Thymidylate Synthase Research Articles (Page 1)

The ability to precisely engineer Agrobacterium strains is crucial for advancing their utility in plant biotechnology. We recently implemented the CRISPR RNA-guided transposase system, INTEGRATE, as an efficient tool for genetic modification in Agrobacterium . Despite its promise, the practical application of INTEGRATE in Agrobacterium strain engineering remains underexplored. Here, we present a standardized and optimized workflow that enables researchers to harness INTEGRATE for targeted genome modifications. By addressing common challenges, such as crRNA design, transformation efficiency, and vector eviction, this protocol expands the genetic toolkit available for Agrobacterium , facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticated Agrobacterium rhizogenes K599 strain by deleting the 15-kb T-DNA region from its root-inducing plasmid pRi2659 and inactivating a thymidylate synthase gene to render the strain auxotrophic for thymidine. The protocol provides detailed guidance for each step, including target site selection, crRNA spacer cloning, Agrobacterium transformation, screening for targeted insertion and Cre/ loxP -mediated deletion, and vector removal. This resource will empower new users to perform efficient and reproducible genome engineering in Agrobacterium using the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology.

Interplay between O-GlcNAcylation and 5-FU response in colorectal cancer: Novel insights from an examination of primary and metastatic cell lines.

The escalating global health crisis of antimicrobial resistance demands the urgent development of novel therapeutic agents with new mechanisms of action. Bioactive peptides (BAPs), and specifically antimicrobial peptides (AMPs), represent a highly promising class of candidates due to their broad-spectrum activity and superior biocompatibility compared to conventional antibiotics. This manuscript presents a novel approach to drug discovery by designing multifunctional hybrid peptides through the strategic fusion of conserved domains from cecropin and cathepsin-derived sequences. We established an integrated in silico pipeline, utilizing machine learning for activity prediction and comprehensive ADMET profiling to rationally select three lead candidates with optimal physicochemical properties. Experimental validation confirmed their potent efficacy invitro, demonstrating significant inhibition of both planktonic cultures and resilient biofilms. Critically, these peptides displayed a high safety profile, with no toxicity in erythrocyte or Galleria mellonella larvae models. To elucidate their mode of action, target fishing and molecular docking studies were conducted, revealing high-affinity interactions with essential E. coli enzymes, DNA gyrase, and thymidylate synthase. By combining computational design with robust biological validation, this work establishes a streamlined framework for accelerating anti-infective discovery and positioning these engineered hybrid BAPs as a promising class of antimicrobial agents.

Retraction Note: Identification of thymidylate synthase as a potential therapeutic target for lung cancer.

Cancer remains one of the most significant health challenges worldwide, characterized by uncontrolled cell growth. However, it can be cured by using anticancer drugs. 5-fluorouracil is a drug used to treat various types of cancer with the inhibitory mechanism of the enzyme thymidylate synthase, which is crucial for DNA replication in cancer cells, leading to errors in their growth. Although effective, the short half-life of 5-FU and individual metabolic differences among patients limit its therapeutic potential. Therefore, it is necessary to monitor the 5-FU concentration during treatment to ensure the treatment efficiency. However, monitoring this compound in plasma is challenging due to the coexistence of complicated matrices. The study optimized and validated a sensitive method to quantify 5-fluorouracil in mice plasma using liquid-liquid extraction combined with a high-performance liquid chromatography-photodiode array detector (HPLC-PAD). The technique achieved excellent linearity, precision, accuracy, detection limits (LOD = 1.12 μg/L), and quantification (LOQ = 3.74μg/L). The optimal extraction solvent was ethyl acetate in two replicates (2x3ml), PSA/C18 was the most effective cleaning agent with 97.9 ± 0.2% recovery and the mass of PSA/C18 did not affect the extraction recovery. However, high plasma volume reduced the recovery of 5-FU in plasma. Finally, in vivo studies in mice were used to confirm this method. The findings of this research underscored the importance of personalized dosing strategies based on 5-FU concentration in blood.

Mycobacterium tuberculosis (M.tb) needs a key enzyme called ThyX to make thymidylate, which is essential for DNA replication and cell survival. In our previous study, carglumic acid (CGA) and mesalazine (MSZ) emerged as promising candidates from a Food and Drug Administration-approved drug library, selected through in silico screening. Our current investigation delves into the impact of CGA and MSZ on ThyX's biophysical properties. Utilizing fluorescence quenching, thermal, chemical denaturation, characterization, and circular dichroism spectroscopy, we probed the interaction between ThyX and the drugs. Our results confirm that both CGA and MSZ effectively quench ThyX's intrinsic fluorescence via a static quenching mechanism, leading to structural alterations in the protein. In subsequent in vitro and ex vivo studies, we determined that MSZ and CGA exhibit minimum inhibitory concentrations of 6.25 and 3.12 µg/mL, respectively, against M.tb. Notably, the survival of M.tb within RAW macrophages significantly decreased upon treatment with CGA and MSZ compared to untreated controls. In summary, our findings support the potential repurposing of CGA and MSZ as anti-tuberculosis (TB) drugs. Further validation in animal and clinical models is essential to assess their suitability for TB treatment.IMPORTANCEThyX (Rv2754c), flavin-dependent thymidylate synthase, is a crucial enzyme required by Mycobacterium tuberculosis for DNA replication and RNA maturation, making it a potential drug target to explore novel anti-tuberculosis (TB) treatments. Given the essentiality of ThyX, it was screened against Food and Drug Administration-approved drugs using molecular docking screening, and carglumic acid (CGA) and mesalazine (MSZ) were selected as potential inhibitors. To validate and explore their anti-mycobacterial potential, molecular dynamic simulation of these drugs in the presence of ThyX was carried out, and these studies were validated using in vitro biophysical characterization to establish their binding kinetics and effects of these drugs on the stability and structural changes of ThyX. Lastly, in vitro and ex vivo anti-mycobacterial activity of CGA and MSZ establish them as probable candidates for management of TB.

Background/Objectives: This study aimed to synthesize a novel, high-molecular-weight acyclic heterocyclic compound, compound 5, via a one-pot reaction between Trichloroisocyanuric acid (TCCA) and ethanolamine, and evaluate its anticancer, antioxidant, and antifungal activities. Methods: Its complex tetrameric structure, assembled through N-N linkages, was unequivocally confirmed by a full suite of spectroscopic techniques including IR, 1H & 13C NMR, 2D-NMR, and high-resolution mass spectrometry (LC/Q-TOF/MS). The MTT assay was used to assess the anticancer activity of compound 5 against four different human cancer cell lines. Results: The findings indicate that human colon (HT29) and ovarian (OVCAR3) cancer cells were sensitive to the treatment, whereas brain (glioblastoma) (T98G) cancer cells were resistant. The most pronounced cytotoxic effect was observed in pancreatic (MiaPaCa2) cancer cells. Notably, compound 5 exhibited potent antifungal properties, achieving 100% inhibition of the pathogenic water mould Saprolegnia parasitica zoospores at 100 µM after 10 min. Molecular docking studies corroborated the biological data, revealing a high binding affinity for key cancer and fungal targets (Thymidylate Synthase and CYP51), providing a strong mechanistic basis for its observed activities. Conclusions: These findings establish compound 5 as a promising dual-action agent with significant potential as both a targeted anticancer lead and an eco-friendly antifungal for applications in aquaculture.

Preventing liver carcinogenesis through the use of inhibitors targeting metabolic enzymes ACLY, G6PD, and TYMS, combined with 9-cis retinoic acid.

Design, synthesis, anticancer evaluation, and In silico studies of 2-thiopyrimidine-5-carbonitrile derivatives as potent thymidylate synthase inhibitors.

Medical drug delivery analysis using molecular dynamics simulation of tomudex; thymidylate synthase inhibitor

Stroke is the second most common cause of death worldwide and predominantly affects individuals over 65 years old. Its prevalence is projected to increase in parallel with the aging global population. Nutrition is a modifiable risk factor for ischemic stroke. Folates, B-vitamins and choline play a central role in one-carbon metabolism (1C), which is a key metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, epigenetics, regulation of cell proliferation, and stress resistance. Our research group has previously shown that deficiencies in 1C lead to worsened stroke outcomes using preclinical models. However, the impact of ischemic stroke on 1C enzymes in affected brain tissue remains unknown. The objective of this study is to investigate whether ischemic stroke contributes to a change in the levels of 1C enzymes after ischemic stroke in male and female patients. Cortical brain tissue sections from ischemic stroke patients and controls were stained for enzymes involved in 1C. All tissue was co-stained with neuronal nuclei (NeuN) and DAPI (4′,6-diamidino-2-phenylindole). The colocalization of all three markers was evaluated by two individuals who were blinded to the experimental groups. Ischemic stroke increased neuronal levels of the folate receptor and 1C enzymes, methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) and serine hydroxy methyltransferase (SHMT). In male stroke brain tissue was observed to have increased levels of MTHFR, TS, and SHMT. Female brain tissue had increases in the folate receptor and TS. The results suggest that ischemic stroke leads to increased demand of 1C and that there are some differences between males and females. Figure

Abstract Thymidylate synthase (TYMS) is a pivotal enzyme for synthesizing 2″‐deoxythymidine‐5″‐monophosphate, an essential component in DNA synthesis which is crucial for cell proliferation. Targeting TYMS has been a critical in cancer treatment strategies due to its significance in DNA biosynthesis. Bioinformatics‐based methodologies have become instrumental in expediting drug discovery processes. Among these, molecular docking, and molecular dynamics (MD) simulations stand out for their efficiency in screening compounds, significantly reducing time and costs compared to experimental approaches. Our study utilized structure‐guided computational techniques, utilizing molecular docking and MD simulations, to assess the potential inhibitory effects of phytochemicals IMPHY006730 and IMPHY007030 on TYMS activity. Notably, these compounds exhibited fair docking score, forming hydrogen bonds with amino acid residues at TYMS' binding site followed by prediction of activity spectra for substances (PASS) analysis reveals anticancer activity. Further, 200 ns MD simulations includes minimal structural deviation and residual fluctuation followed by structural compactness assessment through Rg and SASA calculation. Moreover, hydrogen bond, secondary structure and PCA analysis demonstrated stable protein–ligand interactions without significant structural changes, indicating sustained binding stability. Our findings suggest that IMPHY006730 and IMPHY007030 possess promising potential in inhibiting TYMS, marking a potential pathway for developing cancer therapeutics through targeted inhibition of this crucial enzyme.

3198eP Polymorphisms in the TYMS gene as potential biomarkers of fluoropyrimidine-related toxicity

At least 25% of colorectal cancer (CRC) patients develop liver metastases (CRLM), and chemotherapeutic regimens based on the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) provide a survival advantage, but long-term survival is uncommon. The primary molecular target of FP drugs is thymidylate synthase (TS). A TS/Top1 dual-targeting cytotoxic mechanism for CF10/LV was confirmed by TS ternary complex detection by Western blot and by immunofluorescence detection of Top1 cleavage complexes. CF10/LV activated the ATR/Chk1 pathway consistent with enhanced replication stress and induced apoptosis. In vivo studies showed CF10 and CF10/LV eradicated liver metastasis in a CRLM model without scarring or weight loss, displaying therapeutic advantages relative to legacy FPs. We demonstrated that a nanoscale FP polymer, CF10, displayed greater potency than expected based on FP content in part through more direct conversion to the TS-inhibitory metabolite, FdUMP. In this study, we tested CF10 for potency advantages relative to 5-FU and trifluorothymidine (TFT, the FP component of TAS-102) and confirmed a general potency advantage for CF10 in CRC cell lines in the Broad Institute PRISM screen. We demonstrated that this potency advantage is retained in CRC cells cultured with human-like folate levels and is enhanced by LV co-treatment to a similar extent as that by 5-FU. Our results confirm CF10 development proceeding as a CF10/LV combination. Mechanistically, CF10 cytotoxicity closely correlates with poisons of DNA topoisomerase 1 (Top1) in the PRISM screen relative to 5-FU and TFT. Our pre-clinical data support an early-phase clinical trial for CF10 for treating liver-metastatic CRC.

CAPRIN1/TYMS/MTHFD2 axis promotes EMT process in nasopharyngeal carcinoma development.

Stroke is the second most common cause of death worldwide and predominantly affects individuals over 65 years old. Its prevalence is projected to increase in parallel with the aging global population. Nutrition is a modifiable risk factor for ischemic stroke. Folates, B-vitamins and choline play a central role in one-carbon metabolism (1C), which is a key metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, epigenetics, regulation of cell proliferation, and stress resistance. Our research group has previously shown that deficiencies in 1C lead to worsened stroke outcomes using preclinical models. However, the impact of ischemic stroke on 1C enzymes in brain tissue remains unknown. The objective of this study is to investigate whether ischemic stroke contributes to a change in the levels of 1C enzymes after ischemic stroke in male and female patients. Brain tissue sections from ischemic stroke patients and controls were stained, all tissue was co-stained with neuronal nuclei (NeuN) and DAPI (4′,6-diamidino-2-phenylindole). The colocalization of all three markers was evaluated by two individuals who were blinded to the experimental groups. Ischemic stroke increased neuronal levels of the folate receptor and 1C enzymes, methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) and serine hydroxymethyltransferase (SHMT). In male stroke brain tissue was observed to have increased levels of MTHFR, TS, and SHMT. Female brain tissue had increases in the folate receptor and TS. The results demonstrate that ischemic stroke leads to increased demand on 1C metabolism and that the response is different between male and female patients.

Harringtonine (HT) targets PARP-1 to inhibit TS mRNA m6A modification, blocks STAT3-induced miR-4521/TS axis and attenuates 5-fluorouracil (5-FU) resistance in NSCLC.

Abstract Thymidylate synthase (hTS) is considered an important target for cancer therapy. Novel TS-dimer disruptors, Ddis, inhibit hTS with a different mechanism of action compared to drugs currently used in chemotherapy strategies. Ddis inhibits the activity of hTS, favours its intracellular degradation and inhibits cancer growth. In the present study, we demonstrate the effect of Ddis in HCT116 colon cancer cell line and the role of hTS in DNA damage and DNA damage response. Ddis, unlike other drugs currently in clinical use (e.g. 5-Fluorouracil, 5-FU) reduced hTS protein level, induced replication stress and DNA damage (γ-H2AX) followed by G2/M-phase arrest and apoptosis of HCT116 cell lines. Our immunofluorescence experiment demonstrated that Ddis caused the tumor suppressor P53-binding protein 1 (53BP1) accumulation in the nuclear foci in HCT116 cells after treatment with Ddis with respect to untreated control cells. The 53BP1 is one of the main mediators of DNA Damage Repair (DDR) and plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway. Furthermore, we observed the activation of Checkpoint kinase 1, Chk1 (pChk1-ser345). Chk1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR serine/threonine kinase, leads to inhibition of DNA replication and cell cycle progression. It is noteworthy that through Flow cytometry analysis, we observed a significant increase in the percentage of the G2/M phase of the cell cycle after 24 hours of Ddis treatment with respect to untreated cells. This result was confirmed through the inactivation of nuclear cyclin B/CDK1 activity that halted mitotic entry, allowing time for DNA damage repair. It is well known that the G2/M block prevents cells from entering mitosis when DNA damage has occurred. Overall these results demonstrate a functional link between hTS inhibition and DNA damage that can be exploited therapeutically in a clinical setting. Citation Format: Maria Gaetana Moschella, Michele Lamesta, Daniele Aiello, Gaetano Marverti, Alberto Venturelli, Maria PaolaCosti, Domenico D’Arca. TARGETING THYMIDYLATE SYNTHASE (HTS) SUPPRESSES COLORECTAL CANCER CELL GROWTH BY INDUCING DNA DAMAGE AND APOPTOSIS [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P04.

Synthesis and anticancer evaluation of novel pyrrole-pyrido[2,3-d] pyrimidine-based compounds as thymidylate synthase inhibitors

Structural insights into a plant-conserved DHFR-TS reveal a selective herbicide target.