therapy, as typically practiced in the clinic, could potentially confound this imaging approach since dexamethasone is known to inhibit expression of thymidine kinase 1, a key enzyme in the thymidine salvage pathway. Here we examine the potential impact of dexamethasone on the TS inhibition-mediated thymidine salvage pathway “flare” in NSCLC. Materials and Methods: In order to determine NSCLC cell line sensitivity to dexamethasone and pemetrexed, IC50 studies were performed on NSCLC cell lines H23, H1975, H460, H1299. TS inhibition-mediated “flare” in thymidine salvage pathway activity was then measured at 2hrs of exposure to pemetrexed and cisplatin in NSCLC cells lines following using 3H-thymidine incorporation assays under the following conditions: control (no chemotherapy or dexamethasone), treated with pemetrexed and cisplatin without dexamethasone, with 24 pretreatment of dexamethasone, or with dexamethasone administered together with chemotherapy. These conditions were chosen to model the delivery of pemetrexed-based therapy in the clinic. Results: The IC50 of H23, H1975, H460, H1299 for dexamethasone and pemetrexed were 40, 5.9, 718, 362 uM and 0.22, 0.73, 0.14 and 0.66 uM, respectively. Significant blunting of the thymidine salvage pathway “flare” is observed at 2hrs of pemetrexed-based therapy when dexamethasone-sensitive cell lines H23 and H1975 were pretreated with dexamethasone but not when dexamethasone was given together with pemetrexed therapy or in the setting of dexamethasone resistance (H460 and H1299). Conclusion: Imaging of TS inhibition may be impacted by pretreatment with dexamethasone but not administration of dexamethasone on the same day of therapy delivery. Citation Format: Sharyn I. Katz, Xiao Chen, Yizeng Yang. Dexamethasone pretreatment impairs the TS inhibition-mediated flare in thymidine salvage pathway activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3049.

Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging.

IntroductionInhibition of thymidylate synthase (TS) results in a transient compensatory “flare” in thymidine salvage pathway activity measureable with 18F-thymidine (FLT)- positron emission tomography (PET) at 2hrs. of therapy which may predict non-small cell lung cancer (NSCLC) sensitivity to TS inhibition.Materials and MethodsResistance to TS inhibition by pemetrexed was induced in NSCLC cell lines H460 and H1299 through TS overexpression. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed resistance confirmed with IC50 assays. The presence of a pemetrexed-induced thymidine salvage pathway “flare” was then measured using 3H-thymidine in both pemetrexed sensitive (H460 and H1299) and resistant (H460R, H1299R, CALU-6, H522, H650, H661, H820, H1838) lines in vitro, and validated with FLT-PET in vivo using H460 and H460R xenografts.ResultsOverexpression of TS induced pemetrexed resistance with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Thymidine salvage pathway 3H-thymidine “flare” was observed following pemetrexed in H460 and H1299 but not H460R, H1299R, CALU-6, H522, H650, H661, H820 or H1838 in vitro. Similarly, a FLT “flare” was observed in vivo following pemetrexed therapy in H460 but not H460R tumor-bearing xenografts.ConclusionsImaging of TS inhibition is predictive of NSCLC sensitivity to pemetrexed.

Abstract 177: Thymidine quinoxaline conjugates as a novel selective and effective photosensitizer for anticancer photodynamic therapy

Abstract Thymidine quinoxaline conjugates are a new class anticancer agent that exhibits selective anticancer activity by targeting the abnormally high level of thymidine kinase of the thymidine salvage pathway in cancer cells. In addition, the conjugated quinoxaline moiety could also act as a photosensitizer to generate reactive oxygen species that would significantly enhance the anticancer activity. Therefore, a variety of structural derivatives were synthesized and investigated for the potential of combined anticancer activity. We found that the thymidine moiety was critical for the accumulation of compounds in cancer cells and thus their activity. On the other hand, the substitutions and additional conjugation of quinoxaline moiety had significant impact on the effectiveness to generate reactive oxygen species for enhanced potency. Among all the compounds studied, dT-QP exhibited a potent activity against liver cancer cells with IC50 at 20 nM under photo-irradiation at 400 nm wavelength for 20 min. This potent activity of dT-QP was 12 times more effective than that of the clinically used photosensitizer protoporphyrin IX (IC50 at 250 nM) under the same condition and 5 times more potent than anticancer drug doxorubicin (IC50 at 100 nM). In addition, thymidine quinoxaline conjugates were found to form aggregates with an average size of 200 nm. Further optimization of the aggregating condition led to a highly stable suspension of 90 nm size nanoparticles containing a tumor-specific peptide that could be used for in vivo study. Therefore, thymidine quinoxaline analogs could be a selective anticancer agent and effective photosensitizer for potential photodynamic therapy. Citation Format: Qibing Zhou, Zhiwei Wang, Rong Yang, Ting Qian, Dejun Zhang. Thymidine quinoxaline conjugates as a novel selective and effective photosensitizer for anticancer photodynamic therapy [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 177. doi:10.1158/1538-7445.AM2017-177

Abstract 1783: Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging

Abstract Background: Inhibition of thymidylate synthase (TS) results in a transient compensatory “flare” in thymidine salvage pathway activity which is measureable with 18F-thymidine (FLT) positron emission tomography (PET) at 2 hrs of exposure to therapy. Here we examine the predictive value of the FLT-PET measured “flare” for NSCLC sensitivity to pemetrexed, a commonly used TS inhibitor, in a preclinical model. Experimental Design: Resistance to pemetrexed therapy was induced in two sensitive human NSCLC cell lines, H460 and H1299, by overexpressing TS. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed-resistance confirmed with IC50 assays. The presence of a pemetrexed-induced DNA salvage pathway “flare” was then measured using 3H-thymidine in both pemetrexed-sensitive (H460 and H1299) and resistant (H460R and H1299R) lines in vitro, as well as inherently resistant NSCLC cell lines CALU6, H522, H650, H661, H820, H1838, and validated with FLT-PET in vivo using H460 and H460R xenografts. Results: Overexpression of TS induced resistance to pemetrexed in the H460 and H1299 with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Significant increases in DNA salvage pathway activity (“flare”) was observed at 2hrs of therapy in the pemetrexed-sensitive H460 and H1299 lines but not the resistant H460R and H1299R, CALU6, H522, H650, H661, H820, H1838 cell lines in vitro using 3H-thymidine assays. Similarly, FLT “flare” was observed in the pemetrexed-sensitive H460 xenograft tumors but not the pemetrexed-resistant H460R xenograft tumors with post-therapy increases in measured FLTmax of 58% (STD 12.1) and 10.8% (STD 7.3) respectively. Conclusions: FLT-PET imaging of TS inhibition may provide an early indicator of NSCLC sensitivity to pemetrexed. Citation Format: Xiao Chen, Yizeng Yang, Sharyn Katz. Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging [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 1783. doi:10.1158/1538-7445.AM2017-1783

Early detection of pemetrexed-induced inhibition of thymidylate synthase in non-small cell lung cancer with FLT-PET imaging.

Inhibition of thymidylate synthase (TS) results in a transient flare in DNA thymidine salvage pathway activity measurable with FLT ([18F]thymidine)-positron emission tomography (PET). Here we characterize this imaging strategy for potential clinical translation in non-small cell lung cancer (NSCLC). Since pemetrexed acts by inhibiting TS, we defined the kinetics of increases in thymidine salvage pathway mediated by TS inhibition following treatment with pemetrexed in vitro. Next, using a mouse model of NSCLC, we validated the kinetics of the pemetrexed-mediated flare in thymidine salvage pathway activity in vivo using FLT-PET imaging. Finally, we translated our findings into a proof-of-principle clinical trial of FLT-PET in a human NSCLC patient. In NSCLC cells in vitro, we identified a burst in pemetrexed-mediated thymidine salvage pathway activity, assessed by 3H-thymidine assays, thymidine kinase 1 (TK1) expression, and equilibrative nucleoside transporter 1 (ENT1) mobilization to the cell membrane, that peaked at 2hrs. This 2hr time-point was also optimal for FLT-PET imaging of pemetrexed-mediated TS inhibition in murine xenograft tumors and was demonstrated to be feasible in a NSCLC patient. FLT-PET imaging of pemetrexed-induced TS inhibition is optimal at 2hrs from therapy start; this timing is feasible in human clinical trials.

Imaging Cellular Proliferation in Prostate Cancer with Positron Emission Tomography.

Prostate cancer remains a major public health problem worldwide. Imaging plays an important role in the assessment of disease at all its clinical phases, including staging, restaging after definitive therapy, evaluation of therapy response, and prognostication. Positron emission tomography with a number of biologically targeted radiotracers has been demonstrated to have potential diagnostic and prognostic utility in the various clinical phases of this prevalent disease. Given the remarkable biological heterogeneity of prostate cancer, one major unmet clinical need that remains is the non-invasive imaging-based characterization of prostate tumors. Accurate tumor characterization allows for image-targeted biopsy and focal therapy as well as facilitates objective assessment of therapy effect. PET in conjunction with radiotracers that track the thymidine salvage pathway of DNA synthesis may be helpful to fulfill this necessity. We review briefly the preclinical and pilot clinical experience with the two major cellular proliferation radiotracers, [(18)F]-3'-deoxy-3'-fluorothymidine and [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil in prostate cancer.

Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA.

Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

Incorporation of thymidine analogs for studying replication kinetics in fission yeast.

Labeling DNA during in vivo replication by the incorporation of exogenous thymidine and thymidine analogs has been a mainstay of DNA replication and repair studies for decades. Unfortunately, thymidine labeling does not work in fungi, because they lack the thymidine salvage pathway required for uptake of exogenous thymidine. This obstacle to thymidine labeling has been overcome in yeast by engineering a minimal thymidine salvage pathway consisting of a nucleoside transporter to allow uptake of exogenous thymidine from the medium and a thymidine kinase to phosphorylate the thymidine into thymidine monophosphate, which can be used by the cell. This chapter describes the labeling of fission yeast, Schizosaccharomyces pombe, with the thymidine analog BrdU in order to identify sites and determine kinetics of DNA replication.

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.

29 TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does not diminish therapeutic window in human tumor xenografts

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.

28 Characterization of the type of cell death induced by novel tambjamine analogs in lung cancer

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.

27 TAS-102 treatment results in high trifluridine incorporation into DNA with pyrimidine metabolic pathway markedly up-regulated in cancer

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.