NTPDase8 protects mice from intestinal inflammation by limiting P2Y6 receptor activation: identification of a new pathway of inflammation for the potential treatment of IBD.

OBJECTIVE: Nucleotides are danger signals that activate inflammatory responses via binding P2 receptors. The nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is an ectonucleotidase that hydrolyses P2 receptor ligands. We investigated the role of NTPDase8 in intestinal inflammation. DESIGN: We generated NTPDase8-deficient (Entpd8-/-) mice to define the role of NTPDase8 in the dextran sodium sulfate (DSS) colitis model. To assess inflammation, colons were collected and analysed by histopathology, reverse transcriptase-quantitative real-time PCR (RT-qPCR) and immunohistochemistry. P2 receptor expression was analysed by RT-qPCR on primary intestinal epithelium and NTPDase8 activity by histochemistry. The role of intestinal P2Y6 receptors was assessed by bone marrow transplantation experiments and with a P2Y6 receptor antagonist. RESULTS: NTPDase8 is the dominant enzyme responsible for the hydrolysis of nucleotides in the lumen of the colon. Compared with wild-type (WT) control mice, the colon of Entpd8-/- mice treated with DSS displayed significantly more histological damage, immune cell infiltration, apoptosis and increased expression of several proinflammatory cytokines. P2Y6 was the dominant P2Y receptor expressed at the mRNA level by the colonic epithelia. Irradiated P2ry6-/- mice transplanted with WT bone marrow were fully protected from DSS-induced intestinal inflammation. In agreement, the daily intrarectal injection of a P2Y6 antagonist protected mice from DSS-induced intestinal inflammation in a dose-dependent manner. Finally, human intestinal epithelial cells express NTPDase8 and P2Y6 similarly as in mice. CONCLUSION: NTPDase8 protects the intestine from inflammation most probably by limiting the activation of P2Y6 receptors in colonic epithelial cells. This may provide a novel therapeutic strategy for the treatment of inflammatory bowel disease.

Efficiencies of selected biotreatments for the remediation of PAH in diluted bitumen contaminated soil microcosms.

Unconventional oils such as diluted bitumen from oil sands differs from most of conventional oils in terms of physiochemical properties and PAHs composition. This raises concerns regarding the effectiveness of current remediation strategies and protocols originally developed for conventional oil. Here we evaluated the efficiency of different biotreatment approaches, such as fungi inoculation (bioaugmentation), sludge addition (bioaugmentation/biostimulation), perennial grasses plantation (phytoremediation) and their combinations as well as natural attenuation (as control condition), for the remediation of soil contaminated by synthetic crude oil (a product of diluted bitumen) in laboratory microcosms. We specifically monitored the PAHs loss percentage (alkylated PAHs and unsubstituted 16 EPA Priority PAHs), the residue of PAHs and evaluated the ecotoxicity of soil after treatment. All treatments were highly efficient with more than ~ 80% of ∑PAHs loss after 60 days. Distinctive loss efficiencies between light PAHs (≤ 3 rings, ~ 96% average loss) and heavy PAHs (4-6 rings, ~ 29% average loss) were observed. The lowest average PAHs residue (0.10 ± 0.02 mg·kg-1, for an initial concentration of 0.29 ± 0.12 mg·kg-1) was achieved with the "sludge-plants (grasses)" combination. Sludge addition was the only treatment that achieved significantly lower ecotoxicity (3% ± 4% of growth inhibition of L. sativa) than the control (natural attenuation, 13% ± 4% of inhibition). Sludge addition, grasses plantation and "sludge-fungi combination" treatments could result in lower PAH exposure (than other treatments) in post-treated soil when using the Canadian Soil Quality Guidelines for the protection of environmental and human health for potentially carcinogenic and other PAHs.

Synthesis, biological evaluation, and molecular docking study of sulfonate derivatives as nucleotide pyrophosphatase/phosphodiesterase (NPP) inhibitors.

A new series of sulfonate derivatives 1a-zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC50 value of 0.387 ±â€¯0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with an IC50 value of 0.214 ±â€¯0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC50 value of 0.659 ±â€¯0.007 µM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results.

Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidinediones as selective and efficient non-nucleotide inhibitors of recombinant human ectonucleotidases.

With the aim to discover novel, efficient and selective inhibitors of human alkaline phosphatase and nucleotide pyrophosphatase enzymes, two new series of pyrazolyl pyrimidinetriones (PPTs) (6a-g) and thioxopyrimidinediones (PTPs) (6h-n) were synthesized in good chemical yields using Knoevenagel condensation reaction between pyrazole carbaldehydes (4a-g) and pharmacologically active N-alkylated pyrimidinetrione (5a) and thioxopyrimidinedione (5b). The inhibition potential of the synthesized hybrid compounds was evaluated against human alkaline phosphatase (h-TNAP and h-IAP) and ectonucleotidase (h-NPP1 and h-NPP3) enzymes. Most of the tested analogs were highly potent with a variable degree of inhibition depending on the functionalized hybrid structure. The detailed structure-activity relationship (SAR) of PPT and PTP derivatives suggested that the compound with unsubstituted phenyl ring from PPT series led to selective and potent inhibition (6a; IC50 = 0.33 ±â€¯0.02 µM) of h-TNAP, whereas compound 6c selectively inhibited h-IAP isozyme with IC50 value of 0.86 ±â€¯0.04 µM. Similarly, compounds 6b and 6h were identified as the lead scaffolds against h-NPP1 and h-NPP3, respectively. The probable binding modes for the most potent inhibitors were elucidated through molecular docking analysis. Structure-activity relationships, mechanism of action, cytotoxic effects and druglikeness properties are also discussed.

Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship.

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors. Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1-26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200-0.350 µM) of NTPDase-1, twelve (Ki = 0.071-1.060 µM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700-4.03 µM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ±â€¯0.007, 6.39 ±â€¯0.89 and 1.180 ±â€¯0.002 µM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.

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.

Design, synthesis and biological evaluation of trinary benzocoumarin-thiazoles-azomethines derivatives as effective and selective inhibitors of alkaline phosphatase.

Design, synthesis and characterization of new trinary Benzocoumarin-Thiazoles-Azomethine derivatives having three bioactive scaffolds in a single structural unit were carried out. The newly synthesized molecules were investigated for the inhibitory activity on human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP) isozymes. All the tested compounds exhibited the potent inhibition profile on both isozymes of alkaline phosphatase i.e., h-TNAP and h-IAP. Molecular docking studies were performed to explore the putative binding mode of interactions of selective inhibitors. Moreover, the synthesized derivatives were evaluated against cervical cancer cell line, HeLa and a few compounds exhibited significant inhibition in the range of 21.0-69.7%. The derivatives can be potential and selective alkaline phosphatase inhibitors for future studies.

Palladium-catalyzed synthesis and nucleotide pyrophosphatase inhibition of benzo[4,5]furo[3,2-b]indoles.

A two-step palladium-catalyzed procedure based on Suzuki-Miyaura cross coupling, followed by a double Buchwald-Hartwig reaction, allows for the synthesis of pharmaceutically relevant benzo[4,5]furo[3,2-b]indoles in moderate to very good yield. The synthesized compounds have been analyzed with regard to their inhibitory activity (IC50) of nucleotide pyrophosphatases h-NPP1 and h-NPP3. The activity lies in the nanomolar range. The results were rationalized based on docking studies.

Distinctive inhibition of alkaline phosphatase isozymes by thiazol-2-ylidene-benzamide derivatives: Functional insights into their anticancer role.

In the recent years, the role of alkaline phosphatase (AP) isozymes in the cause of neoplastic diseases such as breast, liver, renal, and bone cancer has been confirmed and, thus they represent a novel target for the discovery of anticancer drugs. In this study different derivatives of thiazol-2-ylidene-benzamide were evaluated for their potential to inhibit alkaline phosphatase (AP) isozymes. Their anticancer potential was assessed using human breast cancer (MCF-7), bone-marrow cancer (K-562), and cervical cancer (HeLa) cell lines in comparison to normal cells from baby hamster kidney BHK-21. The results suggested that in comparison to other derivatives, compounds 2i, 2e, and 2a showed more sensitivity towards human tissue non-specific alkaline phosphatase (h-TNAP). Among these, 2″-chloro-N-(3-(4'-fluorophenyl)-4-methylthiazol-2(3H)-ylidene) benzamide (2e) was found as the most potent and selective inhibitor for h-TNAP with an IC50 value of 0.079 ± 0.002 µM. Moreover, a significant correlation was observed between the enzyme inhibition profile and cytotoxic data. The compounds exhibiting maximum anticancer potential also induced maximum apoptosis in the respective cell lines. Furthermore, the DNA interaction studies exhibited the non-covalent mode of interaction with the herring sperm-DNA. Molecular docking studies also supported the in vitro inhibitory activity of potent compounds. Our findings suggested that potent and selective inhibitors might be useful candidates for the treatment or prevention of those diseases associated with the higher level of AP. Moreover, the study can be useful for the researcher to explore more molecular mechanisms of such derivatives and their analogues with the exact findings.

A domino reaction of 3-chlorochromones with aminoheterocycles. Synthesis of pyrazolopyridines and benzofuropyridines and their optical and ecto-5'-nucleotidase inhibitory effects.

A new and efficient domino reaction of 3-chlorochromones with electron-rich aminoheterocycles was developed which allows for a convenient synthesis of a variety of pyrazolo[3,4-b]pyridines, pyrrolo[2,3-b]pyridines, pyrido[2,3-d]pyrimidines and benzofuro[3,2-b]pyridines. The products exhibit strong fluorescence. In addition, they exhibit significant ecto-5'-nucleotidase inhibition properties and cytotoxic behavior.

4-Aminopyridine based amide derivatives as dual inhibitors of tissue non-specific alkaline phosphatase and ecto-5'-nucleotidase with potential anticancer activity.

Ecto-nucleotidase members i.e., ecto-5'-nucleotidase and alkaline phosphatase, hydrolyze extracellular nucleotides and play an important role in purinergic signaling. Their overexpression are implicated in a variety of pathological states, including immunological diseases, bone mineralization, vascular calcification and cancer, and thus they represent an emerging drug targets. In order to design potent and selective inhibitors, new derivatives of 4-aminopyridine have been synthesized (10a-10m) and their structures were established on the basis of spectral data. The effect of nature and position of substituent was interestingly observed and justified on the basis of their detailed structure activity relationships (SARs) against both families of ecto-nucleotidase. Compound 10a displayed significant inhibition (IC50 ±â€¯SEM = 0.25 ±â€¯0.05 µM) that was found ≈168 fold more potent as compared to previously reported inhibitor suramin (IC50 ±â€¯SEM = 42.1 ±â€¯7.8 µM). This compound exhibited 6 times more selectivity towards h-TNAP over h-e5'NT. The anticancer potential and mechanism were also established using cell viability assay, flow cytometric analysis and nuclear staining. Molecular docking studies were also carried out to gain insight into the binding interaction of potent compounds within the respective enzyme pockets and herring-sperm DNA.

Tricyclic coumarin sulphonate derivatives with alkaline phosphatase inhibitory effects: in vitro and docking studies.

Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was evaluated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC50 = 0.38 ± 0.01 µM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC50 = 0.45 ± 0.02 µM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.

Synthesis, characterization and biological evaluation of novel chalcone sulfonamide hybrids as potent intestinal alkaline phosphatase inhibitors.

Alkaline phosphatase (AP) and ecto-5'-nucleotidase (e5'NT) belong to same family that hydrolyze the extracellular nucleotides and ensure the bioavailability of nucleotides and nucleosides at purinergic receptors. During pathophysiological conditions, the over expression of AP and e5'NT lead to an increased production of adenosine that enhance tumor proliferation, invasiveness, neoangiogenesis and disrupts the body antitumor response. As both enzymes are abundantly expressed in above mentioned conditions, therefore it is of great interest to synthesize and develop potent inhibitors of these enzymes that augment the antitumor therapy. Herein we reported the synthesis and biological activity of a new series of chalcone-sulfonamide hybrids (4a-j). These derivatives were then evaluated for their inhibitory potential against two members of ecto-nucleotidase family, e5'NT (human and rat) and APs isozyme (intestinal and tissue nonspecific). Only six derivatives were found to inhibit both human and rat e5'NT enzymes. Compounds 4e and 4d showed maximum inhibition of human and rat e5'NT with an IC50±SEM=0.26±0.01 and 0.33±0.004µM, respectively. Moreover, on APs, these derivatives were identified as the selective inhibitors of calf intestinal AP (c-IAP). The derivative 4a exhibited maximum inhibition of c-IAP with an IC50±SEM=0.12±0.02µM. In conclusion, these chalcone-sulfonamide hybrids exhibited dual inhibition of both family of isozymes but was more selective towards c-IAP enzyme.

New one-pot synthesis of N-fused isoquinoline derivatives by palladium-catalyzed C-H arylation: potent inhibitors of nucleotide pyrophosphatase-1 and -3.

Various N-fused isoquinoline derivatives were synthesized using a new one-pot reaction of 1-bromo-2-(2,2-difluorovinyl)benzenes with N-H group containing heterocycles followed by intramolecular palladium-catalyzed C-H arylation. The method described gives convenient access to diverse structures of N-fused polycyclic isoquinolines. Sixteen of the synthesized compounds were screened as potential human nucleotide pyrophosphatase/phosphodiesterase 1 and 3 (h-NPP-1 and h-NPP-3) inhibitors. The most effective h-NPP-1 inhibitor showed an IC50 value as high as 0.36 ± 0.06 µM, whereas the most potent h-NPP-3 inhibitor posessed an inhibitory value of 0.48 ± 0.01 µM. Kinetic and molecular docking studies of both most effective inhibitors were carried out.

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 µM ATP or 10 µM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 µM ADP and 1 µM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.

Efficient one-pot synthesis of 5-perfluoroalkylpyrazoles by cyclization of hydrazone dianions.

A highly selective and efficient method for the synthesis of 5-trifluoromethylated and 5-perfluoroalkylated pyrazoles has been developed which relies on the cyclization of hydrazine dianions with ethyl perfluorocarboxylates. The pyrazoles prepared were evaluated as potential inhibitors of alkaline phosphatases, namely human tissue non-specific alkaline phosphatase (h-TNAP) and tissue specific intestinal alkaline phosphatase (IAP). Most pyrazole derivatives inhibited h-IAP more markedly than h-TNAP and had minor effects on nucleotide pyrophosphatase/phosphodiesterases. Therefore, the compounds appear as potential selective inhibitors of h-IAP.

Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the identification of human P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.

Ticlopidine in its prodrug form is a selective inhibitor of human NTPDase1.

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (patho)physiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (K i = 14 µM). Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM) completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10-20%) of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

Purine-metabolizing ectoenzymes control IL-8 production in human colon HT-29 cells.

Interleukin-8 (IL-8) plays key roles in both chronic inflammatory diseases and tumor modulation. We previously observed that IL-8 secretion and function can be modulated by nucleotide (P2) receptors. Here we investigated whether IL-8 release by intestinal epithelial HT-29 cells, a cancer cell line, is modulated by extracellular nucleotide metabolism. We first identified that HT-29 cells regulated adenosine and adenine nucleotide concentration at their surface by the expression of the ectoenzymes NTPDase2, ecto-5'-nucleotidase, and adenylate kinase. The expression of the ectoenzymes was evaluated by RT-PCR, qPCR, and immunoblotting, and their activity was analyzed by RP-HPLC of the products and by detection of Pi produced from the hydrolysis of ATP, ADP, and AMP. In response to poly (I:C), with or without ATP and/or ADP, HT-29 cells released IL-8 and this secretion was modulated by the presence of NTPDase2 and adenylate kinase. Taken together, these results demonstrate the presence of 3 ectoenzymes at the surface of HT-29 cells that control nucleotide levels and adenosine production (NTPDase2, ecto-5'-nucleotidase and adenylate kinase) and that P2 receptor-mediated signaling controls IL-8 release in HT-29 cells which is modulated by the presence of NTPDase2 and adenylate kinase.

Nucleoside-(5'→P) methylenebisphosphonodithioate analogues: synthesis and chemical properties.

Nucleoside-(5'→P) methylenebisphosphonodithioate analogues are bioisosteres of natural nucleotides. The potential therapeutic applications of these analogues are limited by their relative instability. With a view toward improving their chemical and metabolic stability as well as their affinity toward zinc ions, we developed a novel nucleotide scaffold, nucleoside-5'-tetrathiobisphosphonate. We synthesized P1-(uridine/adenosine-5')-methylenebisphosphonodithioate, 2 and 3, and P1,P2-di(uridine/adenosine-5')-methylenebisphosphonodithioate, 4 and 5. Using (1)H and (31)P NMR-monitored Zn(2+)/Mg(2+) titrations, we found that 5 coordinated Zn(2+) by both N7 nitrogen atoms and both dithiophosphonate moieties, whereas 3 coordinated Zn(2+) by an N7 nitrogen atom and Pß. Both 3 and 5 did not coordinate Mg(2+) ions. (31)P NMR-monitored kinetic studies showed that 3 was more stable at pD 1.5 than 5, with t(1/2) of 44 versus 9 h, respectively, and at pD 11 both showed no degradation for at least 2 weeks. However, 5 was more stable than 3 under an air-oxidizing atmosphere, with t1/2 of at least 3 days versus 14 h, respectively. Analogues 3 and 5 were highly stable to NPP1,3 and NTPDase1,2,3,8 hydrolysis (0-7%). However, they were found to be poor ectonucleotidase inhibitors. Although 3 and 5 did not prove to be effective inhibitors of zinc-containing NPP1/3, which is involved in the pathology of osteoarthritis and diabetes, they may be promising zinc chelators for the treatment of other health disorders involving an excess of zinc ions.