Tandem synthesis, cytotoxicity, and in silico study of new 1,3,4-oxadiazoles as potential thymidylate synthase inhibitors.

A new series of pyrrole-linked mono- and bis(1,3,4-oxadiazole) hybrids, attached to various arene units, was prepared using a two-step tandem protocol. Therefore, a benzohydrazide derivative was condensed with the appropriate aldehydes in ethanol at 80°C for 60-150 min to give the corresponding N-(benzoylhydrazones). Without isolation, the previous intermediates underwent intramolecular oxidative cyclization in dimethyl sulfoxide at 180°C for 90-200 min in the presence of chloramine trihydrate to afford the target hybrids. The cytotoxicity of all hybrids was examined in vitro against the MCF-7, HEPG2, and Caco2 cell lines. Arene-linked hybrids 4i and 4j, attached to p-nitro and p-acetoxy units, were the most potent ones, with IC50 values ranging from 5.47 to 8.80 and 12.75 to 21.22 µM, respectively, when tested on the above cell lines. At the tested concentrations of 5 and 7.5 µM, hybrid 4i inhibited thymidylate synthase (TS) with the best inhibition percentages of 72.3 and 91.3, whereas hybrid 4j displayed comparable inhibitory activity to the reference pemetrexed. Hybrid 4j had inhibition percentages of 62.7 and 82.6, whereas pemetrexed had inhibition percentages of 59.2 and 80.2, respectively. The capability of hybrids 4i and 4j as potential TS inhibitors is supported by molecular docking studies, while SwissADME predicts their efficacy as drug-like scaffolds.

Cinobufagin restrains the growth and triggers DNA damage of human hepatocellular carcinoma cells via proteasome-dependent degradation of thymidylate synthase.

Anti-tumor candidate drugs from natural products have gained increasing attention. Cinobufagin is a natural product isolated from the traditional chinese medicine Chansu. Herein, we find that cinobufagin inhibits the proliferation and colony-forming ability of human hepatoma HepG2 and SK-HEP-1 cells. Furthermore, cinobufagin induces G2-phase cell cycle arrest and DNA damage in cancer cells. Thymidylate synthase (TYMS), the major target of chemotherapeutic drugs 5-FU or other fluoropyrimidines, which catalyzes the conversion of dUMP to dTMP and provides the sole de novo source of thymidylate for DNA synthesis. We demonstrate that cinobufagin suppresses TYMS expression via proteasome-dependent degradation in human hepatoma cells, moreover, depletion of TYMS restrains the proliferation and colony formation of tumor cells, and the results of western blotting and immunofluorescence assay indicate DNA damage is induced in tumor cells transfected with TYMS-targeting siRNA (siTYMS), additionally, knockdown of TYMS enhances the inhibitory effect of cinobufagin on the proliferative potential of HepG2 and SK-HEP-1 cells. It is worth noting that cinobufagin in combination with 5-FU exhibits antagonism or synergism combined effects on the proliferation of human hepatoma cells, indicating that Chansu-related preparations such as cinobufacini injection and Huachansu capsules applied to clinical practice should be used with caution in combination with 5-FU for the treatment of liver cancer. Collectively, cinobufagin exerts good anti-hepatoma activity through inhibition of growth and induction of DNA damage by promoting the degradation of TYMS. Our results provide evidence that cinobufagin might be a potential agent for the treatment of cancers such as hepatocellular carcinoma. It can also promote the scientific development of Chansu, and has great significance for enriching the application of TCM in the development of new anti-tumor drugs.

Phase II Study of Pemetrexed as a Salvage Chemotherapy for Thymidylate Synthase-Low Squamous Cell Lung Cancer.

PURPOSE: Squamous cell carcinomas (SqCC) of the lung often express high levels of thymidylate synthase (TS), which is associated with primary resistance to pemetrexed. We explored the efficacy of pemetrexed in a selected population of patients with lung SqCC with low TS expression. MATERIALS AND METHODS: In this single-arm phase II trial, we enrolled 32 previously-treated patients with advanced lung SqCC exhibiting low immunohistochemical staining for TS (i.e., in 10% or less of tumor cells). The primary endpoint was 12-week progression-free survival (PFS) rate. RESULTS: Of 32 patients, eight patients (25%) had an Eastern Cooperative Oncology Group performance status of 2, and seven patients (22%) had previously received three or more lines of chemotherapy. The disease control rate from pemetrexed treatment was 30%, and no objective response was observed. The 12-week PFS rate was 24.5% (95% confidence interval [CI], 13.0 to 46.1). Median PFS was 1.3 months (95% CI, 1.3 to 2.7), and median overall survival was 11.8 months (95% CI, 8.1 to not applicable). Most of adverse events were grade 1 or 2. CONCLUSION: Pemetrexed demonstrated modest activity as a salvage chemotherapy in patients with advanced lung SqCC with low TS expression, although its toxicity was generally manageable.

Design, synthesis and antimalarial screening of some hybrid 4-aminoquinoline-triazine derivatives against pf-DHFR-TS.

Existing antifolate antimalarial drugs have shown resistance due to the mutations at some amino acid positions of Plasmodium falciparum DHFR-TS. In the present study, to overcome this resistance, a new series of hybrid 4-aminoquinoline-triazine derivatives were designed and docked into the active site of Pf-DHFR-TS (PDB i.d. 1J3K) using validated CDOCKER protocol. Binding energy was calculated by applying CHARMm forcefield. Binding energy and the pattern of interaction of the docked compounds were analysed. Fifteen compounds were selected for synthesis based on their binding energy values and docking poses. Synthesized compounds were characterised by FTIR, (1)H NMR, (13)C NMR, mass spectroscopy and were screened for antimalarial activity against 3D7 strain of Plasmodium falciparum.

In vitro antimalarial activity and molecular docking analysis of 4-aminoquinoline-clubbed 1,3,5-triazine derivatives.

AIMS: Present report describes the in vitro antimalarial activity and docking analysis of seven 4-aminoquinoline-clubbed 1,3,5-triazine derivatives on pf-DHFR-TS. METHODS AND RESULTS: The antimalarial activity was evaluated in vitro against chloroquine-sensitive 3D7 strain of Plasmodium falciparum. Compounds were docked onto the active site of pf-DHFR-TS using docking server to explicate necessary structural requirements for antimalarial activity. CONCLUSION: Title molecules demonstrated considerable bioactivity against the malaria parasite. Docking analysis revealed deep engulfment of the molecules into the inner groove of pf-DHFR-TS active site by making stable ligand-receptor posses. Hydrophobic interaction was identified as the only major interacting force playing a role between ligand-receptor interaction and minor with hydrogen bonds. SIGNIfiCANCE AND IMPACT OF THE STUDY: The study provided the novel insight into the necessary structural requirement for rationale-based antimalarial drug discovery.

Single nucleotide polymorphism in the 5' tandem repeat sequences of thymidylate synthase gene predicts for response to fluorouracil-based chemotherapy in advanced colorectal cancer patients.

Thymidylate synthase (TS) is the primary target of 5-fluorouracil (5-FU). A VNTR polymorphism in the TS promoter region is associated with the efficacy of 5-FU-based chemotherapy in colorectal cancer. A common G>C SNP at the 12th nucleotide of the second repeat in the TS*3 alleles has been recently described. The combination of SNP and VNTR allows the definition of 3 TS alleles: *2, *3G and *3C. The aim of our study was to evaluate the predictive value of clinical response and survival of these new defined TS alleles. TS genotypes of 89 patients diagnosed with metastatic colorectal cancer and undergoing 5-FU-based chemotherapy were carried out. The clinical outcome was evaluated according to the genotype (high expression genotype: *2R/*3G; *3C/*3G; *3G/*3G; and low expression genotype: *2R/*2R; *2R/*3C; *3C/*3C. A higher overall response was observed in the group of patients with a low expression genotype (p = 0.035). The probability of achieving a clinical response of patients with a low expression-related genotype was 2.9 higher than that of the other group (95% CI = 1.03-5.6, p = 0.04). The median time to progression was 12 months and 9 months in the low and high expression groups, respectively (p = 0.07, log rank test). Overall survival was significantly longer in the low expression group. In this group the median OS was not achieved at 50 months of follow-up in contrast to the 20 months observed in the high expression group (p = 0.03). TS genotype was an independent predictor of progression-free and overall survival in the Cox regression models after adjustment to the other clinical variables. The selection of patients who are likely to respond to 5-FU therapy may be considerably improved if the TS genotype were to include both the VNTR and the SNP located within the promoter region of the gene.

Molecular biology of sporadic gastric cancer: prognostic indicators and novel therapeutic approaches.

Both the availability of multiple treatment modalities and novel therapeutic targets make the correct prognostic stratification and the identification of truly predictive factors an issue of major debate in gastric cancer. Along with "classic" prognostic factors such as those related to the diffusion of the tumour at diagnosis (i.e., depth of gastric wall infiltration, locoregional lymph nodes or distant metastases) or those concerning the pathologic characteristics of the tumour, other, innovative, factors should be considered if a better definition of the characteristics of the tumour is to be given. These biological factors are often derived from the genetic process, which is thought to represent a crucial step to gastric cancer (DNA copy number changes, microsatellite instability, thymidilate synthase, E-cadherin, beta-catenin, mucin antigen, p53, c-erb B-2, COX-2, matrix metalloproteinases, VEGFR and EGFR). Some of those putative prognostic indicators can also be considered predictive of response to therapy as they are a molecular target either to chemotherapeutics (i.e., thymidilate synthase that is targeted by 5FU) or to a new class of antineoplastic molecules (i.e., c-erb B-2 targeted by trastuzumab, COX-2 by NSAIDs, matrix metalloproteinases, EGFR and VEGFR by specific inhibitors).

Cancer pharmacogenetics.

The large number of active combination chemotherapy regimens for most cancers has led to the need for better information to guide the 'standard' treatment for each patient. In an attempt to individualise therapy, pharmacogenetics and pharmacogenomics (a polygenic approach to pharmacogenetic studies) encompass the search for answers to the hereditary basis for interindividual differences in drug response. This review will focus on the results of studies assessing the effects of polymorphisms in drug-metabolising enzymes and drug targets on the toxicity and response to commonly used chemotherapy drugs. In addition, the need for polygenic pharmacogenomic strategies to identify patients at risk for adverse drug reactions will be highlighted.

Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms: relationships with 5-fluorouracil sensitivity.

The relationship of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on 5-fluorouracil (FU) sensitivity was tested on 19 human cancer cell lines (head and neck, breast, digestive tract) in the absence and presence of folinic acid (FA) supplementation. Thymidylate synthase polymorphisms in the 5' promoter region (double or triple tandem repeats) and 3' untranslated region (6-bp deletion) were analysed by PCR. The C677T and A1298C MTHFR polymorphisms were determined by melting curve analyses (LightCycler). Thymidylate synthase activity and intracellular concentration of the reduced folate 5-10 methylenetetrahydrofolate (CH(2)FH(4)) were measured (biochemical assays). Thymidylate synthase activity was significantly different according to 5' TS genotype, heterozygous cell lines (2R/3R) exhibiting higher TS activities than homozygous ones (P=0.05). However, whether in the absence or presence of FA, FU sensitivity was not statistically associated with either 5' or 3' TS polymorphism. Basal CH(2)FH(4) cellular concentrations were lowest in C677T homozygous wild-type (wt) (C/C) cell lines. FU sensitivity was not linked to C677T polymorphism. In contrast, there was a marked trend for a greater FU efficacy in mutated A1298C variants (C/C+A/C) as compared to wt homozygous cell lines (A/A) (P=0.055 and 0.085 without and with FA supplementation, respectively). These results suggest for the first time a potential role of A1298C MTHFR polymorphism on fluoropyrimidine sensitivity.

Pharmacogenomics in colorectal cancer.

The goal in administering chemotherapeutics is to develop the ability to predict the outcome of therapy in terms of response and toxicity. Technology has been developed to allow tumor profiling with the measurement of protein expression and gene expression levels of markers and even genetic polymorphisms that may predict response to particular chemotherapeutics. The chemotherapeutics for which particular markers have been shown to predict outcome include the fluoropyrimidines and platinums. The next step is to develop clinical trials that will prospectively assess the benefits of profiling a patient's particular tumor, which should translate into an improvement in response and toxicity.

Biología molecular del cáncer colorrectal / Molecular biology of colorectal cancer

La biología molecular del cáncer colorrectal (CCR) abarca una amplísima variedad de aspectos que van desde la conocida teoría de las etapas múltiples del desarrollo del tumor y los síndromes hereditarios hasta la aplicación al tratamiento de determinantes moleculares de sensibilidad y resistencia a citostáticos. En el cáncer hereditario cabe destacar las dos vías de la herencia, la vía supresora mediada por el gen supresor APC de la poliposis múltiple familiar y la vía mutadora mediada por la mutación en genes reparadores y conocida como síndrome de Lynch. No cabe duda del interés de los aspectos de la carcinogénesis y la herencia, pero donde mayor importancia alcanza la biología molecular del CCR en el momento actual es en su aportación al pronóstico y tratamiento de los pacientes.Genes tan importantes como el oncogén k-ras nos proporcionarán información pronóstica sobre la agresividad y capacidad de recaída y metástasis, información que podemos completar con el análisis de la inestabilidad alélica (chromosomyc imbalance) en determinados cromosomas como 8p y 18q, y también con la determinación de los títulos de expresión de timidilato sintetasa. A nuestro entender, todavía es más importante la aportación que hace al tratamiento el análisis de la expresión de genes implicados en los mecanismos de reparación del ADN, como ERCC1, y en los mecanismos de acción de citostáticos y el conocimiento de determinados polimorfismos genéticos tales como TS, UGT1A1, XRCC1, XPD, que van a modificar la sensibilidad o resistencia a determinados fármacos y que serán tratados ampliamente en esta revisión (AU)

Impact of the oxaliplatin-5 fluorouracil-folinic acid combination on respective intracellular determinants of drug activity.

The combination of 5-fluorouracil-folinic acid and oxaliplatin has led to a significant improvement of chemotherapy efficacy in advanced pretreated colorectal cancer. The objective of the present study was, considering the oxaplatin-5-fluorouracil-folinic acid combination, to examine the impact of one given drug on the cellular determinants of cytotoxic activity of the other drug. These cellular factors were analysed on the human colon cancer cell line WiDr in clinically relevant conditions of drug exposure ('De Gramont' schedule) with oxaliplatin-folinic acid during 2 h followed by 5-fluorouracil 48 h. The DNA binding of oxaliplatin was significantly reduced by the presence of 5-fluorouracil but this effect was time-dependent and after 50 h the platinum incorporated into DNA was identical in controls and in the drug combination. In the presence of oxaliplatin, there was less formation of FUH(2) which is the first catabolite produced in the cascade of 5-fluorouracil metabolic degradation. The effects of drugs on cell cycle were quite different from one drug to the other with oxaliplatin inducing a shift towards G(2) accumulation and 5-fluorouracil-folinic acid to a greater proportion of cells in G(1)-S. When oxaliplatin and 5-fluorouracil-folinic acid were combined the cell cycle effects were very similar to that of the 5-fluorouracil-folinic acid sequence alone. Oxaliplatin was able to reduce thymidylate synthase activity with a marked impact 28 h after the beginning of cell exposure to the drug. The 5-fluorouracil-folinic acid drug sequence led to a profound reduction in thymidylate synthase activity and this decrease was not markedly enhanced by the presence of oxaliplatin. Regarding apoptosis, changes in mitochondrial membrane permeability were observed in the presence of the tested drugs and the impact of 5-fluorouracil-folinic acid was greater than that of oxaliplatin. The addition of oxaliplatin did not amplify the action of 5-fluorouracil-folinic acid upon mitochondrial membrane permeability change. The presence of oxaliplatin itself did not modify the intracellular concentration of total reduced folates. The fact that oxaliplatin may reduce 5-fluorouracil catabolism could be central in explaining the supra-additive interaction between these drugs.

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

Role of folic acid in modulating the toxicity and efficacy of the multitargeted antifolate, LY231514.

We studied the effects of folic acid on modulating the toxicity and antitumor efficacy of LY231514. Using several human tumor cell lines adapted to growth in low folate medium, folic acid was shown to be 100- to 1000-fold less active than folinic acid at protecting cells from LY231514-induced cytotoxicity. The lethality of LY231514 was compared in mice maintained on standard diet or low folate diet. The LD50 occurred at 60- and 250-fold lower doses of LY231514 in DBA/2 and CD1 nu/nu mice, respectively, maintained on low folate diet compared to standard diet. The L5178Y/TK-/HX-murine lymphoma was much more sensitive to the antitumor action of LY231514 compared to wild type L5178Y-S tumors. For mice on low folate diet, LY231514 at 0.3 and 1 mg/kg (qd x 10, i.p.) produced 100% inhibition of L5178Y/TK-/HX-lymphoma growth, and significant lethality occurred at > or = 3 mg/kg. For mice on standard diet, LY231514 produced > 95% inhibition of tumor growth at 30 to 300 mg/kg, but all mice died at 800 mg/kg. Folic acid supplementation was demonstrated to preserve the antitumor activity of LY231514 while reducing toxicity. The combination of folic acid with LY231514 may provide a mechanism for enhanced clinical antitumor selectivity.

Synthetic interface peptides as inactivators of multimeric enzymes: inhibitory and conformational properties of three fragments from Lactobacillus casei thymidylate synthase.

Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17-38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic reduction of enzyme activity is observed following incubation of TS with the C 20 peptide. The N 22 and M 17 peptides were unable to cause any loss of enzymatic activity. Addition of the C 20 peptide results in a loss of fluorescence of TS labeled with a dansyl group at Cys 198, following aggregation and precipitation of the protein. The effects are not observed for the N 22 or M 17 peptides. Loss of enzymatic activity is related to the ability of C 20 to promote protein aggregation. The conformations of the peptides have been studied using CD and NMR in order to correlate the observed function with solution structures. Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nascent helical structures or multiple turn conformations has been detected for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v/v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolvent, stabilizes the helical conformation in the C 20 peptide. Under similar conditions, N 22 and M 17 remain largely extended with observations of local beta-turn conformations. Interestingly, the C 20 peptide is a beta-hairpin in the native structure, whereas the other two peptides are individual strand components of a beta-sheet.

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria.

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.

The role of thymidylate synthase in cellular regulation.

Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, studies from our laboratory have shown that thymidylate synthase functions as an RNA binding protein. Specifically, thymidylate synthase binds with high affinity to its own mRNA resulting in translational repression. An extensive series of experiments have now been performed to elucidate the molecular elements underlying the interaction between thymidylate synthase and its own mRNA. These studies have shed new light into the critical nucleotide sequences and/or secondary structure that are important for protein recognition. As well, studies to define the domains on the protein essential for RNA binding are currently underway. In addition to the characterization of the cis- and trans-acting elements underlying the interaction between thymidylate synthase and its own mRNA, we have recently shown that thymidylate synthase has the capacity to specifically bind in vitro and in vivo to other cellular RNA species. In this regard, thymidylate synthase interacts with the mRNAs of the c-myc onocogene and the p53 tumor suppressor gene. These two genes have been shown to play critical roles in cell cycle control, DNA biosynthesis, and apoptosis. In vitro studies reveal that the interaction of TS with these cell-cycle related mRNAs results in their translational repression. While the biological significance of these cellular RNA/TS protein interactions remains to be defined, these studies suggest a potential role for TS as a mediator in the coordinate regulation of several critical aspects of cellular metabolism.

[UFT/CDDP preoperative chemotherapy for progressive gastric cancer--histological antitumor effects and thymidylate synthase inhibition rate].

Pre-operative chemotherapy with concomitant use of UFT and CDDP (UFT: oral administration of 400 mg/day for 2 weeks till one day before operation, CDDP, one intravenous drip of 40 mg/m2 one week before operation) was used for 24 untreated cases of advanced stomach cancer diagnosed as resectable pre-operatively, and the histological antitumor effect analyzed in dissected preparation and the thymidylate synthase inhibition rate (TSIR: %) in tumor tissue were examined. The average administration dose of CDDP was 61.1 mg/body, and the average total administration dose of UFT was 5.0 g/body. The histological antitumor effect was grade 0 in 8 cases (33.3%), grade 1a in 10 cases (41.7%), grade 1b in 5 cases (20.8%), and grade 2 in 1 case (4.2%). TSIR in tumor tissue was under 10% in 2 cases (9.1%); over 10% and under 20% in 4 cases (18.2 %); over 20% and under 30% in 6 cases (27.3%); over 30% and under 40% in 5 cases (22.7%); over 40% and under 50% in 3 cases (13.6%); over 50 % in 2 cases (9.1%), and not measurable in 2 cases, with the average of 29.0%. The correlation was observed between histological anti-tumor effect and TSIR in tumor tissue (p < 0.05). These results suggest the possibility that the anti-tumor effect can be estimated at the in vivo level from measurement of TSIR in tumor tissue.