Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

OBJECTIVE: This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. MATERIALS AND METHODS: Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. RESULTS: BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. CONCLUSION: This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers.

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis.

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.

Electrochemical detection of the oxidative damage of a potential pyrimido[5,4-g]pteridine-derived antitumor agent toward DNA.

In this work, we design and synthesize 2,2'-(7,9-dimethyl-2,4,6,8-tetraoxo-6,7,8,9-tetrahydropyrimido[5,4-g]pteridine-1,3(2H,4H)-diyl)bis(N,N-bis(2-chloroethyl)acetamide) (PT-MCA) as a novel DNA intercalator and potential antitumor agent. Electrochemical analysis reveals the redox process of PT-MCA on the electrode surface. The bioelectrochemical sensors are obtained by modifying the surface of GCE with calf thymus DNA (ctDNA), poly (dG), poly (dA), and G-quadruplex, respectively. The DNA oxidative damage induced by PT-MCA is investigated by comparing the peak intensity change of dGuo and dAdo and monitoring the peaks of the oxidation products of guanine and/or adenine (8-oxoGua and/or 2,8-oxoAde). UV-vis absorption and fluorescence spectra and gel electrophoresis are further employed to understand the intercalation of PT-MCA into DNA base pairs. Moreover, PT-MCA is proved to exhibit stronger anti-proliferation activity than mitoxantrone against both 4T1 and B16-F10 cancer cells. At last, the oxidative damage of PT-MCA toward ctDNA is not interfered by the coexistence of ions and also can be detected in real serums.

Design, synthesis and biological evaluation of pteridine-7(8H)-one derivatives as potent and selective CDK4/6 inhibitors.

Cyclin-dependent kinases play an important role in the regulation of cell cycle and transcription. Selective CDK4/6 inhibitors have been demonstrated to be effective in the treatment of cancer. In this article, we described the design and synthesis of a series of pteridine-7(8H)-one derivatives as dual CDK4/6 inhibitors. Among them, the most promising compound L2 exhibited significant inhibitory activity against CDK4 and CDK6 with IC50 values of 16.7 nM and 30.5 nM respectively and showed excellent selectivity to CDK1/2/7/9. Moreover, compound L2 displayed potent antiproliferative activities at low digital micromolar range via inducing apoptosis in breast and colon cancer cells. In all, we developed a new series of pteridine-7(8H)-one derivatives which exhibited promising antitumor activities as selective CDK4/6 inhibitors.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.

Multitarget, Selective Compound Design Yields Potent Inhibitors of a Kinetoplastid Pteridine Reductase 1.

The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Evidence for Indoxyl Sulfate as an Inducer of Oxidative Stress in Patients With Diabetes.

BACKGROUND/AIM: Indoxyl sulfate is a metabolite of tryptophan and its urinary level reflects the status of bacterial flora in the intestine. Indoxyl sulfate possesses prooxidant properties and is implicated in various diseases including chronic kidney disease and cardiovascular diseases. However, the relation of urinary indoxyl sulfate to oxidative stress is not known. PATIENTS AND METHODS: The association of urinary indoxyl sulfate levels with urinary levels of oxidative stress markers, 15-isoprostane F2t and pteridine derivatives, was investigated in 255 patients with type 2 diabetes. Indoxyl sulfate and pteridine derivatives were measured by using spectrofluorometry. RESULTS: Urinary levels of indoxyl sulfate, pteridines, and 15-isoprostane F2t showed a normal distribution after logarithmic transformation but not before it, and they were thus used for parametric analysis after logarithmic transformation. Urinary indoxyl sulfate levels were significantly correlated (p<0.01) with urinary 15-isoprostane F2t and pteridine levels [Pearson's correlation coefficients: 0.503 (15-isoprostane F2t) and 0.562 (pteridines)]. These associations were also found in multivariable analysis after adjusting for age, sex, insulin therapy for diabetes, body mass index, mean arterial pressure, hemoglobin A1c, estimated glomerular filtration rate, urinary albumin, and histories of smoking and alcohol drinking. CONCLUSION: Urinary indoxyl sulfate levels showed associations with urinary levels of oxidative stress markers, and the associations were independent of age, sex, insulin therapy for diabetes, body mass index, blood pressure, glycemic status, renal function, smoking, and alcohol drinking. Indoxyl sulfate appears to be an important determinant of redox balance in patients with diabetes.

Volasertib as a monotherapy or in combination with azacitidine in patients with myelodysplastic syndrome, chronic myelomonocytic leukemia, or acute myeloid leukemia: summary of three phase I studies.

BACKGROUND: This report summarizes three phase I studies evaluating volasertib, a polo-like kinase inhibitor, plus azacitidine in adults with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, or acute myeloid leukemia. METHODS: Patients received intravenous volasertib in 28-day cycles (dose-escalation schedules). In Part 1 of 1230.33 (Study 1; NCT01957644), patients received 250-350 mg volasertib on day (D)1 and D15; in Part 2, patients received different schedules [A, D1: 170 mg/m2; B, D7: 170 mg/m2; C, D1 and D7: 110 mg/m2]. In 1230.35 (Study 2; NCT02201329), patients received 200-300 mg volasertib on D1 and D15. In 1230.43 (Study 3; NCT02721875), patients received 110 mg/m2 volasertib on D1 and D8. All patients in Studies 1 and 2, and approximately half of the patients in Study 3, were scheduled to receive subcutaneous azacitidine 75 mg/m2 on D1-7. RESULTS: Overall, 22 patients were treated (17 with MDS; 12 previously untreated). Across Studies 1 and 2 (n = 21), the most common drug-related adverse events were hematological (thrombocytopenia [n = 11]; neutropenia [n = 8]). All dose-limiting toxicities were grade 4 thrombocytopenia. The only treated patient in Study 3 experienced 18 adverse events following volasertib monotherapy. Studies 1 and 2 showed preliminary activity (objective response rates: 25 and 40%). CONCLUSIONS: The safety of volasertib with azacitidine in patients with MDS was consistent with other volasertib studies. All studies were terminated prematurely following the discontinuation of volasertib for non-clinical reasons by Boehringer Ingelheim; however, safety information on volasertib plus azacitidine are of interest for future studies in other diseases.

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model - part II.

INTRODUCTION: Determining the biological significance of pteridines in cancer development and progression remains an important step in understanding the altered levels of urinary pteridines seen in certain cancers. Our companion study revealed that several folate-derived pteridines and lumazines correlated with tumorigenicity in an isogenic, progressive breast cancer cell model, providing direct evidence for the tumorigenic origin of pteridines. OBJECTIVES: This study sought to elucidate the pteridine biosynthetic pathway in a progressive breast cancer model via direct pteridine dosing to determine how pteridine metabolism changes with tumorigenicity. METHODS: First, MCF10AT breast cancer cells were dosed individually with 15 pteridines to determine which pteridines were being metabolized and what metabolic products were being produced. Second, pteridines that were significantly metabolized were dosed individually across the progressive breast cancer cell model (MCF10A, MCF10AT, and MCF10ACA1a) to determine the relationship between each metabolic reaction and breast cancer tumorigenicity. RESULTS: Several pteridines were found to have altered metabolism in breast cancer cell lines, including pterin, isoxanthopterin, xanthopterin, sepiapterin, 6-biopterin, lumazine, and 7-hydroxylumazine (p < 0.05). In particular, isoxanthopterin and 6-biopterin concentrations were differentially expressed (p < 0.05) with respect to tumorigenicity following dosing with pterin and sepiapterin, respectively. Finally, the pteridine biosynthetic pathway in breast cancer cells was proposed based on these findings. CONCLUSIONS: This study, along with its companion study, demonstrates that pteridine metabolism becomes disrupted in breast cancer tumor cells. This work highlights several key metabolic reactions within the pteridine biosynthetic pathway that may be targeted for further investigation and clinical applications.

Hypoxia Tumor Microenvironment Activates GLI2 through HIF-1α and TGF-ß2 to Promote Chemotherapy Resistance of Colorectal Cancer.

BACKGROUND: A majority of relapse cases have been reported in colorectal cancer patients due to cancer stem cell progenitors. The factors responsible for chemoresistance have yet to be discovered and investigated as CSCs have reported escaping from chemotherapy's killing action. OBJECTIVE: In this study, we have investigated the effects of HIF-1α and TGF-ß2 in hypoxia conditions on the expression of GLI2, which is a potential factor for causing chemoresistance. Material and Methods. Colorectal samples of treated patients were collected from the Hospital Biological Sample Library. Culture of patient-derived TSs and fibroblasts was performed. The collected patient samples and cells were used for immunohistochemistry, quantitative PCR, and western blotting studies which were performed. RESULTS: It was reported that HIF-1α (hypoxia-inducible factor) and TGF-ß2 secreted from cancer-associated fibroblasts (CAFs) synergistically work to express GLI2 in cancer stem cells. Hence, it increased the stemness as well as resistance to chemotherapy. CONCLUSION: The HIF-1α/TGF-ß2-mediated GLI2 signaling was responsible for causing chemoresistance in the hypoxia environment. High expressions of HIF1α/TGF-ß2/GLI2 cause the relapsing of colorectal cancer, thus making this a potential biomarker for identifying the relapse and resistance in patients. The study uncovers the mechanism involved in sternness and chemotherapy resistance which will help in targeted treatment.

Discovery of Pteridine-7(8H)-one Derivatives as Potent and Selective Inhibitors of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK) is an attractive therapeutic target in the treatment of cancer, inflammation, and autoimmune diseases. Covalent and noncovalent BTK inhibitors have been developed, among which covalent BTK inhibitors have shown great clinical efficacy. However, some of them could produce adverse effects, such as diarrhea, rash, and platelet dysfunction, which are associated with the off-target inhibition of ITK and EGFR. In this study, we disclosed a series of pteridine-7(8H)-one derivatives as potent and selective covalent BTK inhibitors, which were optimized from 3z, an EGFR inhibitor previously reported by our group. Among them, compound 24a exhibited great BTK inhibition activity (IC50 = 4.0 nM) and high selectivity in both enzymatic (ITK >250-fold, EGFR >2500-fold) and cellular levels (ITK >227-fold, EGFR 27-fold). In U-937 xenograft models, 24a significantly inhibited tumor growth (TGI = 57.85%) at a 50 mg/kg dosage. Accordingly, 24a is a new BTK inhibitor worthy of further development.

Efficient Synthesis and Biological Evaluation of 6-Trifluoroethoxy Functionalized Pteridine Derivatives as EGFR Inhibitors.

BACKGROUND: Pteridine-based scaffolds have been widely prevalent in pharmaceuticals, such as kinase inhibitors targeting EGFR, FLT3 and PI3K/mTOR which are attractive targets for the anticancer therapy. OBJECTIVE: This work aimed at designing and synthesizing 6-2,2,2-trifluoroethoxy functionalized pteridine-based derivatives for investigation of their anti-cancer activities as EGFR inhibitor. METHODS: Pteridine-based derivatives were synthesized in 6 steps involving amination, bromination, cyclization, alkoxylation, chlorination and coupling reactions. Cellular anti-proliferative activities and inhibition activities on EGFR signaling of these pteridine derivatives in vitro were determined by the MTT assay and western blot analysis, respectively. Molecular docking simulation studies were carried out by the crystallographic structure of the erlotinib/EGFR kinase domain [Protein Data Bank (PDB) code: 1M17]. RESULTS: The compound 7m, with IC50 values of 27.40 µM on A549 cell line, exhibited comparable anti-proliferative activity relative to the positive control. Besides, western blots showed its obvious down-regulation of p-EGFR and p-ERK expression at 0.8 µM. The molecular docking model displayed a hydrogen bond between Met-769 amide nitrogen and N-1 in pteridine motif of 7m which lied at the ATP binding site of EGFR kinase domain. CONCLUSION: The inhibition of 7m on cellular growth was comparable to that of the positive control. The inhibitory activities of 7m on EGFR phosphorylation and ERK phosphorylation in A549 cell line were relatively superior to that of the positive control. Both results suggested that the antiproliferative activity of 7m against A549 cell line was caused by inhibition of EGFR signaling pathway, providing a new perspective for the modification of pteridine-based derivatives as EGFR inhibitor.

P90 ribosomal S6 kinase confers cancer cell survival by mediating checkpoint kinase 1 degradation in response to glucose stress.

In solid tumors, cancer cells have devised multiple approaches to survival and proliferate in response to glucose starvation that is often observed in solid tumor microenvironments. However, the precise mechanisms are far less known. Herein, we report that glucose deprivation activates 90-kDa ribosomal S6 kinase (p90 RSK), a highly conserved Ser/Thr kinase, and activated p90 RSK promotes cancer cell survival. Mechanistically, activated p90 RSK by glucose deprivation phosphorylates checkpoint kinase 1 (CHK1), a key transducer in checkpoint signaling pathways, at Ser280 and triggers CHK1 ubiquitination mediated by SCFß-TrCP ubiquitin ligase and proteasomal degradation, subsequently suppressing cancer cell apoptosis induced by glucose deprivation. Importantly, we identified an inverse correlation between p90 RSK activity and CHK1 levels within the solid tumor mass, with lower levels of CHK1 and higher activity of p90 RSK in the center of the tumor where low glucose concentrations are often observed. Thus, our study indicates that p90 RSK promotes CHK1 phosphorylation at Ser280 and its subsequent degradation, which allows cancer cells to escape from checkpoint signals under the stress of glucose deprivation, leading to cell survival and thus contributing to tumorigenesis.

Prise en charge médicale de la récidive du cancer épithélial de l'ovaire: Medical management of recurrent epithelial ovarian cancer.

The panel of therapeutic options available for medical treatment of relapsed ovarian cancer increased over the last years. In late, platinum-sensitive relapse, standard treatment remains platinum-based polychemotherapy. The choice between bevacizumab added to chemotherapy followed by maintenance and inhibitors of poly-(ADP-riboses) polymerases (PARPi) after response to platinum-based therapy should be discussed, taking into account prior treatment, contraindications, and disease characteristics (biology, symptoms…). The addition of bevacizumab at first platinum-sensitive relapse can be considered if it has not been administered in first line, and it is optional (rechallenge) if previously administered (but without Marketing Authorization in this setting). PARPi are indicated for maintenance therapy after response to platinum-based chemotherapy (whatever the treatment line), regardless of BRCA mutational status, in case of no prior administration. Early relapses are associated with poor prognosis and therapeutic options are more limited. They are treated by monochemotherapy without platinum agents, associated with bevacizumab if not administered previously. Beyond first early relapse, there is no standard and inclusion in a clinical trial should be proposed if possible. Several clinical studies assessing associations of immunotherapy and chemotherapy and/or antiangiogenic drugs and/or targeted therapies (such as PARPi) are ongoing in early or late relapse.

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model.

INTRODUCTION: Pteridines include folate-derived metabolites that have been putatively associated with certain cancers in clinical studies. However, their biological significance in cancer metabolism and role in cancer development and progression remains poorly understood. OBJECTIVES: The purpose of this study was to examine the effects of tumorigenicity on pteridine metabolism by studying a panel of 15 pteridine derivatives using a progressive breast cancer cell line model with and without folic acid dosing. METHODS: The MCF10A progressive breast cancer model, including sequentially derived MCF10A (benign), MCF10AT (premalignant), and MCF10CA1a (malignant) cell lines were dosed with 0, 100, and 250 mg/L folic acid. Pteridines were analyzed in both intracellular and extracellular contexts using an improved high-performance liquid chromatography-tandem mass spectrometry method. RESULTS: Pteridines were located predominately in the extracellular media. Folic acid dosing increased extracellular levels of pterin, 6-hydroxylumazine, xanthopterin, 6-hydroxymethylpterin, and 6-carboxypterin in a dose-dependent manner. In particular, pterin and 6-hydroxylumazine levels were positively correlated with tumorigenicity upon folate dosing. CONCLUSIONS: Folic acid is a primary driver for pteridine metabolism in human breast cell. Higher folate levels contribute to increased formation and excretion of pteridine derivatives to the extracellular media. In breast cancer, this metabolic pathway becomes dysregulated, resulting in the excretion of certain pteridine derivatives and providing in vitro evidence for the observation of elevated pteridines in the urine of breast cancer patients. Finally, this study reports a novel use of the MCF10A progressive breast cancer model for metabolomics applications that may readily be applied to other metabolites of interest.

Theoretically exploring selective-binding mechanisms of BRD4 through integrative computational approaches.

The origin of cancer is related to the dysregulation of multiple signal pathways and of physiological processes. Bromodomain-containing protein 4 (BRD4) has become an attractive target for the development of anticancer and anti-inflammatory agents since it can epigenetically regulate the transcription of growth-promoting genes. The synthesized BRD4 inhibitors with new chemical structures can reduce the drug resistance, but their binding modes and the inhibitory mechanism remain unclear. Here, we initially constructed robust QSAR models based on 68 reported tetrahydropteridin analogues using topomer CoMFA and HQSAR. On the basis of QSAR results, we designed 16 novel tetrahydropteridin analogues with modified structures and carried out docking studies. Instead of significant hydrogen bondings with amino acid residue Asn140 as reported in previous research, the molecular docking modelling suggested a novel docking pose that involves the amino acid residues (Trp81, Pro82, Val87, Leu92, Leu94, Cys136, Asp144, and Ile146) at the active site of BRD4. The MD simulations, free energy calculations, and residual energy contributions all indicate that hydrophobic interactions are decisive factors affecting bindings between inhibitors and BRD4. The current study provides new insights that can aid the discovery of BRD4 inhibitors with enhanced anti-cancer ability.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.