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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Nonhydrolyzable ATP analogues as selective inhibitors of human NPP1: a combined computational/experimental study
Joanna Lecka1, Gal Ben-David, Luba Simhaev
1Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval , Québec, QC G1V 0A6, Canada.
Abstract:
Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 μM inhibited (66-99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The Ki,app values of the seven most potent and selective inhibitors were in the range of 0.5-56 μM, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor 2 that had the lowest Ki,app (0.5 μM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.
Insights
Researchers developed novel nonhydrolyzable ATP analogues as selective inhibitors for nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). Analogue 2 emerged as the most potent and selective NPP1 inhibitor, offering potential for therapeutic applications in calcification disorders.
Area of Science:
- Biochemistry
- Enzymology
- Medicinal Chemistry
Background:
- Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is linked to pathological calcification.
- Targeted NPP1 inhibitors are crucial for understanding its role in disease and for developing therapeutics.
Purpose of the Study:
- To characterize novel nonhydrolyzable ATP analogues as selective inhibitors of human NPP1.
- To identify potent and selective NPP1 inhibitors for potential therapeutic use.
Main Methods:
- Combined computational (molecular docking) and experimental (enzyme inhibition assays) approaches.
- Screening of 13 nonhydrolyzable ATP analogues against recombinant and cell-surface NPP1.
- Determination of inhibition constants (Ki,app) and selectivity profiling against related ectonucleotidases (NPP3, NTPDases).
Main Results:
- All 13 analogues demonstrated significant inhibition of NPP1 activity (66-99%) at 100 μM.
- Seven analogues exhibited potent and selective inhibition with Ki,app values ranging from 0.5-56 μM.
- Molecular docking revealed ATP-like binding modes, consistent with competitive inhibition.
- Analogue 2 displayed the lowest Ki,app (0.5 μM) and was inactive against P2Y receptors.
Conclusions:
- Nonhydrolyzable ATP analogues represent a promising class of selective NPP1 inhibitors.
- Analogue 2 is the most potent and selective NPP1 inhibitor identified to date.
- These findings support the therapeutic potential of NPP1 inhibitors in treating disorders associated with pathological calcification.
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