Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

Kumar, Meera; Lowery, Robert G.

doi:10.1177/2472555220982321

Cited by 4 publications

(2 citation statements)

References 21 publications

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“…ENTPD2 hydrolyses primarily ATP to ADP, but it does not hydrolyze ADP causing considerable accumulation of ADP before it is further hydrolyzed to AMP ( Zimmermann et al, 2012 ). ENPP1 preferentially hydrolyses ATP, but it also hydrolyses other nucleotides including ADP ( Kumar and Lowery, 2021 ). As aforementioned, NT5E is the main enzyme that converts AMP into ADO.…”

Section: Discussionmentioning

confidence: 99%

Mechanosensitive Hydrolysis of ATP and ADP in Lamina Propria of the Murine Bladder by Membrane-Bound and Soluble Nucleotidases

et al. 2022

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Prior studies suggest that urothelium-released adenosine 5′-triphosphate (ATP) has a prominent role in bladder mechanotransduction. Urothelial ATP regulates the micturition cycle through activation of purinergic receptors that are expressed in many cell types in the lamina propria (LP), including afferent neurons, and might also be important for direct mechanosensitive signaling between urothelium and detrusor. The excitatory action of ATP is terminated by enzymatic hydrolysis, which subsequently produces bioactive metabolites. We examined possible mechanosensitive mechanisms of ATP hydrolysis in the LP by determining the degradation of 1,N6-etheno-ATP (eATP) at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model, using HPLC. The hydrolysis of eATP and eADP was greater in contact with LP of distended than of nondistended bladders whereas the hydrolysis of eAMP remained unchanged during filling, suggesting that some steps of eATP hydrolysis in the LP are mechanosensitive. eATP and eADP were also catabolized in extraluminal solutions (ELS) that were in contact with the LP of detrusor-free bladders, but removed from the organ chambers prior to addition of substrate. The degradation of both purines was greater in ELS from distended than from nondistended preparations, suggesting the presence of mechanosensitive release of soluble nucleotidases in the LP. The released enzyme activities were affected differently by Ca2+ and Mg2+. The common nucleotidase inhibitors ARL67156, POM-1, PSB06126, and ENPP1 Inhibitor C, but not the alkaline phosphatase inhibitor (-)-p-bromotetramisole oxalate, inhibited the enzymes released during bladder distention. Membrane-bound nucleotidases were identified in tissue homogenates and in concentrated ELS from distended preparations by Wes immunodetection. The relative distribution of nucleotidases was ENTPD1 >> ENPP1 > ENTPD2 = ENTPD3 > ENPP3 = NT5E >> ENTPD8 = TNAP in urothelium and ENTPD1 >> ENTPD3 >> ENPP3 > ENPP1 = ENTPD2 = NT5E >> ENTPD8 = TNAP in concentrated ELS, suggesting that regulated ectodomain shedding of membrane-bound nucleotidases possibly occurs in the LP during bladder filling. Mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP diminishes the availability of excitatory purines in the LP at the end of bladder filling. This might be a safeguard mechanism to prevent over-excitability of the bladder. Proper proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.

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Section: Discussionmentioning

confidence: 99%

Mechanosensitive Hydrolysis of ATP and ADP in Lamina Propria of the Murine Bladder by Membrane-Bound and Soluble Nucleotidases

et al. 2022

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“…Briefly, ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING ( 37 ). Accordingly, ENPP1 is intensively investigated as a drug target not only for calcification disorders ( 66 , 67 ), but also for its potential applications in cancer immunotherapy ( 38 , 42 , 68 , 69 , 70 ).…”

Section: Enpps In Physiology and Diseasementioning

confidence: 99%

Structure and function of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family: Tidying up diversity

Borza

Salgado-Polo

Moolenaar

et al. 2022

Journal of Biological Chemistry

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Ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family members (ENPP1–7) have been implicated in key biological and pathophysiological processes, including nucleotide and phospholipid signaling, bone mineralization, fibrotic diseases, and tumor-associated immune cell infiltration. ENPPs are single-pass transmembrane ecto-enzymes, with notable exceptions of ENPP2 (Autotaxin) and ENNP6, which are secreted and glycosylphosphatidylinositol (GPI)-anchored, respectively. ENNP1 and ENNP2 are the best characterized and functionally the most interesting members. Here, we review the structural features of ENPP1–7 to understand how they evolved to accommodate specific substrates and mediate different biological activities. ENPPs are defined by a conserved phosphodiesterase (PDE) domain. In ENPP1–3, the PDE domain is flanked by two N-terminal somatomedin B-like domains and a C-terminal inactive nuclease domain that confers structural stability, whereas ENPP4–7 only possess the PDE domain. Structural differences in the substrate-binding site endow each protein with unique characteristics. Thus, ENPP1, ENPP3, ENPP4, and ENPP5 hydrolyze nucleotides, whereas ENPP2, ENPP6, and ENNP7 evolved as phospholipases through adaptions in the catalytic domain. These adaptations explain the different biological and pathophysiological functions of individual members. Understanding the ENPP members as a whole advances our insights into common mechanisms, highlights their functional diversity, and helps to explore new biological roles.

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