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.

A Multifunctional Biocompatible Drug Candidate is Highly Effective in Delaying Pathological Signs of Alzheimer's Disease in 5XFAD Mice.

BACKGROUND: Metal-ion-chelation was suggested to prevent zinc and copper ions-induced amyloid-ß (Aß) aggregation and oxidative stress, both implicated in the pathophysiology of Alzheimer's disease (AD). In a quest for biocompatible metal-ion chelators potentially useful for AD therapy, we previously tested a series of nucleoside 5'-phosphorothioate derivatives as agents for decomposition of Cu(I)/Cu(II)/Zn(II)-Aß-aggregates, and as inhibitors of OH radicals formation in Cu(I) or Fe(II) /H2O2 solution. Specifically, in our recent study we have identified 2-SMe-ADP(α-S), designated as SAS, as a most promising neuroprotectant. OBJECTIVE: To further explore SAS ability to protect the brain from Aß toxicity both in vitro and in vivo. METHODS: We evaluated SAS ability to decompose or inhibit the formation of Aß42-M(II) aggregates, and rescue primary neurons and astrocytes from Aß42 toxicity. Furthermore, we aimed at exploring the therapeutic effect of SAS on behavioral and cognitive deficits in the 5XFAD mouse model of AD. RESULTS: We found that SAS can rescue primary culture of neurons and astrocytes from Aß42 toxicity and to inhibit the formation and dissolve Aß42-Zn(II)/Cu(II) aggregates. Furthermore, we show that SAS treatment can prevent behavioral disinhibition and ameliorate spatial working memory deficits in 5XFAD mice. Notably, the mice were treated at the age of 2 months, before the onset of AD symptoms, for a duration of 2 months, while the effect was demonstrated at the age of 6 months. CONCLUSION: Our results indicate that SAS has the potential to delay progression of core pathological characteristics of AD in the 5XFAD mouse model.

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.

Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the identification of human P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.

ATP-γ-S-(α,ß-CH2) protects against oxidative stress and amyloid beta toxicity in neuronal culture.

Amyloid beta (Aß) oligomers and oxidative stress, typical of Alzheimer's disease, are highly neurotoxic. Previously we identified ATP-γ-S as a most promising antioxidant and neuroprotectant. To further improve both potency and metabolic stability of ATP-γ-S, we designed a related analogue, ATP-γ-S-(α,ß-CH2). We found that ATP-γ-S-(α,ß-CH2) effectively inhibited ROS formation in PC12 cells subjected to Fe(II)-oxidation, slightly better than ATP-γ-S (IC50 0.18 and 0.20 µM, respectively). Moreover, ATP-γ-S-(α,ß-CH2) rescued primary neurons from Aß42 toxicity, 4-fold more potently than ATP-γ-S, (IC50 0.2 and 0.8 µM, respectively). In addition, the metabolic stability of ATP-γ-S-(α,ß-CH2) in PC12 cells during 4 h of incubation, was up to 20% greater than that of ATP-γ-S and ATP. Previously, we found that ATP-γ-S-(α,ß-CH2) resisted hydrolysis by ecto-nucleotidases such as, NPPs and TNAP, and was found to be ∼7-fold more potent agonist than ATP at P2Y11 receptor. Therefore, we propose ATP-γ-S-(α,ß-CH2) as a promising agent for rescue of neurons from insults typical of Alzheimer's disease.

Nucleoside 5'-phosphorothioate derivatives as oxidative stress protectants in PC12 cells.

Iron-induced oxidative damage of mitochondria contributes to cellular death seen in neurodegenerative diseases, therefore, there is a demand for nontoxic, biocompatible, and effective Fe-ion chelators. We evaluated the chelation of Fe(II) by phosphate derivatives using ferrozine as an indicator. We studied the effect of phosphate derivatives on inhibiting Fe(II)-induced oxidative stress in PC12 cells, and metabolic stability in PC12 cells was evaluated. Nucleotides containing phosphorothioate moieties inhibited ROS formation better than natural nucleotides and were more metabolically stable in PC12 cells. Finally, we elucidated that these nucleotides activate the MAP-kinase pathway that contributes to protection of PC12 cells under oxidative stress.

Highly efficient biocompatible neuroprotectants with dual activity as antioxidants and P2Y receptor agonists.

Currently, there is a need for novel, biocompatible, and effective neuroprotectants for the treatment of neurodegenerative diseases and brain injury associated with oxidative damage. Here, we developed nucleotide-based neuroprotectants acting dually as antioxidants and P2Y-R agonists. To improve the potency, selectivity, and metabolic stability of ATP/ADP, we substituted adenine C2-position by Cl and Pα/Pß position by borano group, 6-9. Nucleotides 6-9 inhibited oxidation in cell-free systems (Fe(II)-H2O2), as detected by ESR (IC50 up to 175 µM), and ABTS assay (IC50 up to 40 µM). They also inhibited FeSO4-induced oxidative stress in PC12 cells (IC50 of 80-200 nM). 2-Cl-ADP(α-BH3), 7a, was found to be the most potent P2Y1-R agonist currently known (EC50 7 nM) and protected primary cortical neurons from FeSO4 insult (EC50 170 nM). In addition, it proved to be metabolically stable in human blood serum (t(1/2) 7 vs 1.5 h for ADP). Hence, we propose 7a as a highly promising neuroprotectant.