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, 15 (2), 247-263

Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 Inhibitor Attenuates NPPase Activity in Human Osteoarthritic Chondrocytes

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Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 Inhibitor Attenuates NPPase Activity in Human Osteoarthritic Chondrocytes

Molhm Nassir et al. Purinergic Signal.

Erratum in

Abstract

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,β-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3 μM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6 μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 μM.

Keywords: ADP analogs; AMP analogs; Acyclic nucleotide; Calcium pyrophosphate dihydrate (CPPD); Ecto-nucleotide pyrophosphatase/phosphodiesterase1 (NPP1); Human chondrocytes.

Conflict of interest statement

Molhm Nassir declares that he has no conflict of interest.

Uri Arad declares that he has no conflict of interest.

Sang-Yong Lee declares that he/she has no conflict of interest.

Shani Journo declares that he has no conflict of interest.

Salahuddin Mirza declares that he has no conflict of interest.

Christian Renn declares that he has no conflict of interest.

Christa E. Müller declares that she has no conflict of interest.

Bilha Fischer declares that she has no conflict of interest.

Figures

Fig. 1
Fig. 1
Selection of known NPP1 inhibitors
Fig. 2
Fig. 2
Adenine nucleotide analogs synthesized and evaluated here as NPP1 inhibitors
Scheme 1
Scheme 1
Synthesis of adenosine-5′-(phosphoryl)methylene-sulfonate, 8, and adenosine-5′-(sulfonyl)methylene-sulfonate, 9. Reagents and conditions: a (1) chloromethylene phosphorus dichloride, dry pyridine, Et3N, 0 °C, 5 h; (2) 0.2 M triethylammonium bicarbonate (TEAB), pH 8, 2 h, RT. b Na2SO3, microwave, 120 °C, 4.5 h. c (1) 10% HCl (pH 2.3), RT, 3 h; (2) 24% NH4OH, RT, 45 min; (3) Dowex 50WX8 hydrogen form, 10% NaOH. d (1) Methanedisulfonyl dichloride, dry pyridine, dry dichloromethane (DCM), 0 °C, 4 h; (2) 0.2 M TEAB, 2 h, RT
Scheme 2
Scheme 2
Synthesis of adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, and adenine-N9-(methoxy)ethyl-β-((dichloromethylene)bisphosphonate), 11. Reagents and conditions: a (1) CH2(PO2Cl)2, dry pyridine, dry DCM, − 30 °C, 4 h; (2) 0.2 M TEAB, 2 h. b Dowex 50WX8 hydrogen form, 10% NaOH. c 4-Toluene-sulfonyl chloride, dry pyridine, − 30 °C, 4 h. d (1) Cl2P2O6((Bu)3NH)2, acetonitrile, microwave, 120 °C, 45 min
Scheme 3
Scheme 3
Synthesis of adenine-N9-(methoxy)ethyl-β-phosphate, 12. Reagents and conditions: a (1) POCl3, POMe3, − 15 °C, 1.5 h; (2) 0.2 M TEAB, 1 h, RT. b Dowex 50WX8 hydrogen form, 10% NaOH
Scheme 4
Scheme 4
Synthesis of adenine-(methoxy)ethyl-β-(H-phosphonate), 13, and adenine-(methoxy)ethyl-β-(thiophosphate), 14. Reagents and conditions: a (1) dry DMF, dry pyridine, − 15 °C, 4 h; (2) 0.25 M TEAB (pH 8). b Dowex 50WX8 hydrogen form, 10% NaOH. c (1) S8, overnight; (2) 0.25 M TEAB (pH 8)
Scheme 5
Scheme 5
Synthesis of benzimidazol-2-yl-methanol-bisphosphonate, 15, and benzimidazole-phosphonate, 16. Reagents and conditions: a (1) 1 eq CH2(PO2Cl)2, dry pyridine, dry DCM, − 30 °C, 4 h; (2) 0.2 M TEAB, 1 h. b Dowex 50WX8 hydrogen form, 10% NaOH. c (1) 0.5 eq CH2(PO2Cl)2, dry pyridine, dry DCM, − 30 °C, 4 h; (2) 0.2 M TEAB, 1 h
Fig. 3
Fig. 3
Previously reported NPP1 inhibitors
Fig. 4
Fig. 4
Concentration-inhibition curve of 10 at human NPP1 vs. 100 μM p-Nph-5′-TMP as a substrate (Ki = 16.3 ± 2.5 μM) and Lineweaver-Burk plot of NPP1 inhibition by 10. S, substrate concentration of p-Nph-5′-TMP (μM); v, velocity of enzyme (nmol/min/mg protein). Concentration of 10: green circle, 0 μM; blue triangle, 7.5 μM; violet triangle, 30 μM. Each experiment was performed in triplicates
Fig. 5
Fig. 5
Concentration-inhibition curve of 10 at human NPP1 vs. 100 μM ATP as a substrate (Ki = 9.60 ± 2.84 μM) and Lineweaver-Burk plot of NPP1 inhibition by 10. S, substrate concentration of ATP (μM); v, velocity of enzyme (nmol/min/mg protein). Concentration of 10: green circle, 0 μM; blue triangle, 50 μM; violet triangle, 150 μM. Each experiment was performed in triplicates
Fig. 6
Fig. 6
a Evaluation of the ability of analogs 8–16 to inhibit NPPase activity in human chondrocytes. NPPase activity was assayed by measuring the hydrolysis of the chromogenic substrate, p-Nph-5′-TMP. Analogs 8–16 and the natural substrate, ATP, were added at equimolar concentrations (100 μM). All values related to untreated human chondrocytes. b Human chondrocytes were incubated with or without analogs 8–16 and assayed for alkaline phosphatase activity by hydrolysis of p-nitrophenyl phosphate
Fig. 7
Fig. 7
Analogs 8–16 are not toxic to primary human chondrocytes. Chondrocytes were incubated with analogs 8–16 at the indicated concentrations for 24 h, and then cell viability was assessed by the XTT assay

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