Thymidine Synthesis Research Articles

Selectivity analysis of diaminopyrimidine-based inhibitors of MTHFD1, MTHFD2 and MTHFD2L

The mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2) is involved in purine and thymidine synthesis via 1C metabolism. MTHFD2 is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. However, the two close homologs of MTHFD2 known as MTHFD1 and MTHFD2L are expressed in healthy adult human tissues and share a great structural resemblance to MTHFD2 with 54% and 89% sequence similarity, respectively. It is therefore notably challenging to find selective inhibitors of MTHFD2 due to the structural similarity, in particular protein binding site similarity with MTHFD1 and MTHFD2L. Tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported till date. Nanomolar potent diaminopyrimidine-based inhibitors of MTHFD2 have been reported recently, however, they also demonstrate significant inhibitory activities against MTHFD1 and MTHFD2L. In this study, we have employed extensive computational modeling involving molecular docking and molecular dynamics simulations in order to investigate the binding modes and key interactions of diaminopyrimidine-based inhibitors at the substrate binding sites of MTHFD1, MTHFD2 and MTHFD2L, and compare with the tricyclic coumarin-based selective MTHFD2 inhibitor. The outcomes of our study provide significant insights into desirable and undesirable structural elements for rational structure-based design of new and selective inhibitors of MTHFD2 against cancer.

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Targeting SHMTs and MTHFDs in cancer: attractive opportunity for anti-tumor strategy.

One-carbon metabolism is a universal metabolic process that mediates the transfer of one-carbon units for purine and thymidine synthesis. One-carbon metabolism has been found to be dysregulated in various cancer types due to its role in production of purine and pyrimidine nucleotides, epigenetic program, and redox homeostasis. One-carbon metabolism is composed a network of one-carbon metabolic enzymes. Disturbing the expression and enzymatic activity of these one-carbon metabolic enzymes could lead to fluctuations of metabolites in the tumor microenvironment. Serine hydroxymethyltransferases (SHMTs) and methylenetetrahydrofolate dehydrogenases (MTHFDs) are gradually recognized as important one-carbon metabolic enzymes for regulating tumor initiation and development, representing potential therapeutic targets for anti-tumor strategies. In the review, we primarily focused on the role of SHMTs and MTHFDs in cancer. Several inhibitors targeting MTHFDs and SHMTs have exert its potential to decrease tumor burden and inhibit tumor proliferation, highlighting the potential of targeting one-carbon metabolic enzymes for anti-cancer strategies.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

Vital role of SHMT2 in diverse disease

One-carbon metabolism is essential for our human cells to carry out nucleotide synthesis, methylation, and reductive metabolism through one-carbon units, and these pathways ensure the high proliferation rate of cancer cells. Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon metabolism. This enzyme can convert serine into a one-carbon unit bound to tetrahydrofolate and glycine, ultimately supporting the synthesis of thymidine and purines and promoting the growth of cancer cells. Due to SHMT2's crucial role in the one-carbon cycle, it is ubiquitous in human cells and even in all organisms and highly conserved. Here, we summarize the impact of SHMT2 on the progression of various cancers to highlight its potential use in the development of cancer treatments.

Binding Analysis and Structure-Based Design of Tricyclic Coumarin-Derived MTHFD2 Inhibitors as Anticancer Agents: Insights from Computational Modeling.

Unfolded protein response (UPR)-dependent metabolic reprogramming diverts metabolites from glycolysis to mitochondrial 1C metabolism, highlighting pharmacological resistance to folate drugs and overexpression of certain enzymes. Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme that plays a key role in 1C metabolism in purine and thymidine synthesis and is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. To the best of our knowledge, tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported until the present date. The current study aims at the investigation of the available structural data of MTHFD2 in complex with potent and selective inhibitors that occupy the substrate binding site, further providing insights into binding mode, key protein-ligand interactions, and conformational dynamics, that correspond to the experimental binding affinities and biological activities. In addition, we carried out structure-based drug design on the substrate binding site of MTHFD2, by exploiting the cocrystal structure of MTHFD2 with the tricyclic coumarin-based inhibitor. The structure-based drug design campaign involves R-group enumeration, bioisostere replacement, molecular docking, ADME prediction, MM-GBSA binding free energy calculations, and molecular dynamics simulations, that led to a small library of new and potential compounds, capable of selectively inhibiting MTHFD2. The results reported herein are expected to benefit medicinal chemists working on the development of selective MTHFD2 inhibitors for cancer treatment, although experimental validation by biochemical and/or pharmacokinetic assays is required to substantiate the outcomes of the study.

Association between folate and non-alcoholic fatty liver disease among US adults: a nationwide cross-sectional analysis.

Association between folate and non-alcoholic fatty liver disease among US adults: a nationwide cross-sectional analysis.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4mM D-glucose + 2mM [U-13C]serine; 2) 6mM glycine + 4mM D-glucose + 2mM [U-13C]serine; 3) 4mM D-fructose + 2mM [U-13C]serine; 4) 6mM glycine + 4mM D-fructose + 2mM [U-13C]serine; 5) 4mM D-glucose + 4mM D-fructose + 2mM [U-13C]serine; or 6) 6mM glycine + 4mM D-glucose + 4mM D-fructose + 2mM [U-13C]serine. After 2h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

SYNTHESIS OF TRITIUM LABELLED THYMIDINE AND URIDINE

This study present tritium labeling methods for uridine and thymidine in the methyl position. The synthesis of tritiated thymidine has been performed using two different methods: catalytic hydrogenation of brominated derivative and respectively of hydroxymethyl deoxiuridine. The tritiated uridine has been synthesized by catalytic hydrogenation of 5-I Uridine. The labelled compounds were purified by Thin Layer Chromatography and characterized by determination of radioactive concentration, chemical concentration and radiochemical purities. The obtained tritiated thymidine and uridine have been used for in vitro studies for evaluation of radiation effects in cell cultures.

One-Carbon Metabolism: Pulling the Strings behind Aging and Neurodegeneration.

One-carbon metabolism (OCM) is a network of biochemical reactions delivering one-carbon units to various biosynthetic pathways. The folate cycle and methionine cycle are the two key modules of this network that regulate purine and thymidine synthesis, amino acid homeostasis, and epigenetic mechanisms. Intersection with the transsulfuration pathway supports glutathione production and regulation of the cellular redox state. Dietary intake of micronutrients, such as folates and amino acids, directly contributes to OCM, thereby adapting the cellular metabolic state to environmental inputs. The contribution of OCM to cellular proliferation during development and in adult proliferative tissues is well established. Nevertheless, accumulating evidence reveals the pivotal role of OCM in cellular homeostasis of non-proliferative tissues and in coordination of signaling cascades that regulate energy homeostasis and longevity. In this review, we summarize the current knowledge on OCM and related pathways and discuss how this metabolic network may impact longevity and neurodegeneration across species.

Protective Effects of Petroselinum crispum (Parsley) Extract Against Methotrexate-Induced Hepatotoxicity

Objective: By inhibiting the synthesis of thymidine and purine, and thereby DNA synthesis, Methotrexate (MTX), suppresses the proliferation of cancer cells. It is thought that the side-effect mechanism is related to oxidant molecules derived from MTX metabolism. In this study, we examined whether the Petroselinum crispum extracts (PCr; parsley) of which the antioxidant properties have been previously shown, was protective against MTX induced liver damage. Materials and Methods: Sprague Dawley rats (female/male; 200-250 g) were used. MTX was injected intraperitoneally and PCr extract was given orally. A single dose of 20mg/kg MTX was administered to the groups that were to experience hepatotoxicity. Then, a physiological saline (MTX group) or PCr (2 g/kg, MTX + PCr group) treatment was applied for 5 days. The same treatments were applied to the other groups (control group, PCr group) for 5 days after a single dose saline injection. At the end of the study, the biochemical parameters were examined in the blood and liver tissues taken from animals sacrificed by decapitation. Results: MTX caused a significant increase in malondialdehyde and collagen levels and myeloperoxidase and caspase-3 activities, while glutathione levels were found to have decreased. PCr treatment showed protective efficacy by preventing these increases. Conclusion: It appears that the administration of PCr to MTX treated rats prevented the accumulation of lipid peroxides, inflamatory reactions and depletion of antioxidant glutathione, and thus protected liver tissues against oxidative stress.

Roles of Mitochondrial Serine Hydroxymethyltransferase 2 (SHMT2) in Human Carcinogenesis.

In the last few years, cellular metabolic reprogramming has been acknowledged as a hallmark of human cancer and evaluated for its crucial role in supporting the proliferation and survival of human cancer cells. In a variety of human tumours, including hepatocellular carcinoma (HCC), breast cancer and non-small-cell lung cancer (NSCLC), a large amount of carbon is reused in serine/glycine biosynthesis, accompanied by higher expression of the key glycine synthetic enzyme mitochondrial serine hydroxymethyltransferase 2 (SHMT2). This enzyme can convert serine into glycine and a tetrahydrofolate-bound one-carbon unit, ultimately supporting thymidine synthesis and purine synthesis and promoting tumour growth. In tumour samples, elevated expression of SHMT2 was found to be associated with poor prognosis. In this review, the pivotal roles of SHMT2 in human carcinogenesis are described, highlighting the underlying regulatory mechanisms through promotion of tumour progression. In conclusion, SHMT2 may serve as a prognostic marker and a target for anticancer therapies.

The toxic side of one-carbon metabolism and epigenetics

One-carbon metabolism is a central metabolic hub that provides one-carbon units for essential biosynthetic reactions and for writing epigenetics marks. The leading role in this hub is performed by the one-carbon carrier tetrahydrofolate (THF), which accepts formaldehyde usually from serine generating one-carbon THF intermediates in a set of reactions known as the folate or one-carbon cycle. THF derivatives can feed one-carbon units into purine and thymidine synthesis, and into the methionine cycle that produces the universal methyl-donor S-adenosylmethionine (AdoMet). AdoMet delivers methyl groups for epigenetic methylations and it is metabolized to homocysteine (Hcy), which can enter the transsulfuration pathway for the production of cysteine and lastly glutathione (GSH), the main cellular antioxidant. This vital role of THF comes to an expense. THF and other folate derivatives are susceptible to oxidative breakdown releasing formaldehyde, which can damage DNA -a consequence prevented by the Fanconi Anaemia DNA repair pathway. Epigenetic demethylations catalysed by lysine-specific demethylases (LSD) and Jumonji histone demethylases can also release formaldehyde, constituting a potential threat for genome integrity. In mammals, the toxicity of formaldehyde is limited by a metabolic route centred on the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which oxidizes formaldehyde conjugated to GSH, lastly generating formate. Remarkably, this formate can be a significant source of one-carbon units, thus defining a formaldehyde cycle that likely restricts the toxicity of one-carbon metabolism and epigenetic demethylations. This work describes recent advances in one-carbon metabolism and epigenetics, focusing on the steps that involve formaldehyde flux and that might lead to cytotoxicity affecting human health.

Revitalizing antifolates through understanding mechanisms that govern susceptibility and resistance.

In prokaryotes and eukaryotes, folate (vitamin B9) is an essential metabolic cofactor required for all actively growing cells. Specifically, folate serves as a one-carbon carrier in the synthesis of amino acids (such as methionine, serine, and glycine), N-formylmethionyl-tRNA, coenzyme A, purines and thymidine. Many microbes are unable to acquire folates from their environment and rely on de novo folate biosynthesis. In contrast, mammals lack the de novo folate biosynthesis pathway and must obtain folate from commensal microbiota or the environment using proton-coupled folate transporters. The essentiality and dichotomy between mammalian and bacterial folate biosynthesis and utilization pathways make it an ideal drug target for the development of antimicrobial agents and cancer chemotherapeutics. In this minireview, we discuss general aspects of folate biosynthesis and the underlying mechanisms that govern susceptibility and resistance of organisms to antifolate drugs.

107 Roles of Amino Acids in One-carbon Metabolism and Nutition.

The critical importance of non-essential and conditionally-essential amino acids for physiological function is perhaps best exemplified in one-carbon metabolism (OCM), a complex metabolic process whereby chemical groups containing a single carbon atom are used as biosynthetic building blocks. OCM is required for the synthesis of three of the building blocks of nucleic acids (adenine, guanine, thymidine). In addition, OCM provides methyl groups for many methyltransferases. OMC involves two vitamins (folate, Vitamin B12) and amino acids such as serine and glycine which serve as sources of the one-carbon groups. As much as 70% of the whole-body labile methyl groups (S-adenosylmethionine) produced by OMC in piglets is utilized for creatine synthesis. Formate plays a critical role in OCM. Formate, produced in mitochondria from serine and other substrates is released to the cytosol where it is incorporated into 10-formyl-THF. This may be directly incorporated into purines or reduced to 5,10-methylene-THF which is used either for thymidine synthesis or for the production of methyl groups. We have measured formate in serum from a variety of animals (mice, rats, sheep, pigs, humans) and find substantial concentrations, (25–50 µM), in all cases. An important exception is in late pregnant fetal lambs and fetal rats, where we found very high formate concentrations (200–400 µM) in the fetal circulation. Serine concentrations in the fetal circulation are also very high almost 10-fold higher than in the pregnant ewe. Formate concentrations in the human umbilical cord are 70% higher than in the mother. These data suggest an important role for formate in fetal development. We examined this possibility by examining the fate of formate in fetal sheep. Label from 13C-formate infused into the fetal circulation is incorporated into methionine. This requires that formate serves as a substrate for OCM in fetal sheep. Formate is a new frontier in OCM.

Pharmacogenetic variation influences sensory neuropathy occurrence in Southern Africans treated with stavudine-containing antiretroviral therapy.

BackgroundThe use of the HIV antiretroviral drug stavudine (d4T), a thymidine analogue, is associated with the development of mitochondrial toxicities such as sensory neuropathy (SN). Genetic variation in genes relating to d4T transport and metabolism, as well as genetic variation in the thymidine synthesis pathway, could influence occurrence of d4T-related toxicity.MethodsWe examined this hypothesis in a cohort of HIV-positive South African adults exposed to d4T, including 143 cases with SN and 120 controls without SN. Ten SNPs in four genes associated with stavudine transport, and 16 SNPs in seven genes of the thymidine synthesis / phosphorylation pathway were genotyped using Agena mass spectrometry methods. Associations between sensory neuropathy and genetic variants were evaluated using PLINK by univariate and multivariable analyses.ResultsAge and height were significantly associated with SN occurrence. Using logistic regression with age and height as covariates, and uncorrected empirical p-values, genetic variation in SLC28A1, SAMHD1, MTHFR and RRM2B was associated with SN in South Africans using d4T.ConclusionVariation in genes relating to d4T transport and metabolism, as well as genetic variation in the thymidine synthesis pathway may influence occurrence of d4T-related SN. These data contribute to the characterisation of African pharmacogenetic variation and its role in adverse response to antiretroviral therapy.

Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M−1cm−1 in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives.

Abnormal folate metabolism causes age-, sex- and parent-of-origin-specific haematological defects in mice.

Key points Folate (folic acid) deficiency and mutations in folate‐related genes in humans result in megaloblastic anaemia.Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one‐carbon methyl groups for cellular methylation.In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late‐onset and sex‐specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia.Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific.Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one‐carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age‐, sex‐ and parent‐specific manner.

Abstract 1424: DNA repair capacity in colon cancer patients - The effect on the response to treatment and long-term survival

Abstract Colorectal carcinoma (CRC) is the third most common cancer worldwide with the highest incidence in Central Europe. It is the fourth leading cause of cancer related deaths mainly due to the late diagnosis and low efficacy of treatment. CRC diagnosed in early stage has a five-year survival rate about 90% which drops to near 12% once distant metastases occur. It is a heterogenous disease with different molecular and clinicopathological features depending on the tumor location. Therefore, different treatment strategies are required. A standard treatment of locally advanced rectal cancer includes neoadjuvant chemoradiotherapy followed by surgery, whereas colon cancer treatment consists of surgical resection of the tumor and/or subsequent adjuvant chemotherapy based on disease characteristics. 5-fluorouracil (5-FU) alone or in combination with other compounds is the most used treatment in CRC. The mechanism of 5-FU on molecular level is either its incorporation into DNA or it imbalances the synthesis of thymidine from uracil resulting in false uracil DNA incorporation. These DNA lesions are repaired by base excision (BER) and mismatch repair (MMR) pathways. An effective DNA damage response (DDR) is essential for the maintenance of genome stability in healthy cells, whereas in malignant cells, the suppression of DNA repair capacity (DRC) would increase the effectiveness of chemotherapy through DNA damage accumulation and consequent apoptosis. In contrary the cells with high DRC may show better survival and therefore patients with these molecular characteristics may contend with poor response, resistance to treatment and decreased survival. The aim of our present study was to investigate DRC of BER and MMR in target tissue as a predictive marker for a treatment strategy and long-term survival. In order to minimize a bias by heterogenous therapy we focused only on patients with newly diagnosed colon cancer. Our set of patients was selected based on the criteria of follow-up at minimum 30 months, subsequent treatment with 5-FU and microsatellite stable tumor tissue characteristics. Tumor and adjacent non-affected tissue samples were obtained from one hundred patients at surgical resection. Protein extracts from tissues were isolated both for protein expression analysis and for measurement of DRC. DNA repair and DDR protein expression levels were tested by Western Blot. Functional assessments of DRC were performed by comet assay-based in vitro DNA repair assay. The analysis is running and the data will be statistically analyzed and compared with clinical data (TNM stage, type and course of treatment, presence of recurrence, patient´s performance, etc.). Understanding DRC effect on the treatment response might contribute to the concept assuming that targeted modulating of DNA repair processes can achieve clinical benefit in cancer treatment. Acknowledgements: GA UK 800216, COST LD14050, GACR P303/15/14789S and AZV 15-27580A. Citation Format: Sona Vodenkova, Michal Kroupa, Katerina Jiraskova, Alessio Naccarati, Alena Opattova, Pavel Vodicka. DNA repair capacity in colon cancer patients - The effect on the response to treatment and long-term survival [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 1424. doi:10.1158/1538-7445.AM2017-1424