Thymidylate Synthase Inhibitor Research Articles

The discovery of 1, 3-diamino-7H-pyrrol[3, 2-f]quinazoline compounds as potent antimicrobial antifolates

The shortage of new antibiotics makes infections caused by gram-negative (G−) bacteria a significant clinical problem. The key enzymes involved in folate biosynthesis represent important targets for drug discovery, and new antifolates with novel mechanisms are urgently needed. By targeting to dihydrofolate reductase (DHFR), a series of 1,3-diamino-7H-pyrrol[3,2-f]quinazoline (PQZ) compounds were designed, and exhibited potent antibacterial activities in vitro, especially against multi-drug resistant G− strains. Multiple experiments indicated that PQZ compounds contain a different molecular mechanism against the typical DHFR inhibitor, trimethoprim (TMP), and the thymidylate synthase (TS) was identified as another potential but a relatively weak target. A significant synergism between the representative compound, OYYF-175, and sulfamethoxazole (SMZ) was observed with a strong cumulative and significantly bactericidal effect at extremely low concentrations (2 μg/mL for SMZ and 0.03 pg/mL for OYYF-175), which could be resulted from the simultaneous inhibition of dihydropteroate synthase (DHPS), DHFR and TS. PQZ compounds exhibited therapeutic effects in a mouse model of intraperitoneal infections caused by Escherichia coli (E. coli). The co-crystal structure of OYYF-175-DHFR was solved and the detailed interactions were provided. The inhibitors reported represent innovative chemical structures with novel molecular mechanism of action, which will benefit the generation of new, efficacious bactericidal compounds.

The Effects of 5-Fluorouracil/Leucovorin Chemotherapy on Cognitive Function in Male 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, which catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. While 5-Fu has been shown to cause cognitive impairment, 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. Six-month-old male mice were used in this study; 15 mice received saline injections and 15 mice received 5-Fu/LV injections. One month after treatment, the elevated plus maze, Y-maze, and Morris water maze behavioral tasks were performed. Brains were then extracted, cryosectioned, and stained for CD68 to assay microglial activation and with tomato lectin to assay the vasculature. All animals were able to locate the visible and hidden platform locations in the water maze. However, 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 5-Fu/LV, but these animals showed spatial memory retention by day 5. There were no significant increases in inflammation as measured by CD68, but 5-Fu/LV treatment did modulate blood vessel morphology. Tandem mass tag proteomics analysis identified 6,049 proteins, 7 of which were differentially expressed with a p-value of <0.05 and a fold change of >1.5. The present data demonstrate that 5-Fu/LV increases anxiety and significantly impairs spatial memory retention.

FOXM1 inhibitor, Siomycin A, synergizes and restores 5-FU cytotoxicity in human cholangiocarcinoma cell lines via targeting thymidylate synthase

Aims5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. Main methodsHuman CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. Key findingSingle drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. SignificanceTargeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.

Cardiotoxicity of Fluoropyrimidines: Epidemiology, Mechanisms, Diagnosis, and Management.

Cancer is a growing public health problem; it is responsible annually for millions of deaths worldwide. Fluoropyrimidines are highly effective and commonly prescribed anti-neoplastic drugs used in a wide range of chemotherapy regimens against several types of malignancies. 5-fluorouracil and its prodrugs affect neoplastic cells in multiple ways by impairing their proliferation, principally through the inhibition of thymidylate synthase. Fluoropyrimidine-induced cardiotoxicity was described more than 50 years ago, but many details such as incidence, mechanisms, and treatment are unclear and remain disputed. Severe cardiotoxicity is not only life-threatening, but also leads to withdrawal from an optimal chemotherapy regimen and decreases survival rate. Differences in the frequency of cardiotoxicity are explained by different chemotherapy schedules, doses, criteria, and populations. Proposed pathophysiological mechanisms include coronary vasospasm, endothelial damage, oxidative stress, Krebs cycle disturbances, and toxic metabolites. Such varied pathophysiology of the cardiotoxicity phenomenon makes prevention and treatment more difficult. Cardiovascular disturbances, including chest pain, arrhythmias, and myocardial infarction, are among the most common side effects of this class of anti-neoplastic medication. This study aims to summarize the available data on fluoropyrimidine cardiotoxicity with respect to symptoms, incidence, metabolism, pathophysiological mechanism, diagnosis, management, and resistance.

475P A phase Ib study of NUC-3373, a targeted inhibitor of thymidylate synthase, in combination with standard therapies in patients with advanced colorectal cancer (NuTide:302)

NUC-3373 is a novel phosphoramidate nucleotide analogue that generates high intracellular levels of the anti-cancer metabolite fluorodeoxyuridine-monophosphate (FUDR-MP). Fluoropyrimidines, such as 5-FU and capecitabine, exert their main anti-cancer activity via FUDR-MP, which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. Fluoropyrimidines also result in the generation of the toxic metabolites FBAL and FUTP. NUC-3373, a targeted TS inhibitor, is designed to bypass the 5-FU and capecitabine resistance mechanisms associated with transport, activation and breakdown and to reduce the generation of toxic metabolites such as FBAL and FUTP.

549P Results of a first-in-human study of the ProTide thymidylate synthase inhibitor NUC-3373, in patients with advanced solid tumours (NuTide:301)

The anti-cancer activity of 5-FU requires conversion to its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP) to inhibit thymidylate synthase (TS), a critical enzyme in de novo nucleotide synthesis and cell survival. Although 5-FU based chemotherapies remain the cornerstone of cancer treatment, their clinical utility is limited by resistance mechanisms including conversion to FUDR-MP, catabolism by DPD and short plasma half-life. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass these mechanisms and enhance TS inhibition.

Synthesis and Biological Evaluation of 1,2,3-Triazole Tethered Thymol-1,3,4-Oxadiazole Derivatives as Anticancer and Antimicrobial Agents.

A library of 1,2,3-triazole-incorporated thymol-1,3,4-oxadiazole derivatives (6–18) hasbeen synthesized and tested for anticancer and antimicrobial activities. Compounds 7, 8, 9, 10, and 11 exhibited significant antiproliferative activity. Among these active derivatives, compound 2-(4-((5-((2-isopropyl-5-methylphenoxy)methyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)phenol (9) was the best compound against all three tested cell lines, MCF-7 (IC50 1.1 μM), HCT-116 (IC50 2.6 μM), and HepG2 (IC50 1.4 μM). Compound 9 was found to be better than the standard drugs, doxorubicin and 5-fluorouracil. These compounds showed anticancer activity through thymidylate synthase inhibition as they displayed significant TS inhibitory activity with IC50 in the range 1.95–4.24 μM, whereas the standard drug, Pemetrexed, showed IC50 7.26 μM. The antimicrobial results showed that some of the compounds (6, 7, 9, 16, and 17) exhibited good inhibition on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The molecular docking and simulation studies supported the anticancer and antimicrobial data. It can be concluded that the synthesized 1,2,3-triazole tethered thymol-1,3,4-oxadiazole conjugates have both antiproliferative and antimicrobial potential.

Uncovering cancer vulnerabilities by machine learning prediction of synthetic lethality

BackgroundSynthetic lethality describes a genetic interaction between two perturbations, leading to cell death, whereas neither event alone has a significant effect on cell viability. This concept can be exploited to specifically target tumor cells. CRISPR viability screens have been widely employed to identify cancer vulnerabilities. However, an approach to systematically infer genetic interactions from viability screens is missing.MethodsHere we describe PAn-canceR Inferred Synthetic lethalities (PARIS), a machine learning approach to identify cancer vulnerabilities. PARIS predicts synthetic lethal (SL) interactions by combining CRISPR viability screens with genomics and transcriptomics data across hundreds of cancer cell lines profiled within the Cancer Dependency Map.ResultsUsing PARIS, we predicted 15 high confidence SL interactions within 549 DNA damage repair (DDR) genes. We show experimental validation of an SL interaction between the tumor suppressor CDKN2A, thymidine phosphorylase (TYMP) and the thymidylate synthase (TYMS), which may allow stratifying patients for treatment with TYMS inhibitors. Using genome-wide mapping of SL interactions for DDR genes, we unraveled a dependency between the aldehyde dehydrogenase ALDH2 and the BRCA-interacting protein BRIP1. Our results suggest BRIP1 as a potential therapeutic target in ~ 30% of all tumors, which express low levels of ALDH2.ConclusionsPARIS is an unbiased, scalable and easy to adapt platform to identify SL interactions that should aid in improving cancer therapy with increased availability of cancer genomics data.

Ethanolic Artichoke Extract improves outcome in a rodent model of 5- Fluorouracil Induced Cardio toxicity

Background: During treatment, many drugs may become cause of cardiac system toxicity, cytotoxic drugs therapy cause cardiac toxicity, including 5- Fluorouracil (5- FU). It is regarded as antimetabolite which cause its toxic effects during S phase of the cell cycle and got its activation by conversion of thymidine phosphorylase into fluorodeoxyuridylate (5 fluoro 2'deoxyuridine 5'monophosphate, 5-FdUMP) which cause inhibition of thymidylate synthase, which ultimately cause prevention of synthesis of DNA. Aim: To focus on evaluation of ethanolic artichoke extract (Cynara scolymus L) with respect to its cardio protective properties against 5-fluorouracil (5-FU) induced cardio-toxicity in rabbits by estimation of Alanine aminotransferase, creatine kinase and aspartate aminotransferase enzymes in serum. Methods: 4 groups consisting of 8 rabbits each were made for collected 32 rabbits who were albinos. Group I: (negative control) administered dimethyl sulfoxide (DMSO) (2 ml/kg /day) orally on daily basis for duration of 10 days. Group II: (positive control) administered DMSO (2 ml/kg /day) daily via oral route for duration of 10 days and subsequently received dose of 5-FU (150 mg/kg) (single) by intraperitoneal injection, on day 8th day in connection with DMSO. Groups III: administered ethanolic artichoke extract (200 mg/kg/day) orally on daily basis for duration of 10 days. Groups IV: administered ethanolic artichoke extract (200 mg/kg/day) daily on oral basis for 10 days with subsequently single intraperitoneal dose of 5-FU (150 mg/kg) on day 8th day. Results: Before intoxication via 5-FU, treatment of ethanolic artichoke extract note worthily reduces the increase serum levels of AST,CK &amp; ALT enzymes due to cardio toxicity induced via 5-FU- in case of rabbits . Conclusions: With respect to present scenario, extracts of ethanolic artichoke serve as powerful modulator in reducing or masking cardiac toxicity cause by induction of 5-FU in case of rabbits. Keywords: Artichoke Extract, Fluorouracil Induced Cardio toxicity, Ethanolic

GZ17-6.02 and Pemetrexed Interact to Kill Osimertinib-Resistant NSCLC Cells That Express Mutant ERBB1 Proteins.

We determined the molecular mechanisms by which the novel therapeutic GZ17-6.02 killed non-small cell lung cancer (NSCLC) cells. Erlotinib, afatinib, and osimertinib interacted with GZ17-6.02 to kill NSCLC cells expressing mutant EGFR proteins. GZ17-6.02 did not interact with any EGFR inhibitor to kill osimertinib-resistant cells. GZ17-6.02 interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing mutant ERBB1 proteins or mutant RAS proteins or cells that were resistant to EGFR inhibitors. The drugs interacted to activate ATM, the AMPK, and ULK1 and inactivate mTORC1, mTORC2, ERK1/2, AKT, eIF2α; and c-SRC. Knockdown of ATM or AMPKα1 prevented ULK1 activation. The drugs interacted to cause autophagosome formation followed by flux, which was significantly reduced by knockdown of ATM, AMPKα1, and eIF2α, or by expression of an activated mTOR protein. Knockdown of Beclin1, ATG5, or [BAX + BAK] partially though significantly reduced drug combination lethality as did expression of activated mTOR/AKT/MEK1 or over-expression of BCL-XL. Expression of dominant negative caspase 9 weakly reduced killing. The drug combination reduced the expression of HDAC2 and HDAC3, which correlated with lower PD-L1, IDO1, and ODC levels and increased MHCA expression. Collectively, our data support consideration of combining GZ17-6.02 and pemetrexed in osimertinib-resistant NSCLC.

Predicting Agents That Can Overcome 5-FU Resistance in Colorectal Cancers via Pharmacogenomic Analysis.

5-Fluorouracil (5-FU) is one of several chemotherapeutic agents in clinical use as a standard of care to treat colorectal cancers (CRCs). As an antimetabolite, 5-FU inhibits thymidylate synthase to disrupt the synthesis and repair of DNA and RNA. However, only a small proportion of patients benefit from 5-FU treatment due to the development of drug resistance. This study applied pharmacogenomic analysis using two public resources, the Genomics of Drug Sensitivity in Cancer (GDSC) and the Connectivity Map, to predict agents overcoming 5-FU resistance in CRC cells based on their genetic background or gene expression profile. Based on the genetic status of adenomatous polyposis coli (APC), the most frequent mutated gene found in CRC, we found that combining a MEK inhibitor with 5-FU exhibited synergism effects on CRC cells with APC truncations. While considering the gene expression in 5-FU resistant cells, we demonstrated that targeting ROCK is a potential avenue to restore 5-FU response to resistant cells with wild-type APC background. Our results reveal MEK signaling plays a pivotal role in loss-of-function, APC-mediated 5-FU resistance, and ROCK activation serves as a signature in APC-independent 5-FU resistance. Through the use of these available database resources, we highlight possible approaches to predict potential drugs for combinatorial therapy for patients developing resistance to 5-FU treatment.

Abstract CT140: NUC-3373, a targeted inhibitor of thymidylate synthase, in patients with advanced colorectal cancer

Abstract Background: NUC-3373 is a novel anti-cancer agent developed to replace 5-FU, one of the most widely used anti-cancer drugs globally. Used across a broad range of tumors, including colorectal cancer (CRC), 5-FU (and oral formulation, capecitabine) exerts its main anti-cancer activity via conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373, a targeted inhibitor of TS, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown and avoid the generation of toxic metabolites. NUC-3373 has a longer plasma t1/2 (~10 hours) than 5-FU (8-14 minutes), generating substantially higher intracellular levels of FUDR-MP and lower levels of toxic metabolites FUTP and FBAL. Methods: NuTide:302 is a 3-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of fluoropyrimidine-containing therapies. Part 1: patients receive NUC-3373 with or without leucovorin (LV). Part 2: NUC-3373 + LV is administered in dose-escalation cohorts with either oxaliplatin (NUFOX) or irinotecan (NUFIRI). Part 3: NUFOX and NUFIRI regimens from Part 2 will be combined with biologics targeting VEGF and EGFR pathways. Results: As of 26 November 2020, 37 patients have been treated in Part 1: 21 received 1500 mg/m2 NUC-3373 ± LV q2w; 11 received 1500 mg/m2 NUC-3373 + LV q1w; and 5 received 2500 mg/m2 NUC-3373 + LV q1w. Patients had a median of 4 prior lines of therapy (range: 2-13). NUC-3373 was well tolerated, with 10 patients experiencing Grade 3 treatment-related AEs (2 ALT elevations, 1 AST elevation, 1 ALP elevation, 1 hyponatremia, 1 anemia, 1 fatigue, 1 nausea, 1 fever, 1 bilirubin elevation) and 1 patient experiencing a Grade 4 treatment-related AE (bilirubin elevation). NUC-3373's favorable plasma PK profile was unaffected by LV. Clinical activity observed to date includes a 4th-line patient who achieved a partial response (40% reduction in tumor volume) and a fluoropyrimidine-refractory patient who achieved a 28% reduction in tumor volume and over 5 months disease stabilization. Among the efficacy evaluable population, a disease control rate of 62% was achieved. The RP2D and schedule has not yet been defined. Conclusions: NUC-3373 ± LV (Part 1) has shown encouraging clinical activity in heavily pre-treated CRC patients, including a partial response in a 4th-line patient and a 28% tumor shrinkage and 5-month disease stabilization in a fluoropyrimidine-refractory patient. The safety profile of NUC-3373 ± LV is very encouraging with no NUC-3373 induced neutropenia or hand-foot syndrome of any grade. NUFOX and NUFIRI combinations are currently being investigated in Part 2.Clinical trial information: NCT03428958 Citation Format: Farasat Kazmi, Kristen Ciombor, Janet Graham, Andrew Coveler, Michelle Myers, Jordan Berlin, David Harrison, Sarah Blagden, TR Jeffry Evans. NUC-3373, a targeted inhibitor of thymidylate synthase, in patients with advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT140.

Abstract 1655: NUC-3373 induced DAMPs release in CRC cells promotes natural killer cell activation

Abstract Background: NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used anti-cancer drugs globally. Used across a broad range of tumors, including colorectal cancer (CRC), 5-FU (and oral formulation, capecitabine) exerts its main anti-cancer activity via conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is a targeted inhibitor of TS is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce the generation of toxic metabolites. Drug-induced cellular stress leads to the release of damage associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, from cancer cells. These DAMPs can be detected by immune cells in the surrounding tumor microenvironment and promote immunogenic cell death (ICD) of the cancer cells. Previous data showed that NUC-3373 induces the release of DAMPs in the human CRC lines HCT116 and SW480. This study aims to demonstrate the association between DAMPs release caused by NUC-3373 and the activation of natural killer (NK) cells. Methods: CRC cells HCT116, HT-29 and SW480 were exposed to several sub-IC50 doses of NUC-3373. The negative control was DMSO in medium, and the positive controls were oxaliplatin, a known inducer of ICD, and PMA/ionomycin, which causes NK cell degranulation. After 24 h, the culture medium was changed to wash out NUC-3373 and subsequently, cells were co-cultured with NK-92 MI cells for 4 h. NK cells were harvested from the supernatant and labelled with anti-CD56 for analysis by flow cytometry. NK cell activation was assessed by measuring expression of LAMP1 on the cell surface as an indication of degranulation, and intracellular accumulation of interferon gamma (IFNγ) in cells treated with GolgiStop. Results: Following treatment of HCT116 cells with sub-IC50 doses of NUC-3373, co-cultured NK-92 MI cells displayed a 125% increase in cell surface LAMP1 and a 96% increase in intracellular IFNγ expression compared to the DMSO-treated control. PMA/ionomycin-treated samples displayed a 463% and 271% upregulation in LAMP1 and IFNγ respectively. Degranulation was also observed in NK cells that had been co-cultured with HT-29 and SW480 cells treated at NUC-3373 doses closer to the respective IC50. Conclusions: NUC-3373 induces endoplasmic reticulum stress in CRC cells, followed by release of DAMPs. This promotes NK activation as shown by increased degranulation and upregulation of IFNγ. In an in vivo system, activation of NK cells can recruit and activate other immune cell subtypes to create a more immunogenic environment. This study suggests that NUC-3373 can modulate the tumor microenvironment to promote ICD, making it an attractive combination partner for immunotherapies such as PD-1/PD-L1 blockade therapy. Citation Format: Oliver James Read, David James Harrison, Jennifer Bré. NUC-3373 induced DAMPs release in CRC cells promotes natural killer cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1655.

Abstract 225: Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model

Abstract The purpose of this study is to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively analyze the treatment outcome of 5-fluorouracil (5-FU) in colon cancer. Predicting the outcome of chemotherapeutic agents plays an essential role in improving dose strategies. To that end, we have developed an integrated mechanistic PK/PD model to analyze the kinetics of tumor response to 5-FU treatment. Compared to the existing PK/PD models, our model is novel in the way it incorporates the 5-FU metabolism pathway and downstream antiproliferative events to provide a comprehensive model of treatment outcome. 5-FU has been widely used to treat cancers, including colon cancer. 5-FU metabolites can be incorporated into RNA, DNA and also interfere with thymidylate synthase (TS) catalytic activity, which is the main mechanism of 5-FU cytotoxicity. TS activity has been shown to be the predictor for treatment efficacy. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate catalyzed by TS is the sole intracellular de novo source of thymidylate. The formation of a ternary complex between fluorodeoxyuridylate (FdUMP) and TS obstructs the access of dUMP to the TS nucleotide-binding site. Resultant thymidine deprivation and dUMP accumulation give rise to the deoxynucleotide triphosphate (dNTP) pool imbalance, which could trigger S-phase specific DNA fragmentation, cell cycle arrest and apoptosis. In our PK model, compartmental analysis is developed to track the distribution of 5-FU from its entry into body to interstitial fluid outside the tumor cells and to its cellular uptake. In the cell model, the connection of TS inhibition and dNTP pool imbalance is implemented. In addition to the central plasma and peripheral tissue compartments, our model includes compartments for: 5-FU concentration in the extracellular fluids; intracellular 5-FU; 5-FU anabolites; genomic 5-FU in RNA, and in DNA; and, TS - FdUMP complex. Posttreatment free TS level is used to compute the perturbed TS catalytic activity. Using the model outcome on TS inhibition, we modeled the kinetics of dNTP pool perturbation. The dNTP pool imbalance leads to double strand break (DSB) formation that initiates DNA damage repair response, kinetics of which are also modeled. Our model uses the law of mass action and Michaelian kinetics and each molecular reaction is expressed as a nonlinear differential equation. Time course data from the literature are used in parameter estimation. Our simulation results successfully captured the kinetics of treatment outcome from the administration of the 5-FU to downstream effects in the tumor. The next step in our effort is to expand the model to include the tumor growth kinetics. Citation Format: Chenhui Ma, Alexandru Almasan, Evren Gurkan-Cavusoglu. Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 225.

Design, Molecular docking, Synthesis and Biological evaluation of 5, 7 dimethyl pyrido(2, 3-d)pyrimidin-4-one and 4,5 dihydro pyrazolo (3, 4-d) pyrimidines for cytotoxic activity

The fused pyrimidine derivatives are potent tyrosine kinase and thymidylate synthase inhibitors. The compound 3-(4-sulphonyl amino)-2-methyl thio-6-phenyl azo-5, 7-dimethyl pyrido(2,3-d)pyrimidin-4-one was synthesized from Ethyl 2-amino-4,6-dimethylpyridine-3-carboxylate, benzene diazonium chloride, benzene sulphonyl amino isothiocyanate in subsequent reactions. 1-(1, 3-benzothiazol-2-yl)-3-methyl-4-phenyl-1H-pyrazolo[3,4-d]pyrimidines were synthesized from 1, 3-benzothiazole, 2-thiol, Hydrazine Hydrate, 2-hydrazinyl-1, 3-benzothiazole and aldehydes in subsequent reactions. Twenty-five derivatives pyrimidine scaffolds were designed and performed molecular docking studies for the ability to inhibit the target protein using molecular docking simulation, selective compounds were synthesized and characterized by spectral methods. All the synthesized compounds evaluated for their antioxidant activity and MTT assay exhibited compounds 13c, 13e and 14d can be potential anticancer candidates against MCF-7, Hep G2 and Hela cell lines respectively. Based on all the studies conclude that good agreement was observed between the top-ranked docking scores and top experimental inhibitors when compared with standards ascorbic acid and imatinib. Hence, the compounds could be considered as new anticancer hits for further lead optimization.

Folic acid alleviates age-related cognitive decline and inhibits apoptosis of neurocytes in senescence-accelerated mouse prone 8: deoxythymidine triphosphate biosynthesis as a potential mechanism

Disturbed deoxythymidine triphosphate biosynthesis due to the inhibition of thymidylate synthase (TS) can lead to uracil accumulation in DNA, eventually, lead to neurocytes apoptosis and cognitive decline. Folic acid supplementation delayed cognitive decline and neurodegeneration in senescence-accelerated mouse prone 8 (SAMP8). Whether folic acid, one of nutrition factor, the effect on the expression of TS is unknown. The study aimed to determine if folic acid supplementation could alleviate age-related cognitive decline and apoptosis of neurocytes by increasing TS expression in SAMP8 mice. According to folic acid concentration in diet, four-month-old male SAMP8 mice were randomly divided into three different diet groups by baseline body weight in equal numbers. Moreover, to evaluate the role of TS, a TS inhibitor was injected intraperitoneal. Cognitive test, apoptosis rates of neurocytes, expression of TS, relative uracil level in telomere, and telomere length in brain tissue were detected. The results showed that folic acid supplementation decreased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, alleviated telomere length shorting, increased expression of TS, then decreased apoptosis rates of neurocytes, and alleviated cognitive performance in SAMP8 mice. Moreover, at the same concentration of folic acid, TS inhibitor raltitrexed increased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, and exacerbated telomere length shorting, decreased expression of TS, then increased apoptosis rates of neurocytes, and decreased cognitive performance in SAMP8 mice. In conclusion, folic acid supplementation alleviated age-related cognitive decline and inhibited apoptosis of neurocytes by increasing TS expression in SAMP8 mice.

Osimertinib-resistant NSCLC cells activate ERBB2 and YAP/TAZ and are killed by neratinib

We performed additional mechanistic analyses to redefine neratinib biology and determined the mechanisms by which the multi-kinase inhibitor neratinib interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing either mutant KRAS (G12S; Q61H; G12A; G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M; exon 19 deletion). Neratinib rapidly reduced KRASG12V and RAC1G12V nanoclustering which was followed by KRASG12V, but not RAC1G12V, being extensively mislocalized away from the plasma membrane. This correlated with reduced levels of, and reorganized membrane localization of phosphatidylserine and cholesterol. Reduced nanoclustering was not associated with inactivation of ERBB1, Merlin or Ezrin. The drug combination killed cells expressing mutant KRAS, NRAS or mutant ERBB1 proteins. Afatinib or osimertinib resistant cells were killed with a similar efficacy to non-resistant cells. Compared to osimertinib-resistant cells, sensitive cells had less ERBB2 Y1248 phosphorylation. In osimertinib resistant H1975 cells, the drug combination was less capable of inactivating AKT, mTOR, STAT3, STAT5, ERK1/2 whereas it gained the ability to inactivate ERBB3. In resistant H1650 cells, the drug combination was less capable of inactivating JAK2 and STAT5. Sensitive cells exhibited elevated basal phosphorylation of YAP and TAZ. In resistant cells, portions of YAP and TAZ were localized in the nucleus. [Neratinib + pemetrexed] increased phosphorylation of YAP and TAZ, caused their nuclear exit, and enhanced ERBB2 degradation. Thus, neratinib targets an unidentified protein whose functional inhibition directly results in RAS inactivation and tumor cell killing. Our data prove that, albeit indirectly, oncogenic RAS proteins are druggable by neratinib.

Cellworks Omics Biology Modeling (CBM) to predict therapy response and identifies novel biomarkers for carboplatin/cisplatin along with pemetrexed in NSCLC patients.

e21211 Background: Cancer management using cytotoxic drugs is hampered by limited efficacy. Hence, a personalized treatment approach matching chemotherapy with appropriate patients remains a persistent and unmet need in the clinic. Genomic heterogeneity among patients creates an opportunity to discern key genomic aberrations and pathways that confer resistance and response to standard treatment options. Pemetrexed, an antifolate, primarily inhibits thymidylate synthase (TYMS) while platinum compounds (carboplatin/cisplatin) trigger DNA damage similar to alkylating agents. When the DNA damage exceeds repair, apoptosis results. We conducted a pilot study using CBM to novel genomic biomarkers of response and resistance to pemetrexed–platinum treatment. Methods: 25 patients who received pemetrexed–platinum therapy were selected from TCGA data. Mutation and copy number aberrations from each case served as input into the CBM (generated from PubMed and other online resources) to create a patient-specific protein network map. Disease-biomarkers unique to each patient were identified within protein network maps. Drug impact on the disease network was digitally simulated to determine treatment efficacy by measuring effect of chemotherapy on the cell growth score, i.e., a composite of cell proliferation, viability, apoptosis, metastasis, DNA damage and other cancer hallmarks. Effectively, the mechanism of action of each drug was mapped to each patient’s genome and biological consequences due to genomic abnormalities were correlated with response. Results: Among the 25 patients, 23 responders (R) and 2 non-responders (NR). The computer simulation correctly predicted response in 19/25 with 76% accuracy, 100% specificity and 73.91% sensitivity. CBM identified co-occurrence of deleted segments of chromosome 6q, 13q, 14q and 17q were responsible for pemetrexed-platinum response. The key genes governing response on these chromosomes included drug transporters and DNA repair pathways. ATP7B (13q) which exports platinum-based drugs was deleted in responders. The loss of REV3L (6q ), MBD1 (6q ), ERCC1 (6q), BRCA2 (13q), BRCA1 (17q), FANCM (14q), RAD51B (14q), XRCC3 (14q ) led to failure of DNA repair. RB1 (13q) del also led to failure of BRG1 mediated DNA repair . Combinations of these repair genes and transporters formed the major response criteria among the 23 responders while these characteristics were absent in non-responders. Conclusions: This pilot study highlights how CBM simulation platform can identify patients for therapy response prediction. Copy number changes impact responsiveness to chemotherapy and should be routinely assessed. We suggest that this approach should be validated prospectively in a larger patient cohort.

Design, synthesis, and molecular docking studies of thiazolidinediones as PPAR-γ agonists and thymidylate synthase inhibitors.

New thiazolidine-2,4-dione hybrids were designed and synthesized as potential peroxisome proliferator-activated receptor (PPAR)-γ agonists and thymidylate synthase inhibitors. All the synthesized compounds follow Lipinski's and Veber's rules and possess the desired pharmacokinetics properties. The PPAR-γ transactivation results displayed that compounds 12 (78.9%) and 11 (73.4%) were the most active compounds and they increased PPAR-γ gene expression by 2.2- and 2.4-fold, respectively. Compounds 12, 11, and 8 showed promising cytotoxicity, with IC50 values ranging from 1.4 to 4.5 μM against MCF-7 cells and from 1.8 to 8.4 μM against HCT-116 cells. Compounds 11 and 12 also inhibited thymidylate synthase with IC50 values of 5.1 and 3.2 μM, respectively, confirming their mode of action as thymidylate synthase inhibitors. Finally, molecular docking studies supported the in vitro biological activity results.

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.