Potent Thymidylate Synthase Inhibitor Research Articles

Abstract C029: Phase Ib/II open label, multi-arm, parallel cohort dose finding and expansion study of NUC-3373 in combination with pembrolizumab in patients with advanced solid tumors or docetaxel in patients with lung cancer (NuTide:303)

Abstract Background: NUC-3373 is a phosphoramidate transformation of fluorodeoxyuridine monophosphate (FUDR-MP) designed to replace fluoropyrimidines, such as 5-FU, which exert their activity via inhibition of thymidylate synthase (TS) and disruption of DNA synthesis and repair. Intracellular cleavage of NUC-3373 releases the anti-cancer metabolite FUDR-MP, thereby potentially improving the therapeutic ratio of 5-FU whilst reducing the practical barriers associated with 5-FU administration. In addition to generating high intracellular levels of FUDR-MP and being a potent inhibitor of TS, pre-clinical data have shown that NUC-3373 can enhance the activity of immunotherapy agents by promoting the release of damage associated molecular patterns, activating a natural killer cell response and reducing inhibitory signals on T-cells. Methods: NuTide:303 is a Phase Ib/II open label, multi-arm, parallel cohort dose finding and expansion study to assess the safety, PK and efficacy of NUC-3373 given in combination with leucovorin (LV) and either (i) pembrolizumab in patients (pts) with solid tumors in Module 1 or (ii) docetaxel in pts with non-small cell lung cancer (NSCLC) or pleural mesothelioma in Module 2. Both modules consist of dose-validation (Phase Ib) and dose-expansion (Phase II). The Phase Ib part will determine the MTD, safety and tolerability along with preliminary efficacy of the combinations (in 6 - 20 pts). In the Phase II part, an additional 20-40 pts will be added. In both modules, pts with measurable disease and ECOG PS 0-1 are eligible. For Module 1, pts diagnosed with any solid tumor who have progressed on ≤2 prior lines of therapy for advanced/metastatic disease (including 1 prior line of an immunotherapy-containing regimen), and for whom pembrolizumab treatment would be appropriate, are eligible. For Module 2, pts with advanced/metastatic NSCLC or pleural mesothelioma who have progressed on, or were unable to tolerate, ≤2 prior lines of chemotherapy-containing regimens for advanced/metastatic disease are eligible. Additional modules combining other agents with NUC-3373 + LV are being planned. Clinical trial information: NCT05714553 Citation Format: Richard H Wilson, Fiona Thistlethwaite, Fiona G McKissock, Elisabeth Oelmann, Jeffrey D Bloss, James F Spicer, Gary Middleton. Phase Ib/II open label, multi-arm, parallel cohort dose finding and expansion study of NUC-3373 in combination with pembrolizumab in patients with advanced solid tumors or docetaxel in patients with lung cancer (NuTide:303) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C029.

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent

IntroductionFluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU.MethodsHuman colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage.ResultsWe demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU.ConclusionTaken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Identification of potential inhibitors of thymidylate synthase (TS) (PDB ID: 6QXH) and nuclear factor kappa-B (NF–κB) (PDB ID: 1A3Q) from Capsicum annuum (bell pepper) towards the development of new therapeutic drugs against colorectal cancer (CRC)

Abstract Colorectal cancer is the third most deadly cancer globally. Drug resistance and attendant side effects make the available standard anti-colorectal cancer drugs against target receptors inefficient. Phytochemicals from medicinal plants are safer, cheaper, effective, and heal diseases from the cellular level. This study is aimed at identifying potential inhibitors of thymidylate synthase (TS) and nuclear factor kappa-B (NF–κB) target receptors from Capsicum annuum towards the development of new therapeutic drugs against colorectal cancer via in silico approach. One hundred and fifty (150) ligands previously reported from Capsicum annuum were downloaded from the PubChem database and were subjected to chemo-informatics analyses such as ADMET, drug-likeness, oral bioavailability, bioactivity, and PASS prediction to ascertain their therapeutic and safety profile before docking. The ligands that passed the analyses were docked against TS and NF–κB in duplicate using a creditable docking tool (PyRx). Raltitrexed and emetine were used as the standard drug inhibitors for TS and NF–κB, respectively. The results obtained from this study showed that feruloyl-beta-D-glucose (8.45 kcal/mol), 5-O-caffeoylquinic acid (−8.40 kcal/mol), 5-O-caffeoylquinic acid methyl ester (−7.89 kcal/mol), feruloyl hexoside (−7.40 kcal/mol), O-glucopyranoside (−7.55 kcal/mol), and quercetin (−7.00 kcal/mol) shared the same binding pocket with TS while feruloyl-beta-D-glucose (−7.00 kcal/mol), chlorogenic acid (−6.90 kcal/mol), 5-O-caffeoylquinic acid (−6.90 kcal/mol) and feruloyl hexoside (−6.50 kcal/mol) shared the same pocket with NF–κB. These compounds were selected as best hits due to their excellent inhibitory efficiency and chemoinformatic profiles. Thus, the compounds may function as prospective lead compounds for developing a new anti-colorectal cancer drug.

New thieno[2,3-b]pyridine-fused pyrimidin-4(3H)-ones as potential thymidylate synthase inhibitors: Synthesis, SAR, in vitro and in silico study

Cancer is the second leading cause of death, following cardiovascular disease. Thymidylate synthase (TS) has long been identified as a significant target for chemotherapy due to its critical role in DNA biosynthesis. New thieno[2,3-b]pyridine-fused pyrimidinones were prepared utilizing a three-component tandem protocol. One equivalent of 3-aminothieno[2,3-b]pyridine-2-carboxylate and 1.5 equivalents of dimethylformamide-dimethylacetal in toluene was microwave irradiated at 110 °C for 15 min. Next, the crude enamine was dissolved in dioxane and one equivalent of the respective aromatic amines or 3-aryl-1H-pyrazol-5-amines was added. The mixture was irradiated by microwaves at 100 °C for 20–45 min to produce the desired pyrimidinones in 72–89% yields. Examination of the in vitro cytotoxicity of the new products revealed a wide range of activity. Pyrazole-linked pyrimidinones 2b and 2c, attached to p-Cl and p-NO2, demonstrated the best cytotoxicity with IC50 values up to 6.59 and 3.65 µM, respectively, against MCF-7, HEPG2, and Caco2. The in vitro TS inhibitory activity of 2b and 2c was also assessed. 2b demonstrated comparable activity to the standard pemetrexed with inhibition percentages up to 86.3, whereas 2c inhibited TS the best with inhibition percentages up to 91.5. The Ames test revealed that 2b and 2c had no or weakly mutagenic potential to the Salmonella TA 98 or TA 100 strain in the presence or absence of metabolic activation. Docking study predicted the promising TS inhibitory activity of pyrimidinones 2b and 2c. According to SwissADME and Lipinski's rule of five, all new pyrimidinones could be classified as drug-like.

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.

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil.

The major metabolite of the anticancer agent 5-fluorouracil (5-FU) is 5-fluorodeoxyuridine monophosphate (FdUMP), which is a potent inhibitor of thymidylate synthase (TS). Recently, we hypothesized that 5-FU-resistant colorectal cancer (CRC) cells have increased levels of TS protein relative to 5-FU-sensitive CRC cells and use a fraction of their TS to trap FdUMP, which results in resistance to 5-FU. In this study, we analyzed the difference between the regulation of the balance of the free, active form of TS and the inactive FdUMP-TS form in 5-FU-resistant HCT116 cells and parental HCT116 cells. Silencing of TYMS, the gene that encodes TS, resulted in greater enhancement of the anticancer effect of 5-FU in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. In addition, the trapping of FdUMP by TS was more effective in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. Our observations suggest that the regulation of the balance between the storage of the active TS form and the accumulation of FdUMP-TS is responsible for direct resistance to 5-FU. The findings provide a better understanding of 5-FU resistance mechanisms and may enable the development of anticancer strategies that reverse the sensitivity of 5-FU resistance in CRC cells.

Design, synthesis, anticancer activity and molecular docking studies of new benzimidazole derivatives bearing 1,3,4-oxadiazole moieties as potential thymidylate synthase inhibitors

Compounds 10 and 14 arrest the cell cycle at the G1 phase and induce apoptosis without any necrosis in MDA-MB-231 cells.

Abstract P025: NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites

Abstract Background NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used therapies across a broad range of tumors, including colorectal cancer (CRC). 5-FU exerts its main anti-cancer activity via the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), preventing the conversion of dUMP into dTMP and disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce generation of toxic metabolites including FUTP, which is incorporated into RNA causing myelosuppression and gastrointestinal toxicity, and FBAL which causes hand-foot syndrome. Thymidine supplementation rescues cells from NUC-3373 induced cytotoxicity, supporting a DNA targeted mode of action. The aim of this study was to further evaluate the differences between NUC-3373 and 5-FU. Methods CRC cell lines HCT116 and SW480 were exposed to sub-IC50 doses of NUC-3373 or 5-FU (0.1-25 μM) for 6h. At specific time-points, cells were harvested and analyzed as follows: TS inhibition by western blot, metabolite levels by mass spectrometry (LC-MS & LC-MS/MS) and cell cycle by flow cytometry. In rescue experiments, NUC-3373 and 5-FU were supplemented with thymidine (8 μg/mL) for 24h and cell survival assessed at 96h post-treatment. Results In both cell lines, NUC-3373 was a more potent inhibitor of TS than 5-FU with a higher proportion of TS protein bound to FUDR-MP at low concentrations. At 24h, 10 µM 5-FU was required to achieve the same level of TS binding as 0.1 µM NUC-3373 in HTC116 cells and as 0.5 µM NUC-3373 in SW480 cells. TS inhibition by NUC-3373 was almost maximal by 6 hours and was sustained for at least 48 hours. NUC-3373 generated significantly higher levels (50x) of free FUDR-MP compared to 5-FU, resulting in a more pronounced increase in dUMP levels (5x compared to 5-FU). At 48h, NUC-3373 treated cells remained arrested in S phase, while 5-FU treated cells had reverted to a normal cell cycle. FUTP was present in cells exposed to low doses of 5-FU (0.5 μM) but was not detectable in NUC-3373 treated cells. Finally, thymidine supplementation did not alter cell sensitivity to 5-FU but rescued cells treated with NUC-3373. Conclusion NUC-3373 generates higher intracellular levels of FUDR-MP and is a more potent inhibitor of TS than 5-FU, leading to more pronounced effects on cell cycle arrest and perturbation of the nucleotide pool that can result in misincorporation of uracil into DNA. Furthermore, NUC-3373 did not generate FUTP, consistent with the observation that patients treated with NUC-3373 in clinical studies have experienced much lower rates of FUTP-related toxicities. NUC-3373 is a potent TS inhibitor with a favorable safety profile. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Peter Mullen, Clarissa M. Czekster, David J. Harrison. NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P025.

In Silico Investigation: Opening Doors to Novel Thymidylate Synthase Inhibitors

Introduction: Lung cancer is presumed to be the most notable cause of morbidity and impermanency in human beings caused by uncontrolled cell proliferation of lung tissue which results in abrupt synthesis of DNA. Methods: Prevention of DNA synthesis can show distinctive effect on lung cancer by utilizing Thymidylate synthase (TS), a key rate-limiting enzyme in the DNA synthesis process. However, the available finite aggregate of clinically approved blockers and their corresponding side effects lead to the urgent origination of novel inhibitors. Results and Discussion: In silico approaches (QSAR and molecular docking) have been accomplished to discover new potential inhibitors of TS providing a new strategy to evolve novel thymidylate synthase inhibitors functional in lung cancer. Conclusion: In the present study chemical features of a series of compounds alongside their activities alternating over numerous orders of magnitudes was utilized to generate QSAR models, and these could be further employed to predict the activity of new designed compounds. 3D‒QSAR kNNSW based model with decent statistical data having q2 approximately 95% (internal validation) and 80% (external validation) has validated the importance of steric feature. Further docking analysis using D‒score and ligand receptor interactions indicated that all the studied compounds are well accommodated in the binding pocket of TS and disparities in the activity are controlled by hydrogen and hydrophobic interactions.

Exposure-response analysis of Raltitrexed assessing liver toxicity.

Raltitrexed (RTX) is a thymidylate synthase inhibitor with large pharmacokinetics (PK) variability that can be administered in case of 5-fluorouracil (5FU) intolerance or dihydropyrimidine dehydrogenase deficiency. While it is a more potent thymidylate synthase inhibitor than 5FU, RTX failed to replace this drug for colorectal cancer patients, mainly due to its toxicity at the recommended dose of 3 mg/m2 every 3 weeks. However, every 2 weeks administration at 2 mg/m2 demonstrated a favourable toxicity profile. We performed a randomized crossover comparative population PK study between every 2 weeks TOMOX (RTX 2 mg/m2 ) and every 3 weeks TOMOX (RTX 3 mg/m2 ). A three-compartment model and a proportional error model best describe the data. Creatinine clearance and sex, but not body surface area (BSA), were covariates of RTX clearance leading to decrease of its interindividual variability of 28%. Weight and body surface area were covariates of central and peripheral volumes of distribution, respectively, leading to decreases of interindividual variability of 34.6% and 100%, respectively. In contrast to the dose, AUC was a good predictor of liver toxicity (P = 0.006, OR = 3.91, 95%CI = [1.48-10.34]). Using covariates to compute individual clearance and a threshold AUC (1.639, determined in this study), a covariates-based dose was calculated, leading to less variability in AUC than observed with the actual BSA-based or fixed doses. These results advocate for the use of creatinine clearance and sex to determine the RTX dose instead of BSA.

Design, synthesis, anticancer evaluation and docking studies of new pyrimidine derivatives as potent thymidylate synthase inhibitors

Cancer is a perplexing and challenging problem for researchers. In this study, a series of 6-aryl-5-cyano-pyrimidine derivatives were designed, synthesized and evaluated for their anticancer activity against HePG-2, MCF-7 and HCT-116 cell lines. Compounds 2, 3d, 4a-c, 5, 8 and 12 displayed high anticancer activity, comparable to that of 5-fluorouracil. Additionally, those compounds with effective anticancer activity were further assessed for their ability to inhibit thymidylate synthase (TS) enzyme. All the tested compounds demonstrated a marked TS inhibitory activity (33.66–74.98%), with IC50 ranging from 3.89 to 15.74 nM. Moreover, apoptosis studies were conducted on the most potent compound 8, to evaluate its proapoptotic potential. Interestingly, compound 8 induced the level of active caspase 3, and elevated the Bax/Bcl2 ratio 44 folds in comparison to the control. Finally, a molecular docking study was conducted to detect the probable interaction between the active compounds and the thymidylate synthase active site.

Abstract 5213: A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines

Abstract Overexpression of the “activating” transcription factors, E2F1,2 and-3a induces genes involved in DNA synthesis and leads to cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. Dysregulation/overexpression of E2Fs is also controlled by deletion or mutations in the retinoblastoma protein in tumors. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) inhibited transcription of the E2F1, 2 and 3a by bounding tightly to E2F promoters. The PEP was cytotoxic at low micro molar concentrations to several malignant cell lines. As the PEP was unstable in vivo, the PEP was encapsulated in PEGylated liposomes (PL-PEP). Treatment of xenografts of the pRB negative small cell lung cancer H-69 and castrate resistant prostate cancer DU145 tumors propagated in mice with the PL- PEP, caused tumor regression. To increase stability and potency of the PEP, L-Arg in the PEP was replaced by D-Arg. Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L- Arg peptide structure in water. In vitro studies showed that the D-Arg PEP was more potent and more resistant to degradation by serum proteases than the L- form. As E2F is important for DNA repair, and for transcription of thymidylate synthase, we tested the effects of the PEP in combination with cisplatin, a DNA damaging drug and pemetrexed, a potent thymidylate synthase inhibitor. Combination studies of the D-ARG pep with cisplatin (in DU145, PC3, LnCap and 4T1) cells and with pemetrexed (in non-small cell lung cancer, H2009, H441, H1975 and H2228) resulted in synergistic cytotoxicity as determined by Chou-Talalay analysis. Citation Format: Gulam M. Rather, Michael Anyanwu, John E. Kerrigan, Kathleen W. Scotto, Olga Garbuzenko, Tamara Minko, Zoltan Szekely, Joseph R. Bertino. A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5213.

Synthesis, characterization and molecular docking studies of thiouracil derivatives as potent thymidylate synthase inhibitors and potential anticancer agents.

Thymidylate synthase (TS), one of folate-dependent enzymes, is a key and well-recognized target for anticancer agents. In this study, a series of 6-aryl-5-cyano thiouracil derivatives were designed and synthesized in accordance with essential pharmacophoric features of known TS inhibitors. Nineteen compounds were screened in vitro for their anti-proliferative activities toward HePG-2, MCF-7, HCT-116, and PC-3 cell lines. Compounds [Formula: see text], [Formula: see text], and 24 exhibited high anti-proliferative activity, comparable to that of 5-fluorouracil. Additionally, ten compounds with potent anti-proliferative activities were further evaluated for their ability to inhibit TS enzyme. Six compounds ([Formula: see text], [Formula: see text], [Formula: see text], 22, 23 and 24) demonstrated potent dose-related TS inhibition with [Formula: see text] values ranging from 1.57 to [Formula: see text]. The in vitro TS activity results were consistent with those of the cytotoxicity assay where the most potent anti-proliferative compounds of the series showed good TS inhibitory activity comparable to that of 5-fluorouracil. Furthermore, molecular docking studies were carried out to investigate the binding pattern of the designed compounds with the prospective target, TS (PDB-code: 1JU6).

Phase II trial of pemetrexed and carboplatin in platinum-sensitive recurrent ovarian, fallopian tube, and primary peritoneal cancer

Objectives: Despite improvements in median and overall survival (OS) using adjuvant platinum and paclitaxel-based therapy, recurrence of epithelial ovarian cancer (EOC) remains challenging. Pemetrexed is a new-generation antifolate that is a potent inhibitor of thymidylate synthase. Based on prior studies, pemetrexed is a promising agent for targeting ovarian cancer, which generally overexpresses folate receptors, and has shown synergistic preclinical efficacy in combination with platinum.

Good Response to Pemetrexed after Treatment Failure with Another Thymidylate Synthase Inhibitor in Lung Adenocarcinoma: A Case Report

Pemetrexed is an antimetabolic agent and is well-known as a potent inhibitor of thymidylate synthase (TS). It also inhibits dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). We reported an intriguing case in which a non-small cell lung cancer patient who was refractory to pretreatment with S-1, a strong TS inhibitor, showed good response to pemetrexed treatment.

The PARAMOUNT trial: Implications for Maintenance Therapy in Lung Cancer Patients

243 ISSN 1758-1966 10.2217/LMT.13.28 © 2013 Future Medicine Ltd Lung Cancer Manage. (2013) 2(4), 243–246 The maintenance strategic approach has been extensively investigated in hematologic malignancies and then in solid tumors. Among thoracic cancers, small-cell lung cancer has been the setting for several clinical trials of maintenance treatment. The most recent systematic review pooled the available trials evaluating maintenance therapy in small-cell lung cancer, producing a thorough picture of efficacy related to different pharmacological strategies. Despite an overall survival (OS) advantage reported for maintenance chemo therapy and interferon treatment, the results are not clinically relevant [1,2,101]. Maintenance therapy has only recently been accepted as a treatment strategy for advanced non-smallcell lung cancer (NSCLC) with the main goal of prolonging a favorable clinical state after induction first-line platinum-based combination chemo therapy in respect of an adequate tolerability and without impairing quality of life (QoL). This renewed interest has been raised with the availability of active and tolerable new drugs. Among these, pemetrexed, a new cytotoxic agent, is a potent inhibitor of thymidylate synthase and other folate-dependent enzymes. Based on its mechanism of action, pemetrexed was the first chemotherapeutic to be studied as a targeted agent. In fact, due to the evidence of the differential expression of thymidylate synthase between adenocarcinoma and squamous NSCLC, pemetrexed reported an increased efficacy in nonsquamous histology, and thus it is now registered only for the treatment of this histotype worldwide [3,4]. With adequate vitamin supplementation, pemetrexed was shown to be active and well tolerated, with grade ≥3 toxicities, when administered as a single agent, not exceeding 5–10% [5]. First, pemetrexed was tested as switch maintenance within the randomized Phase III trial JMEN. Advanced NSCLC patients of any histology not progressing after four cycles of platinum-based doublets, not including pemetrexed, were randomized to pemetrexed or placebo. Pemetrexed showed a statistically better progressionfree survival (PFS) and OS than placebo, which were more significant for nonsquamous histology [6]. These results led to the design of the PARAMOUNT study, a double-blind, multicenter, Phase III, randomized, placebocontrolled trial. The primary end point was PFS, chosen because this measure is not

Novel 1,3,4-oxadiazole thioether derivatives targeting thymidylate synthase as dual anticancer/antimicrobial agents

A series of novel 1,3,4-oxadiazole thioether derivatives (compounds 9–44) were designed and synthesized as potential inhibitors of thymidylate synthase (TS) and as anticancer agents. The in vitro anticancer activities of these compounds were evaluated against three cancer cell lines by the MTT method. Among all the designed compounds, compound 18 bearing a nitro substituent exhibited more potent in vitro anticancer activities with IC50 values of 0.7±0.2, 30.0±1.2, 18.3±1.4μM, respectively, which was superior to the positive control. In the further study, it was identified as the most potent inhibitor against two kinds of TS protein (for human TS and Escherichia coli TS, IC50 values: 0.62 and 0.47μM, respectively) in the TS inhibition assay in vitro and the most potent antibacterial agents with MIC (minimum inhibitory concentrations) of 1.56–3.13μg/mL against the tested four bacterial strains. Molecular docking and 3D-QSAR study supported that compound 18 can be selected as dual antitumor/antibacterial candidate in the future study.

Assessment of thymidylate synthase expression in biopsy specimens and corresponding resection specimens of non‐small‐cell lung cancer

Pemetrexed (Pem) is a potent inhibitor of thymidylate synthase (TS), and has shown efficacy in the treatment of non-small-cell lung cancer (NSCLC). TS expression in NSCLC biopsy specimens may correlate with the tumour responses to TS-inhibiting agents. As TS is not homogeneously expressed, our aim was to test whether TS expression in biopsy specimens may be representative for the whole tumour and can be used for therapeutic assessment. We immunohistochemically analysed TS expression in biopsy and corresponding resection specimens of 100 consecutive NSCLCs. The samples were subgrouped according to TS expression levels, and the degree of agreement between the biopsy and the corresponding resection specimen was calculated. TS expression-based grouping according to one cut-off criterion (high or low expression) led to concordant results between biopsy and resection specimens. When more than one cut-off criterion was used, the results were significantly different. Thymidylate synthase expression levels in NSCLC biopsy specimens may correspond to TS expression of the whole tumour when robust scoring systems are applied. Further studies are needed to determine whether high or low TS expression in NSCLC biopsy specimens is of predictive value, and whether it may allow the selection of patients who will benefit from Pem treatment.