Thymidylate Synthase Inhibitor Research Articles

Design, synthesis and biological evaluation of novel uracil derivatives bearing 1, 2, 3-triazole moiety as thymidylate synthase (TS) inhibitors and as potential antitumor drugs

Research on thymidylate synthase inhibitors has been a hot spot for anticancer drug development. Here, based on the structures and pharmacological properties of two types of TS inhibitors, through a molecular assembly principle of drugs design, we designed and synthesized a series of 30 novel uracil derivatives as TS inhibitors. The antiproliferative ability of these compounds was evaluated against four cancer cell lines (A549, OVCAR-3, SGC-7901, and HepG2) by the MTT assay. Most of them showed excellent activities against all the tested cell lines. Furthermore, hTS assay results showed that these compounds have the unique ability to inhibit hTS activity in vitro. Notably, compound 13j exhibited the most potent activity against A549 cells (IC50 = 1.18 μM) and extremely prominent enzyme inhibition (IC50 = 0.13 μM), which was superior to the pemetrexed (PTX, IC50 = 3.29 μM and IC50 = 2.04 μM). Flow cytometric analysis showed the compound 13j could inhibit A549 cells proliferation by arresting the cell cycle in the G1/S phase, then induced the cell apoptosis. Further western blot analysis showed that compound 13j could down-regulate the cycle checkpoint proteins cyclin D1 and cyclin E to inhibit the cell cycle progression, and then induce intrinsic apoptosis by activating caspase-3, and reducing the ratio of bcl-2/bax. All of these results demonstrated that this new structure has potential drug-making properties and provides new ideas for drug development.

The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast

ABSTRACT5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3ʹ-5ʹ exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU’s cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets.

Ex vivo resistance in childhood acute lymphoblastic leukemia: Correlations between BCRP, MRP1, MRP4 and MRP5 ABC transporter expression and intracellular methotrexate polyglutamate accumulation

Chemoresistance is an important factor in the treatment failure of childhood acute lymphoblastic leukemia (ALL). One underlying mechanism of chemoresistance involves (over)expression of ATP-dependent drug efflux transporters such as multidrug resistance protein 1–5 (MRP1–5) and breast cancer resistance protein (BCRP), which can extrude the important antileukemia drug methotrexate (MTX). Survival of childhood ALL critically depends on the leukemic blasts' capacity for intracellular retention of MTX and MTX-polyglutamates. This pilot study assessed whether expression of MRP1, MRP4, MRP5 and BCRP (real-time PCR) in primary childhood ALL blasts (n = 23) correlated with ex vivo resistance to MTX (assayed by in situ thymidylate synthase inhibition assay (TSIA)), ex vivo accumulation of (radioactive) MTX polyglutamates, and patient survival. Results show that high MRP4 expression is correlated with ex vivo MTX resistance assayed by TSIA (P = 0.01). Moreover, elevated MRP4 and BCRP expression correlated with lower accumulation of MTX-PGs (P = 0.004 and P = 0.03, respectively). Combined high expression of BCRP and MRP4 even further impacted reduced MTX-PG accumulation (P = 0.02). Overall survival was lower (P logrank = 0.04) in children with ALL cells which featured a relatively high expression of both BCRP and MRP4 transporters. These results underscore the impact of high drug efflux transporter expression, notably MRP4 and BCRP, in diminished MTX response in childhood ALL.

Synthesis and Structure-Activity Relationship Studies of Benzo[b][1,4]oxazin-3(4H)-one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase X.

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.

Synthesis, Docking, and Anticancer Activity of New Thiazole Clubbed Thiophene, Pyridine, or Chromene Scaffolds

2‐Cyanoacetamido‐4‐methylthiazole (1) was utilized as a versatile precursor for the construction of new thiazole clubbed thiazolidine, thiophene, pyridine, or chromene scaffold. The base‐catalyzed addition of 1 to phenyl isothiocyanate followed by subsequent treatment of the produced thiocarbamoyl intermediate with ethyl chloroacetate or chloroacetonitrile furnished the corresponding thiazolyl‐thiazolidine and thiazolyl‐thiophene hybrids. The reactions of compound 1 with chemical reagents, namely, acetylacetone, malononitrile, and/or 2‐(4‐anisylidene)‐malononitrile have been studied and furnished the corresponding thiazole‐pyridine hydrides 8–10. Furthermore, treatment of the precursor 1 with salicylaldehyde, various aryl diazonium chlorides, and/or aromatic aldehydes afforded their corresponding thiazolyl‐chromene hybrid 12, arylhydrazono‐nitriles 13, and unsaturated nitriles 14, respectively. The cytotoxicity of the synthesized compounds was screened against the cell lines HepG2, HCT‐116, and MCF‐7. Compounds 8, 10, and 12 recorded the best results, which was illustrated by molecular docking. Molecular Operating Environment molecular docking calculations carried out here is to rationalize correlation between docking results and biological data of thymidylate synthase (Protein Data Bank code: IHVY) inhibition. Docking has been carried out in the same co‐crystallographic inhibitor binding site to predict if the binding mode of active compounds is analogous to that of native inhibitor.

NuTide:302: A phase Ib study to assess the safety, pharmacokinetics and clinical activity of the ProTide NUC-3373 when combined with standard agents used in colorectal cancer.

TPS719 Background: Although 5-fluorouracil-based chemotherapies (5-FU, capecitabine, and floxuridine) remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. These agents require intracellular conversion to the active metabolite fluorodeoxyuridine-monophosphate (FUDR-MP) before they can exert their core anti-cancer activity through inhibition of the enzyme thymidylate synthase (TS). NUC-3373 is a phosphoramidate transformation of FUDR-MP designed to bypass the key resistance mechanisms associated with 5-FU. Results from the first-in-human study of NUC-3373 (NuTide:301) in patients with advanced solid tumours demonstrated a favourable PK/PD profile for NUC-3373, with a longer plasma t1/2 (9.7 hours) than 5-FU (8-14 minutes) and much higher levels of the active anti-cancer metabolite, FUDR-MP (Ghazaly et alESMO, 2017). TS is efficiently inhibited and sequestered into TS-ternary complexes (TS-T), depleting the pool of dTMP within 2-4 hours. Methods: NuTide:302 is a two-part, Phase Ib study in patients with CRC who have relapsed after ≥ 2 prior lines of 5-FU-containing therapies. The primary objective is to identify a recommended NUC-3373 dose when administered every 2 weeks in combination with standard agents used in CRC treatment. Secondary objectives include safety, PK/PD, and anti‐tumour activity. In Part 1, approximately 12 patients will be administered NUC-3373 with leucovorin (LV) to determine if LV is beneficial in augmenting the formation of TS-T. If so, it will be administered in Part 2. In Part 2, the following combination agents will be administered with NUC-3373 (±LV): oxaliplatin; oxaliplatin + bevacizumab; oxaliplatin + panitumumab; irinotecan; and irinotecan + cetuximab. Up to 62 patients will be enrolled in cohorts of 3-6, in a modified 3+3 design. Enrollment to Part 1 initiated in September 2018. Clinical trial information: NCT03428958.

Germline pharmacogenomics of thymidylate synthase gene in patients with gastrointestinal malignancies treated with fluoropyrimidines-based chemotherapy regimens.

545 Background: Fluoropyrimidines are antimetabolites that target the S phase of the cell cycle. The active metabolite, 5-fluorodeoxyuridine monophosphate inhibits thymidylate synthase (TS) enzyme, thus preventing DNA synthesis and ultimately cell death. While controversy exists in the literature, polymorphism in the promoter region of thymidylate synthase gene (TYMS) that decrease TS expression has been associated with increased fluoropyrimidines-associated toxicities. This study explored the association between polymorphism in the promoter region of TYMS gene and fluoropyrimidines-associated toxicities in patients with gastrointestinal malignancies with mixed racial background. Methods: Between 2011 and 2018, 126 patients were genotyped for TYMS. Patients with known high-risk dihydropyrimidine dehydrogenase gene variants were excluded. Fluoropyrimidines-associated toxicity was graded according to the National Cancer Institiute Common Terminology Criteria for Adverse Events (v 5.0). Fisher’s exact test was used for statistical analysis. Results: TYMS genotypes that predict increased TS expression (3RG/3RG, 3RG/3RC, 2R/3RG, 2R/4R, 3R/4R, 4R/3RG) were identified in 55 patients (44%). TYMS genotypes that predict decreased TS expression (2R/2R, 2R/3RC, 3RC/3RC) were seen in 71 patients (56%). Among patients with genotypes that predict increased TS expression (N = 55), 12 patients had grade 3-4 toxicity (22%) while among patients with genotypes that predict decreased TS expression, 30 patients had grade 3-4 toxicities (42%) (P = 0.0219). Compared to patients with genotypes predicting increased TS expression, 17 out of 31 patients (55%) with 2R/2R TYMS genotype had grade 3-4 toxicity (P = 0.0039) and 15 out 40 patients (38%) with 2R/3RC and 3RC/3RC TYMS genotype had grade 3-4 toxicity (P = 0.1108). Among patients with 2R/2R TYMS, Caucasians represented 61% and African Americans represented 39%. Females represented 65% of the patients. Conclusions: Polymorphism in the promoter region of TYMS gene that predict decreased TS expression due to 2R/2R variant was associated with grade 3-4 fluoropyrimidines-associated toxicities.

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.

17-allylamino-17-demethoxygeldanamycin impeded chemotherapy through antioxidant activation via reducing reactive oxygen species-induced cell death.

Hyperthermia enhances the anticancer effects of thymidylate synthase (TYMS) inhibitors (raltitrexed, RTX) and improves the precise biochemical mechanisms partially through enhancement of intracellular drug absorption. Recent research focuses on the potential anticancer drug target Heat Shock Protein 90 (HSP90), which could increase the sensitivity of cancer cells to TYMS inhibitors; however, with different HSP90 inhibitors, several research studies finally showed a poor efficacy in preclinical or clinical research. Here, we showed that 17-allylamino-17-demethoxygeldanamycin (17-AAG, HSP90 inhibitor) affects the efficacy of chemotherapy through antioxidant activation-induced resistance. In this study, we found that RTX, alone or in combination with hyperthermia, triggers reactive oxygen species (ROS) exposure and thus induces cell death. Also, the addition of hyperthermia showed more ROS exposure and function. The pharmacologic inhibition of HSP90 reversed the effects of chemotherapeutical treatments, while the overexpression of HSP90 showed no relation with these effects, which demonstrated that dysregulation of HSP90 might have a significant impact on chemotherapeutic treatments. The addition of 17-AAG increased the activation of antioxidant with increased antioxidant enzymes, thus affecting the RTX efficacy.

PH-Promoted Release of a Novel Anti-Tumour Peptide by "Stealth" Liposomes: Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells.

To evaluate the potential effects of PEGylated pH-sensitive liposomes on the intracellular activity of a new peptide recently characterized as a novel inhibitor of the human thymidylate synthase (hTS) over-expressed in many drug-resistant human cancer cell lines. Peptide-loaded pH-sensitive PEGylated (PpHL) and non-PEGylated liposomes (nPpHL) were carefully characterized and delivered to cis-platinum resistant ovarian cancer C13* cells; the influence of the PpHL on the drug intracellular activity was investigated by the Western Blot analysis of proteins involved in the pathway affected by hTS inhibition. Although PpHL and nPpHL showed different sizes, surface hydrophilicities and serum stabilities, both carriers entrapped the drug efficiently and stably demonstrating a pH dependent release; moreover, the different behavior against J774 macrophage cells confirmed the ability of PEGylation in protecting liposomes from the reticuloendothelial system. Comparable effects were instead observed against C13* cells and biochemical data by immunoblot analysis indicated that PEGylated pH-sensitive liposomes do not modify the proteomic profile of the cells, fully preserving the activity of the biomolecule. PpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.

Quantitative structure–activity relationship of the thymidylate synthase inhibitors of Mus musculus in the series of quinazolin-4-one and quinazolin-4-imine derivatives

A quantitative structure–activity relationship analysis of the 2-methylquinazolin-4-one and quinazolin-4-imine derivatives, well-known antifolate thymidylate synthase (TYMS) inhibitors, has been performed in the range IC50 = 0.4÷380000.0 nmoL/L using the GUSAR 2013 program. Based on the MNA and QNA descriptors using the self-consistent regression, 6 statistically significant consensus models for predicting the IC50 numerical values have been constructed. These models demonstrate high and moderate prognostic accuracies for the training and external validation test sets, respectively. The molecular fragments of TYMS inhibitors regulating their antitumor activity are identified. The obtained data open opportunities for developing novel promising inhibitors of TYMS.

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.

Effect of Simultaneous Inhibition of Protein Kinase CK2 and Thymidylate Synthase in Leukemia and Breast Cancer Cells.

Protein kinase CK2 was recently identified as a promising therapeutic target for combination therapy. Our study aims to investigate the anticancer effect of a simultaneous inhibition of thymidylate synthase (TS) and CK2 in MCF-7 breast cancer and CCRF-CEM leukemia cells. The type of interaction between CK2 inhibitors: CX-4945, 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), or recently obtained 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile (2b) and TS-directed anticancer drug, 5-fluorouracil (5-FU) was determined using the MTT assay and a combination index method. The influence of the combined treatment on apoptosis in leukemia cells, as well as on cell-cycle progression and the levels of TS, CK2α and P-Ser529-p65 were determined in both cell lines, using flow cytometry and western blot analysis, respectively. The best synergistic effect was observed in CCRF-CEM cell line with the combination of 5-FU and 2b which correlated with a decrease in the endocellular CK2 activity and enhancement of the pro-apoptotic effect. The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-FU in anticancer treatment, indicating a different molecular mechanism of the studied CK2 inhibitors interaction with 5-FU.

Evaluation of pemetrexed and etoposide as therapeutic regimens for human papillomavirus-positive oral and oropharyngeal cancer.

Although human papillomavirus (HPV) positive oral and oropharyngeal cancers have distinct epidemiologic and molecular characteristics compared to HPV-negative cancers, all patients with oral and oropharyngeal cancers received same standard regimen regardless of HPV status. For these reasons, specific regimens for patients with HPV-positive oral and oropharyngeal cancer are needed. Differentially expressed genes (DEG) between HPV-positive and HPV-negative oropharyngeal cancers were re-analyzed and categorized from public database. Then, druggable targets to HPV-positive oral and oropharyngeal cancer were identified and were validated with E6/E7, which is oncogene of HPV, transfected oral and oropharyngeal cancer cell lines and HPV infected cell lines. In DEG analysis, HPV-positive oral and oropharyngeal cancer showed distinct disease entity from HPV-negative cancers. Unlike HPV-negative oral and oropharyngeal cancer, thymidylate synthase (TS) and topoisomerase II (Topo II) were overexpressed in HPV-positive cancers. Transfection of Lenti-virus containing E6/ E7 to HPV-negative oral and oropharyngeal cancer cells induced upregulation of TS and Topo II in those cells. Although cisplatin, which is standard regimen in head and neck cancers, showed more effectiveness in HPV-negative cells, 5-FU and pemetrexed, which are TS inhibitors, or etoposide, which is Topo II inhibitors, worked more effectively in HPV-positive cells. In addition, cisplatin/etoposide and cisplatin/pemetrexed combination regimens showed synergic effects in HPV-positive cells. Pemetrexed or etoposide alone, or in combination with other chemotherapeutic agents such as cisplatin, can be used as novel substitutes in a regimen of concurrent chemoradiotherapy or a palliative regimen for HPV-positive oral and oropharyngeal cancer patients. However, a well-designed clinical trial is needed.

Abstract 4027: Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells

Abstract Pemetrexed (MTA) is a folate antimetabolite used for treatment of non-small cell lung cancer and plural mesothelioma. Although several molecules such as thymidylate synthase (TS) or ATP-binding cassette transporter 11 (ABCC11) have been reported to be involved in the mechanisms of resistance to MTA, precise mechanisms underlying resistance to MTA remains unknown. To elucidate the mechanisms of resistance to MTA in lung cancer, we established MTA resistant clones in PC9 and H1993 lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Then, gene expression profiles were compared between PC9 and PC9-MTA cells by using microarray analyses, and several genes including fibroblast growth factor 2 (FGF2) were identified. Elevated expression of FGF2 in PC9-MTA cells and H1993-MTA cells were confirmed by western blot analyses. Knockdown of FGF2 by siRNA partially restored the sensitivity to MTA both in PC9-MTA and H1993-MTA cells. In addition, anti-FGFR1 inhibitor (PD173074) restored MTA sensitivity particularly in PC9-MTA cells. On the other hand, the expression of TS was increased in PC9-MTA and H1993-MTA cells, and the inhibition of TS with the TS-targeting siRNA restored the sensitivity to MTA. However, with regard to the expression of FGF2 and TS, the inhibition of FGF2 did not modify the expression of TS, and vise versa. Furthermore, a simultaneous inhibition of FGF2 and TS by siRNAs in the MTA-resistant cells restored MTA sensitivity to approximately the level observed for the parental cell lines. With regard to the factors related to the signal transduction pathway, the phosphorylation of Erk and Akt in H1993-MTA cells was decreased compared with those in the parental cells. When H1993-MTA cells were treated with FGF2-targeting siRNA or anti-FGFR1 inhibitor, the increase of phosphorylation of ERK and AKT were induced. These data indicated the possibility that both FGF2 and TS could confer MTA resistance in the lung cancer cell lines in an independent manner, and that modification of signal transduction through FGF2-FGFR1 pathway might be involved in the development of MTA resistance in lung cancer cells. Citation Format: Kentaro Miura, Ken-ichi Ito, Takaaki Oba, Asumi Iesato. Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells [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 4027.

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.

Enhancement of 5-Fluorouracil Cytotoxicity by Pyridoxal 5'-Phosphate and Folinic Acid in Tandem.

The current study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (H4PteGlu) into methylene tetra hydro pteroylglutamate (CH2-H4PteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyzes formation of CH2-H4PteGlu by transfer of the Cβ of serine to H4PteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The median-effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29 and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the KD for binding to SHMT, and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.

Design, synthesis and biological evaluation of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)benzoyl hydrazide derivatives as thymidylate synthase (TS) inhibitors and as potential antitumor drugs

The Inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. Thymidylate synthase (TS) is a key rate-limiting enzyme in the initiation of DNA synthesis in cell. Here, we presented two types of thymidylate synthase inhibitors, and, the key pharmacological properties of these two types of thymidylate synthase inhibitor were extracted and combined to design new compounds with inhibitory activity. Therefore, two series of 42 new compounds with the common biological effect of promoting apoptosis are designed and synthesized by combination principle. Most of the compounds had good anti-proliferative activity on A549, OVCAR-3, SGC7901 and MDA-MB-231 cells. The IC50 of compound 10l on A549 cells was 1.26 μM, which was better than that of pemetrexed (PTX, IC50 = 3.31 μM), furthermore, the selection index of compound 10l was higher than PTX. Flow cytometry analysis showed that compound 10l (the apoptosis rate is 39.4%) could induce A549 cell apoptosis and effectively inhibit tumor cell proliferation. Further western blot analysis showed that compound 10l could induce intrinsic apoptosis by activating caspase-3, increasing expression of cleaved caspase-3 and reducing the ratio of bcl-2/bax. All of this makes compound 10l to be a promising compound in future animal tumor models.

Disparate effects of cytotoxic chemotherapy on the antiviral activity of antiretroviral therapy: implications for treatments of HIV-infected cancer patients.

Cancer is a leading cause of death in HIV-infected patients in the era of combination antiretroviral therapy (cART). Yet, there are no specific guidelines for the combined use of cART and chemotherapy in HIV-infected cancer patients. The cellular enzyme thymidylate synthase (TS) catalyses the conversion of dUMP to TMP, which is converted to TDP and ultimately to TTP, a building block in DNA synthesis. TS inhibitors are recommended in some cancers, particularly non-small cell lung cancer (NSCLC). Because TS inhibitors modulate intracellular concentrations of endogenous 2'-deoxynucleotides, we hypothesized that TS inhibitors could impact the anti-HIV activity of nucleoside analogue reverse transcriptase inhibitors (NRTIs). We evaluated gemcitabine and pemetrexed, two approved TS inhibitors, on the anti-HIV activities of NRTIs in infectivity assays using peripheral blood mononuclear cells (PBMCs) and in humanized mice. Gemcitabine enhanced the anti-HIV activities of tenofovir, abacavir and emtricitabine (FTC) in PBMCs. In contrast, pemetrexed had no effect on tenofovir, enhanced abacavir and, unexpectedly, decreased FTC and lamivudine (3TC) activities. Pemetrexed inhibitory effects on FTC and 3TC may be due to lower concentrations of active metabolites (FTCtp and 3TCtp) relative to their competing endogenous nucleotide (dCTP), as shown by decreases in FTCtp/dCTP ratios. Gemcitabine enhanced tenofovir while pemetrexed abrogated FTC antiviral activity in humanized mice. Chemotherapy with TS inhibitors can have opposing effects on cART, potentially impacting control of HIV and thereby development of viral resistance and size of the reservoir in HIV-infected cancer patients. Combinations of cART and chemotherapy should be carefully selected.

The Effects of 5‐Fluorouracil/Leucovorin Chemotherapy on Cognitive Function in Mice

5‐fluorouracil (5‐Fu) and leucovorin (LV) are often given in combination to treat colorectal cancer. 5‐Fu/LV prevents cell proliferation by inhibiting thymidylate synthase (TS), which is an enzyme that catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. While 5‐Fu has been shown to cause cognitive impairments, the synergistic effect of 5‐Fu with LV has not been fully explored. The present investigation was designed to assess how the combination of 5‐Fu and LV affect cognition in a murine model. A total of 30 nine‐month‐old male mice were used in this study; 8 mice received 0.9 % saline injections, 6 mice received LV (90 mg/kg) injections, 8 mice received 5‐Fu (50 mg/kg) injections, and 8 mice received 5‐Fu and LV (50 mg/kg and 90 mg/kg) injections. One‐month post‐treatment, Y‐maze and Morris water maze (MWM) behavioral tasks were performed. Brains were then extracted, cryosectioned, and stained with CD68 and tomato lectin. All animals in the MWM task were able to locate the visible and hidden platform locations in the water maze; however, treatment effects were seen when spatial memory retention was assessed in the probe trials (no platform). A significant impairment in spatial memory retention was observed in the probe trial after the first day of hidden platform training (first probe trial) in animals that received LV, 5‐Fu, and 5‐Fu/LV. However, by day 5, animals that received 5‐Fu only and 5‐Fu/LV showed spatial memory retention in the probe trials, whereas the LV only mice were impaired. We are currently performing microscopy to quantify changes in the activated microglia and brain vasculature. In studies conducted by Mustafa et. al and El Betagy et. al, Lister‐hooded rats were given 5‐Fu and LV and performed the novel location recognition task. For both studies, rats that received 5‐Fu/LV failed to discriminate between the two object locations, suggesting a spatial memory deficit. While these studies are promising, there is still a need to determine how this drug combination affects other aspects of memory and brain function. The present data suggests that LV may exacerbate the cognitive deficits seen with 5‐Fu treatment, as mice that received LV treatment only remained impaired by day 5 of the MWM. Mice that received 5‐Fu and 5‐Fu/LV were also impaired but showed spatial memory retention by day 5.Support or Funding InformationThis work was supported by Pilot Grant under NIH P20 GM109005 (ARA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.