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Substrate-Dependence of Competitive Nucleotide Pyrophosphatase/Phosphodiesterase1 (NPP1) Inhibitors.

ABSTRACT: Nucleotide pyrophosphatase/phosphodiesterase type 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its major substrate is ATP which is converted to AMP and diphosphate. NPP1 was proposed as a new therapeutic target in brain cancer and immuno-oncology. Several NPP1 inhibitors have been reported to date, most of which were evaluated vs. the artificial substrate p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP). Recently, we observed large discrepancies in inhibitory potencies for a class of competitive NPP1 inhibitors when tested vs. the artificial substrate p-Nph-5'-TMP as compared to the natural substrate ATP. Therefore, the goal of the present study was to investigate whether inhibitors of human NPP1 generally display substrate-dependent inhibitory potency. Systematic evaluation of nucleotidic as well as non-nucleotidic NPP1 inhibitors revealed significant differences in determined K_i values for competitive, but not for non- and un-competitive inhibitors when tested vs. the frequently used artificial substrate p-Nph-5'-TMP as compared to ATP. Allosteric modulation of NPP1 by p-Nph-5'-TMP may explain these discrepancies. Results obtained using the AMP derivative p-nitrophenyl 5'-adenosine monophosphate (p-Nph-5'-AMP) as an alternative artificial substrate correlated much better with those employing the natural substrate ATP.

SUBMITTER: Lee SY

PROVIDER: S-EPMC5309242 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Substrate-Dependence of Competitive Nucleotide Pyrophosphatase/Phosphodiesterase1 (NPP1) Inhibitors.

Lee Sang-Yong SY Sarkar Soumya S Bhattarai Sanjay S Namasivayam Vigneshwaran V De Jonghe Steven S Stephan Holger H Herdewijn Piet P El-Tayeb Ali A Müller Christa E CE

Frontiers in pharmacology 20170215

Nucleotide pyrophosphatase/phosphodiesterase type 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its major substrate is ATP which is converted to AMP and diphosphate. NPP1 was proposed as a new therapeutic target in brain cancer and immuno-oncology. Several NPP1 inhibitors have been reported to date, most of which were evaluated vs. the artificial substrate <i>p</i>-nitrophenyl 5'-thymidine monophosphate (<i>p</i>-Nph-5'-TMP). Recently, we observed l ...[more]

PMID: 28261095

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Project description:BackgroundNucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM.ResultsThe catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding.ConclusionsSubstrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.

Dataset Information

Substrate-Dependence of Competitive Nucleotide Pyrophosphatase/Phosphodiesterase1 (NPP1) Inhibitors.

Publications

Substrate-Dependence of Competitive Nucleotide Pyrophosphatase/Phosphodiesterase1 (NPP1) Inhibitors.

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