Quinazoline‐4‐piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells

Abstract

Background and Purpose Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline‐4‐piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. Experimental Approach In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1‐2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate‐induced mineralization of human primary aortic valve interstitial cells (VICs). Key Results The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non‐competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1–3), ecto‐5′‐nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 μM) prevented phosphate‐induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. Conclusions and Implications We have provided evidence that QPS1 is a potent and selective non‐competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.

Crystal structures of human ENPP1 in apo and bound forms

“…The ENPP1 inhibitors Ex54 (Gallatin et al, 2019) and QPS2 (Shayhidin et al, 2015) have a modified quinoline or quinazoline core (monomethoxylated or dimethoxylated, respectively) that packs between Phe257 and Tyr340 and forms a hydrogen bond to Lys295 (2.8-3.2 Å ), analogous to the adenine in the AMP-bound structure (Figs. 3c,3d,4c,4d and 5).…”

2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

“…Full length human ENPP1 was cloned into pcDNA6 vector. QS1 was synthesized as previously described 31 . The following monoclonal antibodies were used for western blotting: rabbit anti-cGAS (D1D3G Cell Signaling, 1:1,000) rabbit antimouse cGAS (D2O8O Cell Signaling, 1:1,000), mouse anti-tubulin (DM1A Cell Signaling, 1:2,000), and rabbit anti-STING (D2P2F Cell Signaling, 1:1,000), IRDye 800CW goat anti-rabbit (LI-COR, 1:15,000), and IRDye 680RD goat anti-mouse (LI-COR, 1:15,000).…”

“…To study the physiological relevance of extracellular cGAMP, we sought to develop cell impermeable ENPP1 inhibitors that only affect extracellular ENPP1 activity. We first tested a nonspecific ENPP1 inhibitor QS1 30,31 (Extended Data Fig. 3a, b).…”

Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways

“…NPP1 inhibitors have been developed as potential therapeutics for diabetes mellitus and brain cancers [ 32 ]. Additionally, a recent in vitro study showed that treating mineralized VICs—which overexpress NPP1—with a specific non-competitive NPP1 inhibitor led to a significant ( p < 0.05) decrease in mineral content, apoptosis and osteo/chondrogenic transdifferentiation, as compared to VICs on a pro-calcifying medium without inhibitor supplementation [ 33 ]. In conclusion, it is key to maintain the NPP1 activity in a well-defined range as overexpression, as well as reduced expression of NPP1 activity, have been linked to arterial calcification.…”