Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.

Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.

Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC50 (µM); 0.05 ± 0.008) and 5g (IC50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme.

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Testicular adenosine acts as a pro-inflammatory molecule: role of testicular peritubular cells.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Discovery of natural product ellagic acid as a potent CD73 and CD39 dual inhibitor.

The ATP-adenosine pathway has been recently identified as an attractive immune-oncology target and several drug candidates have been entered clinic trials. Inspired by the report of the first small-molecule CD73inhibitor AB680, we describe the discovery of natural product ellagic acid as a dual CD73 and CD39 inhibitor with an IC50 value of 1.85 ± 0.21 µM and 0.50 ± 0.22 µM, respectively. The result of cytotoxicity assays indicated that ellagic acid is a valuable lead compound with low cytotoxicity effect for immune therapy.

Divergent synthesis and elaboration of structure activity relationship for quinoline derivatives as highly selective NTPDase inhibitor.

Quinoline derivatives have interesting biological profile. In continuation for the comprehensive evaluations of substituted quinoline derivatives against human nucleoside triphosphate diphosphohydrolases (h-NTPDases) a series of substituted quinoline derivatives (2a-g, 3a-f, 4, 5a-c, 6) was synthesized. The inhibitory activities of the synthesized compounds were evaluated against four isoenzymes of human nucleoside triphosphate diphosphohydrolases (h-NTPDases). These quinoline derivatives had IC50 (µM) values in the range of 0.20-1.75, 0.77-2.20, 0.36-5.50 and 0.90-1.82 for NTPDase1, NTPDase2, NTPDase3 and NTPDase8, respectively. The derivative 3f was the most active compound against NTPDase1 (IC50, 0.20 ± 0.02 µM) that also possessed selectivity towards NTPDase1. Similarly, derivative 3b (IC50, 0.77 ± 0.06), 2h (IC50, 0.36 ± 0.01) and 2c (IC50, 0.90 ± 0.08) displayed excellent activity corresponding to NTPDase2, NTPDase3 and NTPdase8. The compound 5c emerged as a selective inhibitor of NTPDase8. The most active compounds were then investigated to determine their mode of inhibition and finally binding interactions of the active compounds were analyzed through molecular docking studies. The obtained results strongly support the quinoline scaffold's potential as potent and selective NTPDase inhibitor.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

Cardamonin Presents in Vivo Activity against Schistosoma mansoni and Inhibits Potato Apyrase.

Schistosomiasis, a neglected tropical disease caused by Schistosoma species, harms over 250 million people in several countries. The treatment is achieved with only one drug, praziquantel. Cardamonin, a natural chalcone with in vitro schistosomicidal activity, has not been in vivo evaluated against Schistosoma. In this work, we evaluated the in vivo schistosomicidal activities of cardamonin against Schistosoma mansoni worms and conducted enzymatic apyrase inhibition assay, as well as molecular docking analysis of cardamonin against potato apyrase, S. mansoni NTPDase 1 and S. mansoni NTPDase 2. In a mouse model of schistosomiasis, the oral treatment with cardamonin (400 mg/kg) showed efficacy against S. mansoni, decreasing the total worm load in 46.8 % and reducing in 54.5 % the number of eggs in mice. Cardamonin achieved a significant inhibition of the apyrase activity and the three-dimensional structure of the potato apyrase, obtained by homology modeling, showed that cardamonin may interact mainly through hydrogen bonds. Molecular docking studies corroborate with the action of cardamonin in binding and inhibiting both potato apyrase and S. mansoni NTPDases.

2-Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors.

The antithrombotic prodrugs ticlopidine and clopidogrel are thienotetrahydro-pyridine derivatives that are metabolized in the liver to produce thiols that irreversibly block adenosine diphosphate (ADP)-activated P2Y12 receptors on thrombocytes. In their native, nonmetabolized form, both drugs were reported to act as inhibitors of ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39). CD39 catalyzes the extracellular hydrolysis of nucleoside tri- and diphosphates, mainly adenosine 5'-triphosphate (ATP) and ADP, yielding adenosine monophosphate, which is further hydrolyzed by ecto-5'-nucleotidase (CD73) to produce adenosine. While ATP has proinflammatory effects, adenosine is a potent anti-inflammatory, immunosuppressive agent. Inhibitors of CD39 and CD73 have potential as novel checkpoint inhibitors for the immunotherapy of cancer and infection. In the present study, we investigated 2-substituted thienotetrahydropyridine derivatives, structurally related to ticlopidine, as CD39 inhibitors. Due to their substituent on the 2-position, they will not be metabolically transformed into reactive thiols and can, therefore, be expected to be devoid of P2Y12 receptor-antagonistic activity in vivo. Several of the investigated 2-substituted thienotetrahydropyridine derivatives showed concentration-dependent inhibition of CD39. The most potent derivative, 32, showed similar CD39-inhibitory potency to ticlopidine, both acting as allosteric inhibitors. Compound 32 showed an improved selectivity profile: While ticlopidine blocked several NTPDase isoenzymes, 32 was characterized as a novel dual inhibitor of CD39 and CD73.

Recent Advances Clarifying the Structure and Function of Plant Apyrases (Nucleoside Triphosphate Diphosphohydrolases).

Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered.

A Body of Circumstantial Evidence for the Irreversible Ectonucleotidase Inhibitory Action of FSCPX, an Agent Known as a Selective Irreversible A1 Adenosine Receptor Antagonist So Far.

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the "paradox" effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.

The Preventive Effect of the Phenotype of Tumour-Associated Macrophages, Regulated by CD39, on Colon Cancer in Mice.

BACKGROUND: This study was designed to investigate the effect of cluster differentiation (CD)39 and CD73 inhibitors on the expresion of tumour-associated macrophages (TAMs), M1- versus M2-tumour phenotypes in mice with colon cancer. METHODS: An in vivo study of co-culture with colon cancer cells and immune cells from the bone marrow (BM) of mice was performed. After the confirmation of the effect of polyoxotungstate (POM-1) as an inhibitor of CD39 on TAMs, the mice were randomly divided into a control group without POM-1 and a study group with POM-1, respectively, after subcutaneous injection of CT26 cells. On day 14 after the injection, the mice were sacrificed, and TAMs were evaluated using fluorescence-activated cell sorting. RESULTS: In the in vivo study, the co-culture with POM-1 significantly increased the apoptosis of CT26 cells. The cell population from the co-culture with POM-1 showed significant increases in the expression of CD11b+ for myeloid cells, lymphocyte antigen 6 complex, locus C (Ly6C+) for monocytes, M1-tumour phenotypes from TAMs, and F4/80+ for macrophages. In the in vivo study, tumour growth in the study group with POM-1 was significantly limited, compared with the control group without POM-1. The expressions of Ly6C+ and major histocompatibility complex class II+ for M1-tumour phenotypes from TAMs on F4/80+ from the tumour tissue in the study group had significantly higher values compared with the control group. CONCLUSION: The inhibition of CD39 with POM-1 prevented the growth of colon cancer in mice, and it was associated with the increased expression of M1-tumour phenotypes from TAMs in the cancer tissue.

Combined Blockade of TIGIT and CD39 or A2AR Enhances NK-92 Cell-Mediated Cytotoxicity in AML.

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56dimCD16- and CD56brightCD16- NK cells represent the predominant NK cell subpopulations in AML, while the CD56dimCD16+ NK cells are significantly reduced compared to HDs. Moreover, TIGIT+ and PVRIG+ cells cluster on the CD56dimCD16+ subset whereas CD39+ and CD38+ cells do so on CD56brightCD16- NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

Tumor-infiltrating CD39+CD8+ T cells determine poor prognosis and immune evasion in clear cell renal cell carcinoma patients.

PURPOSE: Tumor microenvironment is important in the progression of clear cell renal cell carcinoma (ccRCC), and its prognostic value is still unclear. Recent reports demonstrated tumor-infiltrating CD39+CD8+ T cells are abundant, but their function remains obscure. We aim to assess clinical value of CD39+CD8+ T cells and seek a potential therapeutic target in ccRCC. EXPERIMENTAL DESIGN: We immunohistochemically evaluated clinical value of CD39+CD8+ T cells in a retrospective Zhongshan Hospital cohort of 243 ccRCC patients. Fresh tumor samples (n = 48), non-tumor tissues and peripheral blood for flow cytometry analyses were collected to analyze immune cell functions from Zhongshan Hospital. The survival benefit of tyrosine kinase inhibitors (TKIs) in this subpopulation was evaluated. Kaplan-Meier analysis and COX regression model were applied for survival analyses. Bioinformatics analysis performed in TCGA KIRC cohort and the scRNA-seq cohort. RESULTS: We found that accumulation of CD39+CD8+ T cells indicated poor prognosis (p < 0.0001) and indicated therapeutic benefit of TKIs therapy (p = 0.015). CD39+CD8+ T cells showed decreased TNF-α and IFN-γ with elevated PD-1 and TIM-3 expression. Further analysis of tumor-infiltrating immune cell landscape in the ccRCC revealed the positive correlation between CD39+CD8+ T cells and Tregs (p = 0.037) and M2-polarized macrophages (p < 0.0001). Finally, inhibition of CD39 partially restores the anti-tumor function of CD8+ T cells. CONCLUSIONS: High CD39+CD8+ T cells indicated poor prognosis in ccRCC, due to impaired anti-tumor function of CD39+CD8+ T cells and indicated therapeutic benefit of TKIs therapy.

Role of inflammasome activation in tumor immunity triggered by immune checkpoint blockers.

Immune checkpoint blockers improve the overall survival of a limited number of patients among different cancers. Identifying pathways that influence the immunological and clinical response to treatment is critical to improve the therapeutic efficacy and predict clinical responses. Recently, a key role has been assigned to innate immune mechanisms in checkpoint blockade-driven anti-tumor responses. However, inflammatory pathways can both improve and impair anti-tumor immunity. In this review, we discuss how different inflammatory pathways, particularly inflammasome activation, can influence the clinical outcome of immune checkpoint blockers. Inflammasome activation may reinforce anti-tumor immunity by boosting CD8+ T cell priming as well as by enhancing T helper type 17 (Th17) responses. In particular, we focus on the modulation of the cation channel transmembrane protein 176B (TMEM176B) and the ectonucleotidase CD39 as potential targets to unleash inflammasome activation leading to reinforced anti-tumor immunity and improved efficacy of immune checkpoint blockers. Future studies should be aimed at investigating the mechanisms and cell subsets involved in inflammasome-driven anti-tumor responses.

Human gingiva-derived mesenchymal stem cells are therapeutic in lupus nephritis through targeting of CD39-CD73 signaling pathway.

Cell specific and cytokine targeted therapeutics have underperformed in systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) have emerged as a novel therapy to address the dysregulation in autoimmune diseases but also have limitations. Human gingiva derived MSCs (GMSCs) are superior in regulating immune responses. Here, we demonstrate that the adoptive transfer of GMSCs homes to and maintains in the kidney and has a robust therapeutic effect in a spontaneous lupus nephritis model. Specifically, GMSCs limits the development of autoantibodies as well as proteinuria, decreases the frequency of plasma cells and lupus nephritis histopathological scores by directly suppressing B cells activation, proliferation and differentiation. The blockage of CD39-CD73 pathway dramatically abrogates the suppressive capacities of GMSCs in vitro and in vivo and highlights the significance of this signaling pathway in SLE. Collectively, manipulation of GMSCs provides a promising strategy for the treatment of patients with SLE and other autoimmune diseases.

Pharmacological blockade of the CD39/CD73 pathway but not adenosine receptors augments disease in a humanized mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is a curative therapy for a number of hematological malignancies, but is limited by the development of graft-versus-host disease (GVHD). CD39 and CD73 form an ectoenzymatic pathway that hydrolyzes extracellular adenosine 5'-triphosphate (ATP) to adenosine, which respectively exacerbate or alleviate disease in allogeneic mouse models of GVHD. The current study aimed to explore the role of the CD39/CD73 pathway and adenosine receptor (AR) blockade in a humanized mouse model of GVHD. Immunodeficient nonobese diabetic-severe combined immunodeficiency-IL-2 receptor γnull mice were injected with human peripheral blood mononuclear cells, and subsequently injected with the CD39/CD73 antagonist αß-methylene-ADP (APCP) (50 mg kg-1 ) or saline for 7 days, or the AR antagonist caffeine (10 mg kg-1 ) or saline for 14 days. Mice predominantly engrafted human CD4+ and CD8+ T cells, with smaller proportions of human regulatory T cells, invariant natural killer T cells, monocytes and dendritic cells. Neither APCP nor caffeine altered engraftment of these human leukocyte subsets. APCP (CD39/CD73 blockade) augmented GVHD as shown through increased weight loss and worsened liver histology, including increased leukocyte and human T-cell infiltration, and increased apoptosis. This treatment also increased serum human IL-2 concentrations and decreased the frequency of human CD39-  CD73-  CD4+ T cells. In contrast, caffeine (AR blockade) did not alter GVHD severity or human serum cytokine concentrations (IL-2, IL-6, IL-10 or tumor necrosis factor-α). In conclusion, blockade of CD39/CD73 but not ARs augments disease in a humanized mouse model of GVHD. These results indicate that CD39/CD73 blockade maintains sufficient extracellular ATP concentrations to promote GVHD in this model.

Inhibition of lung cancer cells and Ras/Raf/MEK/ERK signal transduction by ectonucleoside triphosphate phosphohydrolase-7 (ENTPD7).

BACKGROUND: The aim of this study was to investigate the effects and mechanisms of ectonucleoside triphosphate phosphohydrolase-7 (ENTPD7) on lung cancer cells. METHODS: The expression characteristics of ENTPD7 and its effect on the survival of lung cancer patients were analyzed by referring to The Cancer Genome Atlas (TCGA). Streptavidin-peroxidase (SP) staining was performed to detect the ENTPD7 protein in tumor tissues and adjacent tissues. Plasmid transfection technology was also applied to silence ENTPD7 gene. Crystal violet staining and flow cytometry were performed to determine cell proliferation and apoptosis. Tumor-bearing nude mice model was established to investigate the effect of sh-ENTPD7 on tumors. RESULTS: The results showed that patients with low levels of ENTPD7 had higher survival rates. ENTPD7 was up-regulated in lung cancer tissues and cells. Down-regulation of the expression of ENTPD7 inhibited proliferation but promoted apoptosis of lung cancer cell. Silencing ENTPD7 also inhibited the expression levels of Ras and Raf proteins and the phosphorylation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK). Tumor-bearing nude mice experiments showed that silencing ENTPD7 had an inhibitory effect on lung cancer cells. CONCLUSIONS: ENTPD7 was overexpressed in lung cancer cells. Down-regulating ENTPD7 could inhibit lung cancer cell proliferation and promote apoptosis via inhibiting the Ras/Raf/MEK/ERK pathway.

Investigation of new quinoline derivatives as promising inhibitors of NTPDases: Synthesis, SAR analysis and molecular docking studies.

Nucleoside triphosphate diphosphohydrolases (NTPDases), an important class of ectonucleotidases, are responsible for the sequential hydrolysis of extracellular nucleotides. However, over-expression of NTPDases has been linked with various pathological diseases e.g. cancer. Thus, to treat these diseases, the inhibitors of this class of enzyme are of interest. The significance of this class of enzyme encouraged us to synthesize a new class of quinoline derivatives with the aim to find selective and potent inhibitors of NTPDases. Therefore, a mild and efficient synthetic route was established for the synthesis of quinoline derivatives. The reaction was catalyzed by molecular iodine to afford the substituted quinoline derivatives. All the synthetic derivatives (3a-3w) were evaluated for their potential to inhibit the h-NTPDase1, 2, 3 and 8. Most of the compounds were identified as dual inhibitors of h-NTPDase1 and 8 with lower effects on h-NTPDase2 and 3. Two compounds i.e.3f and 3t were identified as selective inhibitor of h-NTPDase1 whereas the compound 3s inhibited the h-NTPDase8 selectively. Moreover, the compounds 3p (IC50 = 0.23 ±â€¯0.01 µM), 3j (IC50 = 21.0 ±â€¯0.03 µM) 3d (IC50 = 5.38 ±â€¯0.21 µM) and 3c (IC50 = 1.13 ±â€¯0.04 µM) were found to be the most potent inhibitors of h-NTPDase1, 2, 3 and 8, respectively. To determine the binding interaction, molecular docking studies were also carried out.