8-BuS-ATP derivatives as specific NTPDase1 inhibitors

Abstract

Background and purpose: Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3 and -8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer.

Experimental approach: Here, we present the synthesis and enzymatic characterization of five 8-BuS-adenine nucleotide derivatives as potent and selective inhibitors of NTPDase1.

Key results: The compounds 8-BuS-AMP, 8-BuS-ADP and 8-BuS-ATP inhibit recombinant human and mouse NTPDase1 by mixed type inhibition, predominantly competitive with Ki values <1 μM. In contrast to 8-BuS-ATP which could be hydrolyzed by other NTPDases, the other BuS derivatives were resistant to hydrolysis by either NTPDase1, -2, -3 or -8. 8-BuS-AMP and 8-BuS-ADP were the most potent and selective inhibitors of NTPDase1 expressed in human umbilical vein endothelial cells as well as in situ in human and mouse tissues. As expected, as a result of their inhibition of recombinant human NTPDase1, 8-BuS-AMP and 8-BuS-ADP impaired the ability of this enzyme to block platelet aggregation. Importantly, neither of these two inhibitors triggered platelet aggregation nor prevented ADP-induced platelet aggregation, in support of their inactivity towards P2Y1 and P2Y12 receptors.

Conclusions and implications: The 8-BuS-AMP and 8-BuS-ADP have therefore potential to serve as drugs for the treatment of pathologies regulated by NTPDase1.

Figure 1

NTPDase inhibitors: (1), 8-BuS-ATP; (2), N⁶,N⁶-diethyl-D-β-γ-dibromomethylene-ATP (ARL 67156); (3), suramin; (4), polyoxometalates (POM-1); (5), reactive blue 2; (6), pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS); (7), 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone.

Figure 2

Synthesis of 8-BuS-AMP 10 and 8-BuS-ADP, 11. ^a Reagents and conditions: a, POCl₃, proton sponge, −15°C, 2 h; b, [Bu₃NH⁺]₂[(P₂O₅)₂]²⁻/NBu₃,−10°C, 6 min; c, 1M TEAB, RT, 45 min, 50% overall yield for 1 and 35% for 10; d, H₂O, 10 d, RT.

Figure 3

Synthesis of 8-BuS-AMP-PNP, 8^a. ^a Reagents and conditions: a, POCl₃, proton sponge, −15°C, 2 h; b, [Bu₃NH⁺]₂[NH(PO₃)₂]²⁻/NBu₃,−10°C, 6 min; c, 1M TEAB, RT, 45 min, overall yield 35%.

Figure 4

Synthesis of 8-BuS-AMP-PCP, 9. ^a Reagents and conditions: a, POCl₃, proton sponge, −15°C, 2 h; b, [Bu₃NH⁺]₂[CH₂ PO₃)₂]²⁻/NBu₃,−10°C, 6 min; c, 1M TEAB, RT, 45 min; d, 2,2-dimethoxypropane, p-TsOH, DMF, RT, 24 h; e, TFA, RT, 10 min, 60% overall yield.

Figure 5

Influence of adenine nucleotide analogues 1 and 8–11 on recombinant ectonucleotidase activities. Enzymatic assays were carried out with protein extracts from COS-7 cells transfected with an expression vector encoding the respective enzyme. The substrate (ATP, ADP, AMP or pnp-TMP) was added together with each BuS derivative, both at 100 μM. The 8-BuS derivatives are indicated in the top left of each panel or in the legend. (A) The activity (without inhibitor) with the substrate ATP corresponded to 670 ± 35, 928 ± 55, 202 ± 37, and 129 ± 11 nmol P_i·min⁻¹·mg protein⁻¹ for human NTPDase1, −2, −3 and −8, respectively, and with ADP to 530 ± 22, 110 ± 10, and 30 ± 5 nmol P_i·min⁻¹·mg protein⁻¹ for NTPDase1, −3 and −8, respectively. Data are presented as the mean ± SD of 3 experiments carried out in triplicate. (B) Comparative effect of 8-BuS-ATP (1; top panel), 8-BuS-AMP (10; middle panel) and 8-BuS-ADP (11; lower panel) on the ATPase and ADPase activities of human, mouse and rat NTPDase1. Relative activities are expressed as the mean ± SD of 3 independent experiments, each performed in triplicate. The activity with ATP without inhibitors was 670 ± 35, 990 ± 39 and 750 ± 32 nmol P_i·min⁻¹·mg protein⁻¹ for human, mouse and rat NTPDase1, respectively, and with ADP, 530 ± 22, 878 ± 35 and 690 ± 25 nmol P_i·min⁻¹·mg protein⁻¹ for human, mouse and rat NTPDase1, respectively. (C) Compounds 10 and 11 modestly affect NPP and ecto-5′-nucleotidase activities. The activity without inhibitors with pnp-TMP as substrate were 30 ± 2 and 61 ± 4 nmol p-nitrophenol min⁻¹·mg protein for NPP1 and NPP3, respectively. The activity without inhibitors of ecto-5′-nucleotidase was 1.7 ± 0.1 μmoles Pi min⁻¹·mg protein. Data are presented as the mean ± SD of 3 experiments carried out in triplicate.

Figure 6

Concentration dependent inhibition (A) and K_i determination (B) of ATPase activity of human NTPDase1 by 8-BuS-AMP (10), 8-BuS-ADP (11) and 8-BuS-ATP (1). (A) The activity with 100 μM ATP without inhibitors was 650 ± 33 nmol P_i·min⁻¹·mg protein⁻¹. K_i determination for (B, D) 8-BuS-AMP (10) and (C, E) 8-BuS-ADP (11) by Dixon (B, C) and Cornish-Bowden (D, E) plot. ATP concentration range was 50, 100 and 250 μM, and the inhibitor's range was 0, 10, 50 and 100 μM. The data of one representative experiment out of 3 is shown.

Figure 7

Compounds 10 and 11 inhibit the ectonucleotidase activity of intact HUVECs. Hydrolysis of 100 μM ATP (open bars) and 100 μM ADP (solid bars) was evaluated in the presence of 100 μM 8-BuS-AMP (10) or 8-BuS-ADP (11). The activity without inhibitors were 2.25 ± 0.01 and 3.31 ± 0.02 nmol P_i·min⁻¹·well⁻¹ for ATP and ADP respectively. Activities are expressed as the mean ± SD of 3 independent experiments with confluent cells from different donors at passage 2, each performed in triplicate, mean cell number in one well was in the order of 250 000.

Figure 8

Inhibition of ATPase activity in human and mouse cells and tissues by compounds 1, 10 and 11 using enzyme histochemistry. Substrate, ATP, was used at a final concentration of 200 and 500 μM for human and mouse, respectively, and inhibitors at 100 and 500 μM for human and mouse, respectively. (A) ATPase activity is present at the surface of HUVEC due to NTPDase1. (B) In pancreas, ATPase activity is seen as a brown precipitate in Langerhans islets cells due to NTPDase3, and in blood vessels, in the luminal membrane of acinar cells and in zymogen granules due to NTPDase1. (C) In the liver (D for mouse), ATPase activity due to NTPDase1 is present in vascular endothelium, central vein and sinusoids, as well as in Küpffer cells. Inhibition of ATPase activity in mouse liver (D) and kidney (E) by compounds 1 and 11 using enzyme histochemistry. ATPase activity due to NTPDase1 is seen as a brown precipitate in vascular elements, veins and capillaries, which is inhibited by 1 and 11. In contrast, the activity due to NTPDase3 and/or NTPDase8 in kidney tubules remains. Serial sections were used for all tissues. Nuclei were counterstained with Haematoxylin. Scale bar = 50 μm; * = Langerhans islet; V = vein; black arrows = capillaries.

Figure 9

Compounds 10 and 11 inhibit recombinant human NTPDase1 and its ability to block platelet aggregation induced by ADP. Human NTPDase1 was obtained from a protein extract from transfected COS-7 cells (6 μg protein). Proteins from non-transfected COS-7 cells (6 μg) were used as controls. The controls with protein extract from non-transfected COS-7, with and without ADP, were similar to the assays with inhibitors. They were omitted for clarity. Platelet aggregation in the presence or absence of the indicated protein extract and nucleotide analogue was initiated with 8 μM ADP. (A) Platelet aggregation curves of one out of 3 representative experiments with 8-BuS-AMP are shown. (B) Platelet aggregation curves of one out of 3 representative experiments with 8-BuS-ADP are shown. (C) Qualitative assessment of the effect of 8-BuS-AMP (100 μM) and 8-BuS-ADP (100 μM) on platelet aggregation ± hNTPDase1, by light microscopy (40×). The reaction was performed for 10 min. Pictures of one out of 3 representative experiments are shown. The control without platelets is represented by platelet poor plasma (PPP) and with non-aggregated platelets by platelet-rich plasma (PRP). (D) Number of non-aggregated platelets after 10 min of reaction ± nucleotide analogues and hNTPDase1. The 100% value was set at 3.15 × 10⁶ platelets in 1 mL. The significant statistical differences are indicated as *P < 1·10⁻³.