Structure-activity relationship study of NPP1 inhibitors based on uracil-N1-(methoxy)ethyl-β-phosphate scaffold
Molhm Nassir1, Julie Pelletier2, Uri Arad3
1Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900, Israel.
Abstract:
Overexpression of ecto-nucleotide pyrophosphatase-1 (NPP1) is associated with diseases such as calcium pyrophosphate dihydrate deposition disease, calcific aortic valve disease, and type 2 diabetes. In this context, NPP1 inhibitors are potential drug candidates for the treatment of these diseases. The present study focuses on the analysis of the structure-activity relationship of NPP1 inhibitors based on acyclic uracil-nucleotides. For this purpose, we synthesized acyclic uridine-monophosphate analogs, 10-11, uridine-diphosphate analogs, 12-14, and uridine-Pα,α-dithio-triphosphate analogs, 15-17. We evaluated their inhibitory activity and selectivity towards NPP1, -3, NTPDase1, -2, -3, and -8, and P2Y2,4,6 receptors. Analogs 16 and 17 were the most selective and potent NPP1 inhibitors (Ki 0.94 and 0.73 μM, respectively) among the tested molecules. Analogs 10-17 had only minute effect on uracil-nucleotide responding P2Y2,4,6 receptors. Analog 17 (100 μM) displayed 96% inhibition of NPPase activity in osteoarthritic human chondrocytes. Analogs 14-17 displayed weak inhibitory effect on alkaline phosphatase activity at equimolar concentrations in human chondrocytes. All tested analogs showed no toxicity at human chondrocytes. We concluded that ribose-ring to chain transformation, as well as the type of the nucleobase, are parameters of minor significance to NPP1 inhibition, whereas the major parameter is Pα-dithio-substitution. In addition, the length of the phosphate chain also significantly affects inhibition. Overall, the experimental results were well reproduced by molecular docking. A correlation was observed between the activities of the compounds and the number of H-bonds and salt bridges formed between the inhibitors and NPP1 binding site residues. Uracil-N1-(methoxy)ethyl-β-Pα,α-dithio, Pβ,γ-methylene tri-phosphate, 17, was identified as the most potent, selective, and non-toxic NPP1 inhibitor among the tested analogs, and may be used as a lead structure for further drug development.
Insights
New acyclic uracil-nucleotide analogs were synthesized and tested as ecto-nucleotide pyrophosphatase-1 (NPP1) inhibitors. Compound 17 demonstrated potent, selective, and non-toxic NPP1 inhibition, showing promise for treating NPP1-associated diseases.
Area of Science:
- Medicinal Chemistry
- Enzyme Inhibition
- Drug Discovery
Background:
- Ecto-nucleotide pyrophosphatase-1 (NPP1) overexpression is linked to diseases like type 2 diabetes and calcific aortic valve disease.
- NPP1 inhibitors represent a potential therapeutic strategy for these conditions.
Purpose of the Study:
- To explore the structure-activity relationship (SAR) of novel acyclic uracil-nucleotide analogs as NPP1 inhibitors.
- To synthesize and evaluate the inhibitory activity and selectivity of these analogs against NPP1 and related enzymes/receptors.
Main Methods:
- Synthesis of acyclic uridine-monophosphate, -diphosphate, and -Pα,α-dithio-triphosphate analogs.
- Enzyme inhibition assays against NPP1, NPP3, NTPDases, and P2Y receptors.
- Assessment of inhibitory activity in human chondrocytes and evaluation of toxicity.
Main Results:
- Analogs 16 and 17 emerged as the most potent and selective NPP1 inhibitors (Ki values of 0.94 and 0.73 μM).
- Compound 17 exhibited significant NPPase inhibition (96%) in osteoarthritic human chondrocytes with no observed toxicity.
- Molecular docking studies correlated inhibitor activity with binding interactions (H-bonds, salt bridges) within the NPP1 active site.
Conclusions:
- Pα-dithio-substitution and phosphate chain length are critical for potent NPP1 inhibition.
- Acyclic uracil-nucleotide analog 17 is a highly promising lead compound for developing novel NPP1 inhibitors for therapeutic applications.
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