Inhibition of nucleotide pyrophosphatase/phosphodiesterase 1: implications for developing a calcium pyrophosphate deposition disease modifying drug.

Objectives: Calcium pyrophosphate deposition (CPPD) is associated with osteoarthritis and is the cause of a common inflammatory articular disease. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) is the major ecto-pyrophosphatase in chondrocytes and cartilage-derived matrix vesicles (MVs). Thus, eNPP1 is a principle contributor to extracellular pyrophosphate levels and a potential target for interventions aimed at preventing CPPD. Recently, we synthesized and described a novel eNPP1-specific inhibitor, SK4A, and we set out to evaluate whether this inhibitor attenuates nucleotide pyrophosphatase activity in human OA cartilage. Methods: Cartilage tissue, chondrocytes and cartilage-derived MVs were obtained from donors with OA undergoing arthroplasty. The effect of SK4A on cell viability was assayed by the XTT method. eNPP1 expression was evaluated by western blot. Nucleotide pyrophosphatase activity was measured by a colorimetric assay and by HPLC analysis of adenosine triphosphate (ATP) levels. ATP-induced calcium deposition in cultured chondrocytes was visualized and quantified with Alizarin red S staining. Results: OA chondrocytes expressed eNPP1 in early passages, but this expression was subsequently lost upon further passaging. Similarly, significant nucleotide pyrophosphatase activity was only detected in early-passage chondrocytes. The eNPP1 inhibitor, SK4A, was not toxic to chondrocytes and stable in culture medium and human plasma. SK4A effectively inhibited nucleotide pyrophosphatase activity in whole cartilage tissue, in chondrocytes and in cartilage-derived MVs and reduced ATP-induced CPPD. Conclusion: Nucleotide analogues such as SK4A may be developed as potent and specific inhibitors of eNPP1 for the purpose of lowering extracellular pyrophosphate levels in human cartilage with the aim of preventing and treating CPPD disease.

Identification of Highly Promising Antioxidants/Neuroprotectants Based on Nucleoside 5'-Phosphorothioate Scaffold. Synthesis, Activity, and Mechanisms of Action.

With a view to identify novel and biocompatible neuroprotectants, we designed nucleoside 5'-thiophosphate analogues, 6-11. We identified 2-SMe-ADP(α-S), 7A, as a most promising neuroprotectant. 7A reduced ROS production in PC12 cells under oxidizing conditions, IC50 of 0.08 vs 21 µM for ADP. Furthermore, 7A rescued primary neurons subjected to oxidation, EC50 of 0.04 vs 19 µM for ADP. 7A is a most potent P2Y1-R agonist, EC50 of 0.0026 µM. Activity of 7A in cells involved P2Y1/12-R as indicated by blocking P2Y12-R or P2Y1-R. Compound 7A inhibited Fenton reaction better than EDTA, IC50 of 37 vs 54 µM, due to radical scavenging, IC50 of 12.5 vs 30 µM for ADP, and Fe(II)-chelation, IC50 of 80 vs >200 µM for ADP (ferrozine assay). In addition, 7A was stable in human blood serum, t1/2 of 15 vs 1.5 h for ADP, and resisted hydrolysis by NPP1/3, 2-fold vs ADP. Hence, we propose 7A as a highly promising neuroprotectant.

Highly potent and selective ectonucleotide pyrophosphatase/phosphodiesterase I inhibitors based on an adenosine 5'-(α or γ)-thio-(α,ß- or ß,γ)-methylenetriphosphate scaffold.

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-ß,γ-CH2 (1), ATP-α-thio-ß,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 µM inhibited thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 µM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 µM. Analogues 1-3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1-3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.