Substrate-dependence of competitive nucleotide pyrophosphatase / phosphodiesterase1 (NPP1)

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 versus 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 versus 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 Ki values for competitive, but not for non- and un-competitive inhibitors when tested versus 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 p41 nitrophenyl 5’-adenosine monophosphate (p-Nph-5’-AMP) as an alternative artificial substrate correlated much better with those employing the natural substrate ATP.