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Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes

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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.

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  • 20 July 2019

    The original version of the article unfortunately contained an error.

Abbreviations

eNPP:

Ecto-nucleotide pyrophosphatase/phosphodiesterase

NTP:

Nucleoside-5′-triphosphate

p-Nph-5′-TMP:

p-nitrophenyl ester thymidine 5′-monophosphate

SD:

Standard deviation

CPPD:

Calcium pyrophosphate dihydrate

PPi :

Pyrophosphate/diphosphate

TEAB:

Triethylammonium bicarbonate

DCM:

Dichloromethane

References

  1. Yamakawa K, Iwasaki H, Masuda I, Ohjimi Y, Honda I, Iyama K-I, Shono E, Naito M, Kikuchi M (2002) Cartilage intermediate layer protein expression in calcium pyrophosphate dihydrate crystal deposition disease. J Rheumatol 29(8):1746–1753

    CAS  PubMed  Google Scholar 

  2. Gibilisco PA, Schumacher HR, Hollander JL, Soper KA (1985) Synovial fluid crystals in osteoarthritis. Arthritis Rheum 28(5):511–515

    Article  CAS  PubMed  Google Scholar 

  3. Bjelle AO, Sundström BK (1975) An ultrastructural study of the articular cartilage in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease (chondrocalcinosis articularis). Calcif Tissue Int 19(1):63–71

    Article  CAS  Google Scholar 

  4. Nalbant S, Martinez J, Kitumnuaypong T, Clayburne G, Sieck M, Schumacher H Jr (2003) Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies. Osteoarthr Cartil 11(1):50–54

    Article  CAS  PubMed  Google Scholar 

  5. Abhishek A, Neogi T, Choi H, Doherty M, Rosenthal AK, Terkeltaub R (2018) Unmet needs and the path forward in joint disease associated with calcium pyrophosphate crystal deposition. Arthritis Rheum 70:1182–1191

    Article  Google Scholar 

  6. Rachow JW, Ryan LM (1988) Inorganic pyrophosphate metabolism in arthritis. Rheum Dis Clin N Am 14(2):289–302

    CAS  Google Scholar 

  7. Terkeltaub RA (2001) Inorganic pyrophosphate generation and disposition in pathophysiology. A J Phys 281(1):C1–C11

    CAS  Google Scholar 

  8. Goding JW, Grobben B, Slegers H (2003) Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta (BBA) - Mol Basis Dis 1638(1):1–19

    Article  CAS  Google Scholar 

  9. Johnson K, Vaingankar S, Chen Y, Moffa A, Goldring MB, Sano K, Jin-Hua P, Sali A, Goding J, Terkeltaub R (1999) Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes. Arthritis Rheum 42(9):1986–1997

    Article  CAS  PubMed  Google Scholar 

  10. Johnson K, Hashimoto S, Lotz M, Pritzker K, Goding J, Terkeltaub R (2001) Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification. Arthritis Rheum 44(5):1071–1081

    Article  CAS  PubMed  Google Scholar 

  11. Huang R, Rosenbach M, Vaughn R, Provvedini D, Rebbe N, Hickman S, Goding J, Terkeltaub R (1994) Expression of the murine plasma cell nucleotide Pyrophosphohydrolase Pc-1 is shared by human liver, bone, and cartilage cells - regulation of Pc-1 expression in osteosarcoma cells by transforming growth factor-Beta. J Clin Invest 94(2):560–567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Johnson K, Moffa A, Chen Y, Pritzker K, Goding J, Terkeltaub R (1999) Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells. J Bone Miner Res 14(6):883–892

    Article  CAS  PubMed  Google Scholar 

  13. Nadel Y, Lecka J, Gilad Y, Ben-David G, Förster D, Reiser G, Kenigsberg S, Camden J, Weisman GA, Senderowitz H, Sévigny J, Fischer B (2014) Highly potent and selective Ectonucleotide pyrophosphatase/phosphodiesterase I inhibitors based on an adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate scaffold. J Med Chem 57(11):4677–4691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Villa-Bellosta R, Wang X, Millan JL, Dubyak GR, O’Neill WC (2011) Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle. Am J Physiol Heart Circ 301(1):H61–H68

    Article  CAS  Google Scholar 

  15. Bollen M, Gijsbers R, Ceulemans H, Stalmans W, Stefan C (2000) Nucleotide Pyrophosphatases/Phosphodiesterases on the move. Crit Rev Biochem Mol Biol 35(6):393–432

    Article  CAS  PubMed  Google Scholar 

  16. Agresti C, Meomartini M, Amadio S, Ambrosini E, Volonte C, Aloisi F, Visentin S (2005) ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors. Brain Res Rev 48(2):157–165

    Article  CAS  PubMed  Google Scholar 

  17. Burnstock G (2002) Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22(3):364–373

    Article  CAS  PubMed  Google Scholar 

  18. Burnstock G (2002) Potential therapeutic targets in the rapidly expanding field of purinergic signalling. J Clin Respir Med 2(1):45–53

    CAS  Google Scholar 

  19. Sak K, Boeynaems J-M, Everaus H (2003) Involvement of P2Y receptors in the differentiation of haematopoietic cells. J Leukoc Biol Suppl 73(4):442–447

    Article  CAS  Google Scholar 

  20. Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmied Arch Pharmacol 362(4–5):299–309

    Article  CAS  Google Scholar 

  21. Zimmermann H, Zebisch M, Sträter N (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8(3):437–502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lee S-Y, Müller CE (2017) Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) and its inhibitors. MedChemComm 8(5):823–840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Henz SL, Fürstenau CR, Chiarelli RA, Sarkis JJF (2007) Kinetic and biochemical characterization of an ecto-nucleotide pyrophosphatase/phosphodiesterase (EC 3.1. 4.1) in cells cultured from submandibular salivary glands of rats. Arch Oral Biol 52(10):916–923

    Article  CAS  PubMed  Google Scholar 

  24. Jj L, Choi HJ, Yun M, Kang Y, Jung JE, Ryu Y, Kim TY, Cha Y, Cho HS, Min JJ (2015) Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein–drug conjugates for targeted therapy. Angew Chem Int Ed Eng 54(41):12020–12024

    Article  CAS  Google Scholar 

  25. Eliahu S, Lecka J, Reiser G, Haas M, Fo B, Lévesque SA, Pelletier J, Sévigny J, Fischer B (2010) Diadenosine 5′, 5′′-(Boranated) polyphosphonate analogues as selective nucleotide pyrophosphatase/phosphodiesterase inhibitors⊥. J Med Chem 53(24):8485–8497

    Article  CAS  PubMed  Google Scholar 

  26. Müller CE, Iqbal J, Baqi Y, Zimmermann H, Röllich A, Stephan H (2006) Polyoxometalates—a new class of potent ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors. Bioorg Med Chem Lett 16(23):5943–5947

    Article  CAS  PubMed  Google Scholar 

  27. Vollmayer P, Clair T, Goding JW, Sano K, Servos J, Zimmermann H (2003) Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases. FEBS J 270(14):2971–2978

    CAS  Google Scholar 

  28. Lee S-Y, Fiene A, Li W, Hanck T, Brylev KA, Fedorov VE, Lecka J, Haider A, Pietzsch H-J, Zimmermann H (2015) Polyoxometalates—potent and selective ecto-nucleotidase inhibitors. Biochem Pharmacol 93(2):171–181

    Article  CAS  PubMed  Google Scholar 

  29. Khan KM, Fatima N, Rasheed M, Jalil S, Ambreen N, Perveen S, Choudhary MI (2009) 1, 3, 4-Oxadiazole-2 (3H)-thione and its analogues: a new class of non-competitive nucleotide pyrophosphatases/phosphodiesterases 1 inhibitors. Bioorg Med Chem 17(22):7816–7822

    Article  CAS  PubMed  Google Scholar 

  30. Choudhary MI, Fatima N, Khan KM, Jalil S, Iqbal S (2006) New biscoumarin derivatives-cytotoxicity and enzyme inhibitory activities. Bioorg Med Chem 14(23):8066–8072

    Article  CAS  PubMed  Google Scholar 

  31. Grobben B, Claes P, Roymans D, Esmans EL, Van Onckelen H, Slegers H (2000) Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists. Br J Pharmacol 130(1):139–145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hosoda N, Si H, Kanda Y, Katada T (1999) Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans. FEBS J 265(2):763–770

    CAS  Google Scholar 

  33. Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H (2008) Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res 334(2):199–217

    Article  CAS  PubMed  Google Scholar 

  34. Pope MT, Kortz U. 2012. Polyoxometalates. Encyclo Inorg Bioinorg Chem

  35. Shayhidin EE, Forcellini E, Boulanger MC, Mahmut A, Dautrey S, Barbeau X, Lagüe P, Sévigny J, Paquin JF, Mathieu P (2015) Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells. Br J Pharmacol 172(16):4189–4199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Patel SD, Habeski WM, Cheng AC, de la Cruz E, Loh C, Kablaoui NM (2009) Quinazolin-4-piperidin-4-methyl sulfamide PC-1 inhibitors: alleviating hERG interactions through structure based design. Bioorg Med Chem Lett 19(12):3339–3343

    Article  CAS  PubMed  Google Scholar 

  37. Lee S-Y, Perotti A, De Jonghe S, Herdewijn P, Hanck T, Müller CE (2016) Thiazolo [3, 2-a] benzimidazol-3 (2H)-one derivatives: structure–activity relationships of selective nucleotide pyrophosphatase/phosphodiesterase1 (NPP1) inhibitors. Bioorg Med Chem 24(14):3157–3165

    Article  CAS  PubMed  Google Scholar 

  38. Lee SY, Müller CE (2014) Large-volume sample stacking with polarity switching for monitoring of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) reactions by capillary electrophoresis. Electrophoresis 35(6):855–863

    Article  CAS  PubMed  Google Scholar 

  39. Fürstenau CR, Trentin DS, Barreto-Chaves MLM, Sarkis JJ (2007) The effects of angiotensin II and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes. Thromb Res 120(6):877–884

    Article  CAS  PubMed  Google Scholar 

  40. Stefan C, Jansen S, Bollen M (2005) NPP-type ectophosphodiesterases: unity in diversity. Trends Biochem Sci 30(10):542–550

    Article  CAS  PubMed  Google Scholar 

  41. Stefan C, Stalmans W, Bollen M (1996) Threonine autophosphorylation and Nucleotidylation of the hepatic membrane protein PC-1. Eur J Biochem 241(2):338–342

    Article  CAS  PubMed  Google Scholar 

  42. Danino O, Svetitsky S, Kenigsberg S, Levin A, Journo S, Gold A, Drexler M, Snir N, Elkayam O, Fischer B (2018) Inhibition of nucleotide pyrophosphatase/phosphodiesterase 1: implications for developing a calcium pyrophosphate deposition disease modifying drug. Rheumatology 57(8):1472–1480

    Article  CAS  PubMed  Google Scholar 

  43. Lecka J, Ben-David G, Simhaev L, Eliahu S, Oscar J Jr, Luyindula P, Pelletier J, Fischer B, Senderowitz H, Sévigny J (2013) Nonhydrolyzable ATP analogues as selective inhibitors of human NPP1: a combined computational/experimental study. J Med Chem 56(21):8308–8320

    Article  CAS  PubMed  Google Scholar 

  44. Sayer AH, Itzhakov Y, Stern N, Bilha F (2013) characterization of complexes of Nucleoside-5′-phosphorothioate analogues with zinc ions. In Org Chem 52(19):10886–10896

    CAS  Google Scholar 

  45. Chang L, Lee S-Y, Leonczak P, Rozenski J, De Jonghe S, Hanck T, Müller CE, Herdewijn P (2014) Imidazopyridine-and purine-thioacetamide derivatives: potent inhibitors of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). J Med Chem 57(23):10080–10100

    Article  CAS  PubMed  Google Scholar 

  46. Chen A-H, Liu S-C, Chen C-Y, Chen C-Y (2008) Comparative adsorption of cu (II), Zn (II), and Pb (II) ions in aqueous solution on the crosslinked chitosan with epichlorohydrin. J Hazard Mater 154(1):184–191

    Article  CAS  Google Scholar 

  47. van Meeteren LA, Ruurs P, Christodoulou E, Goding JW, Takakusa H, Kikuchi K, Perrakis A, Nagano T, Moolenaar WH (2005) Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate. J Biol Chem 280(22):21155–21161

    Article  CAS  PubMed  Google Scholar 

  48. Meltzer D, Ethan O, Arguin G, Nadel Y, Danino O, Lecka J, Sevigny J, Gendron F-P, Fischer B (2015) Synthesis and structure–activity relationship of uracil nucleotide derivatives towards the identification of human P2Y 6 receptor antagonists. Bioorg Med Chem 23(17):5764–5773

    Article  CAS  PubMed  Google Scholar 

  49. Bhattarai S, Freundlieb M, Pippel J, Meyer A, Abdelrahman A, Fiene A, Lee S-Y, Zimmermann H, Yegutkin GG, Sträter N (2015) α, β-methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5′-nucleotidase (CD73) inhibitors. J Med Chem 58(15):6248–6263

    Article  CAS  PubMed  Google Scholar 

  50. Iqbal J, Lévesque SA, Sévigny J, Müller CE (2008) A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions. Electrophoresis 29(17):3685–3693

    Article  CAS  PubMed  Google Scholar 

  51. Lee S-Y, Sarkar S, Bhattarai S, Namasivayam V, De Jonghe S, Stephan H, Herdewijn P, El-Tayeb A, Müller CE (2017) Substrate-dependence of competitive nucleotide pyrophosphatase/phosphodiesterase1 (NPP1) inhibitors. Front Pharmacol 8:54

    PubMed  PubMed Central  Google Scholar 

  52. Mahmut A, Boulanger M-C, Bouchareb R, Hadji F, Mathieu P (2015) Adenosine derived from ecto-nucleotidases in calcific aortic valve disease promotes mineralization through A2a adenosine receptor. Cardiovasc Res 106(1):109–120

    Article  CAS  PubMed  Google Scholar 

  53. Gendron F-P, Halbfinger E, Fischer B, Duval M, D’Orléans-Juste P, Beaudoin AR (2000) Novel inhibitors of nucleoside triphosphate diphosphohydrolases: chemical synthesis and biochemical and pharmacological characterizations. J Med Chem 43(11):2239–2247

    Article  CAS  PubMed  Google Scholar 

  54. Baykov A, Evtushenko O, Avaeva S (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171(2):266–270

    Article  CAS  PubMed  Google Scholar 

  55. Freundlieb M, Zimmermann H, Müller CE (2014) A new, sensitive ecto-5′-nucleotidase assay for compound screening. Anal Biochem 446:53–58

    Article  CAS  PubMed  Google Scholar 

  56. Baqi Y, Lee S-Y, Iqbal J, Ripphausen P, Lehr A, Scheiff AB, Zimmermann H, Bajorath JR, Müller CE (2010) Development of potent and selective inhibitors of ecto-5′-nucleotidase based on an anthraquinone scaffold. J Med Chem 53(5):2076–2086

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, Bar-Ilan University, 52900, Ramat-Gan, Israel

    Molhm Nassir & Bilha Fischer

  2. Department of Rheumatology, Tel Aviv Medical Center and the Faculty of Medicine, Tel Aviv University, 6997801, Tel Aviv, Israel

    Uri Arad & Shani Journo

  3. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany

    Sang-Yong Lee, Salahuddin Mirza, Christian Renn & Christa E. Müller

  4. Institute of Cell Biology and Neuroscience, Goethe-University, 60438, Frankfurt am Main, Germany

    Herbert Zimmermann

  5. Centre de Recherche du CHU de Québec – Université Laval, Québec City, QC, Canada

    Julie Pelletier & Jean Sévigny

  6. Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université Laval, Québec City, QC, Canada

    Jean Sévigny

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  1. Molhm Nassir
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  2. Uri Arad
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  3. Sang-Yong Lee
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  7. Herbert Zimmermann
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  10. Christa E. Müller
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Correspondence to Bilha Fischer.

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Conflicts of interest

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.

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Nassir, M., Arad, U., Lee, SY. et al. Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes. Purinergic Signalling 15, 247–263 (2019). https://doi.org/10.1007/s11302-019-09649-2

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