Joanna Lecka

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases

ARL 67156, 6‐N,N‐Diethyl‐D‐β‐γ‐dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto‐ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156.

Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration.

The chemokine interleukin 8 (IL-8) is a major chemoattractant for human neutrophils. Here, we demonstrate novel evidence that IL-8-induced neutrophil chemotaxis requires a concurrent activation of P2 receptors, most likely the P2Y(2) which is dominantly expressed in these cells. Indeed, the migration of human neutrophils towards IL-8 was significantly inhibited by the P2Y receptor antagonists, suramin and reactive blue 2 (RB-2) and potentiated by a P2Y(2) ligand, ATP, but insensitive to specific antagonists of P2Y(1), P2Y(6) and P2Y(11) receptors. Adenosine had no effect on neutrophil migration towards IL-8 which contrasted with the stimulatory effect of this molecule on neutrophil chemotaxis caused by formyl-Met-Leu-Phe (fMLP or fMLF). Taken together, these data suggest that extracellular ATP is necessary for IL-8 to exert its chemotactic effect on neutrophils.

NTPDase1 Controls IL-8 Production by Human Neutrophils

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam3CSK4 (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y2 receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam3CSK4-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y2 activation.

Inhibition of vascular ectonucleotidase activities by the pro‐drugs ticlopidine and clopidogrel favours platelet aggregation

BACKGROUND AND PURPOSE After conversion to their active forms by the liver, ticlopidine and clopidogrel exert antiplatelet effects through irreversible inhibition of the P2Y12 receptor. Concentrations of nucleotides such as ADP, the physiological agonist at platelet P2Y1 and P2Y12 receptors, are regulated by vascular ectonucleotidases, mainly nucleoside triphosphate diphosphohydrolase (NTPDase)1 and ecto‐5′‐nucleotidase. Here we evaluate the effect of these pro‐drugs on vascular ectonucleotidase activity and on the natural function of these enzymes in regulating platelet aggregation.

High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.

Identification of hydrolytically stable and selective P2Y1 receptor agonists

P2Y nucleotide receptors (P2YRs) are attractive pharmaceutical targets. Most P2YR agonists proposed as drugs consist of a nucleotide scaffold, but their use is limited due to their chemical and enzymatic instabilities. To identify drug candidates, we developed non-hydrolyzable P2YR agonists. We synthesized ATP-beta,gamma-CH(2) analogues 2-4, and evaluated their chemical and metabolic stabilities and activities at P2Y(1,2,4,6) receptors. Analogues 2-4 exhibited t(1/2) values of 14.5-65 h in gastric juice pH. They were completely resistant to alkaline phosphatase for 30 min at 37 degrees C and slowly hydrolyzed in human blood serum (t(1/2) 12.7-71.9 h). In comparison to ATP, analogues 2-4 were barely hydrolyzed by nucleoside triphosphate diphosphohydrolases, NTPDase1,2,3,8 (< 8% hydrolysis), and nucleotide pyrophosphatases, NPP1,3 (< or = 10% hydrolysis). Analogues 2 and 4B were selective agonists of the P2Y(1)R with EC(50)s of 0.08 and 17.2 microM, respectively. These features make analogues 2 and 4B potential therapeutic agents for health disorders involving the P2Y(1)R.

Nonhydrolyzable ATP analogues as selective inhibitors of human NPP1: a combined computational/experimental study.

Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 μM inhibited (66-99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The Ki,app values of the seven most potent and selective inhibitors were in the range of 0.5-56 μM, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor 2 that had the lowest Ki,app (0.5 μM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.

2-MeS-β,γ-CCl2-ATP is a Potent Agent for Reducing Intraocular Pressure†

Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

Isonicotinohydrazones as Inhibitors of Alkaline Phosphatase and Ecto-5'-nucleotidase.

A series of isonicotinohydrazide derivatives was synthesized and tested against recombinant human and rat ecto‐5′‐nucleotidases (h‐e5′NT and r‐e5′NT) and alkaline phosphatase isozymes including both bovine tissue‐non‐specific alkaline phosphatase (b‐TNAP) and tissue‐specific calf intestinal alkaline phosphatase (c‐IAP). These enzymes are implicated in vascular calcifications, hypophosphatasia, solid tumors, and cancers, such as colon, lung, breast, pancreas, and ovary. All tested compounds were active against both enzymes. The most potent inhibitor of h‐e5′NT was derivative (E)‐N′‐(1‐(3‐(4‐fluorophenyl)‐5‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethylidene)isonicotinohydrazide (3j), whereas derivative (E)‐N′‐(4‐hydroxy‐3‐methoxybenzylidene)isonicotinohydrazide (3g) exhibited significant inhibitory activity against r‐e5′NT. In addition, the derivative (E)‐N′‐(4′‐chlorobenzylidene)isonicotinohydrazide (3a) was most potent inhibitor against calf intestinal alkaline phosphatase and the derivative (E)‐N′‐(4‐hydroxy‐3‐methoxybenzylidene)isonicotinohydrazide (3g) was found to be most potent inhibitor of bovine tissue‐non‐specific alkaline phosphatase. Furthermore, putative binding modes of potent compounds against e5′NT (human and rat e5′NT) and AP (including b‐TNAP and c‐IAP) were determined computationally.