Search Results (19)

Background: Lactate dehydrogenase (LDH) activity, producing high levels of lactate from pyruvate in cancer cells, is often associated with poor patient prognosis. We previously showed enhanced LDH/lactate levels in estrogen receptor (ER) compared to ER + breast cancer cells; lactate or pyruvate supplementation to ER + cells significantly enhanced their motile ability, while LDHB gene knockout (KO) or treatment with LDH inhibitors reduced the motility of the highly aggressive ER breast cancer cells. Aims: To investigate the molecular mechanisms by which lactate, LDHB KO, or treatment with LDH inhibitors can modulate the motile capabilities of breast cancer cell lines. Methods: KO experiments were performed using siRNA, and global expression was determined by proteomic profiling with Proteome Profiler Human XL Oncology arrays, Western blot, and immunofluorescence. Results: Lactate supplementation to ER + breast cancer cells enhanced expression of vimentin, N-cadherin, and snail, while reducing the expression of JAM-A, E-cadherin, and nectin-4. This expression profile was reversed with LDHB KO in ER cells. LDHB KO, or treatment with LDH inhibitors in ER cells, also reduced the expression of IL-6, IL-8, and MMP-2. The expressions of other markers such as PECAM-1, CCL20, and ENPP-2 were differentially modulated with LDH B KO in de novo ER cells (MDA-MB-231) vs. those that had ER knockout (pII). Conclusions: Our data show a novel role for lactate in modulating the EMT status in breast cancer cells and highlight the important role of lactate in breast cancer motility in part through modulating EMT status and the expression profile of cytokines, adhesion molecules, MMP-2, and nectin-4. Full article

The phosphodiester linkage is found in DNA, RNA and many signaling molecules, such as cyclic mononucleotide, cyclic dinucleotides (CDNs) and cyclic oligonucleotides (cONs). Enzymes that cleave the phosphodiester linkage (nucleases and phosphodiesterases) play important roles in cell persistence and fitness and have therefore become targets for various diseased states. While various inhibitors have been developed for nucleases and cyclic mononucleotide phosphodiesterases, and some have become clinical successes, there is a paucity of inhibitors of the recently discovered phosphodiesterases or ring nucleases that cleave CDNs and cONs. Inhibitors of bacterial c-di-GMP or c-di-AMP phosphodiesterases have the potential to be used as anti-virulence compounds, while compounds that inhibit the degradation of 3′,3′-cGAMP, cA₃, cA₄, cA₆ could serve as antibiotic adjuvants as the accumulation of these second messengers leads to bacterial abortive infection. In humans, 2′3′-cGAMP plays critical roles in antiviral and antitumor responses. ENPP1 (the 2′3′-cGAMP phosphodiesterase) or virally encoded cyclic dinucleotide phosphodiesterases, such as poxin, however, blunt this response. Inhibitors of ENPP1 or poxin-like enzymes have the potential to be used as anticancer and antiviral agents, respectively. This review summarizes efforts made towards the discovery and development of compounds that inhibit CDN phosphodiesterases and cON ring nucleases. Full article

Phosphatases are enzymes that catalyze the hydrolysis of phosphate esters. They play critical roles in diverse biological processes such as extracellular nucleotide homeostasis, transport of molecules across membranes, intracellular signaling pathways, or vertebrate mineralization. Among them, tissue-nonspecific alkaline phosphatase (TNAP) is today increasingly studied, due to its ubiquitous expression and its ability to dephosphorylate a very broad range of substrates and participate in several different biological functions. For instance, TNAP hydrolyzes inorganic pyrophosphate (PP_i) to allow skeletal and dental mineralization. Additionally, TNAP hydrolyzes pyridoxal phosphate to allow cellular pyridoxal uptake, and stimulate vitamin B6-dependent reactions. Furthermore, TNAP has been identified as a key enzyme in non-shivering adaptive thermogenesis, by dephosphorylating phosphocreatine in the mitochondrial creatine futile cycle. This latter recent discovery and others suggest that the list of substrates and functions of TNAP may be much longer than previously thought. In the present review, we sought to examine TNAP within the alkaline phosphatase (AP) superfamily, comparing its sequence, structure, and evolutionary trajectory. The AP superfamily, characterized by a conserved central folding motif of a mixed beta-sheet flanked by alpha-helices, includes six subfamilies: AP, arylsulfatases (ARS), ectonucleotide pyrophosphatases/phosphodiesterases (ENPP), phosphoglycerate mutases (PGM), phosphonoacetate hydrolases, and phosphopentomutases. Interestingly, TNAP and several ENPP family members appear to participate in the same metabolic pathways and functions. For instance, extra-skeletal mineralization in vertebrates is inhibited by ENPP1-mediated ATP hydrolysis into the mineralization inhibitor PP_i, which is hydrolyzed by TNAP expressed in the skeleton. Better understanding how TNAP and other AP family members differ structurally will be very useful to clarify their complementary functions. Structurally, TNAP shares the conserved catalytic core with other AP superfamily members but has unique features affecting substrate specificity and activity. The review also aims to highlight the importance of oligomerization in enzyme stability and function, and the role of conserved metal ion coordination, particularly magnesium, in APs. By exploring the structural and functional diversity within the AP superfamily, and discussing to which extent its members exert redundant, complementary, or specific functions, this review illuminates the evolutionary pressures shaping these enzymes and their broad physiological roles, offering insights into TNAP’s multifunctionality and its implications for health and disease. Full article

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that peripheral and central LPA receptors (LPARs) were involved in HCQ-evoked pruriception. To unravel the signaling paths, we assessed the localization of candidate genes and itching behavior in knockout models addressing LPAR5, LPAR2, autotaxin/ENPP2 and the lysophospholipid phosphatases, as well as the plasticity-related genes Prg1/LPPR4 and Prg2/LPPR3. LacZ reporter studies and RNAscope revealed LPAR5 in neurons of the dorsal root ganglia (DRGs) and in skin keratinocytes, LPAR2 in cortical and thalamic neurons, and Prg1 in neuronal structures of the dorsal horn, thalamus and SSC. HCQ-evoked scratching behavior was reduced in sensory neuron-specific Advillin-LPAR5^−/− mice (peripheral) but increased in LPAR2^−/− and Prg1^−/− mice (central), and it was not affected by deficiency of glial autotaxin (GFAP-ENPP2^−/−) or Prg2 (PRG2^−/−). Heat and mechanical nociception were not affected by any of the genotypes. The behavior suggested that HCQ-mediated itch involves the activation of peripheral LPAR5, which was supported by reduced itch upon treatment with an LPAR5 antagonist and autotaxin inhibitor. Further, HCQ-evoked calcium fluxes were reduced in primary sensory neurons of Advillin-LPAR5^−/− mice. The results suggest that LPA-mediated itch is primarily mediated via peripheral LPAR5, suggesting that a topical LPAR5 blocker might suppress “non-histaminergic” itch. Full article

Calcification plays a key role in biological processes, and breakdown of the regulatory mechanism results in a pathological state such as ectopic calcification. We hypothesized that ENPP1, the enzyme that produces the calcification inhibitor pyrophosphate, is transcriptionally regulated by Nrf2, and that Nrf2 activation augments ENPP1 expression to inhibit ectopic calcification. Cell culture experiments were performed using mouse osteoblastic cell line MC3T3-E1. Nrf2 was activated by 5-aminolevulinic acid and sodium ferrous citrate. Nrf2 overexpression was induced by the transient transfection of an Nrf2 expression plasmid. ENPP1 expression was monitored by real-time RT-PCR. Because the promoter region of ENPP1 contains several Nrf2-binding sites, chromatin immunoprecipitation using an anti-Nrf2 antibody followed by real-time PCR (ChIP-qPCR) was performed. The relationship between Nrf2 activation and osteoblastic differentiation was examined by alkaline phosphatase (ALP) and Alizarin red staining. We used mice with a hypomorphic mutation in ENPP1 (ttw mice) to analyze whether Nrf2 activation inhibits ectopic calcification. Nrf2 and Nrf2 overexpression augmented ENPP1 expression and inhibited osteoblastic differentiation, as indicated by ALP expression and calcium deposits. ChIP-qPCR showed that some putative Nrf2-binding sites in the ENPP1 promoter region were bound by Nrf2. Nrf2 activation inhibited ectopic calcification in mice. ENPP1 gene expression was transcriptionally regulated by Nrf2, and Nrf2 activation augmented ENPP1 expression, leading to the attenuation of osteoblastic differentiation and ectopic calcification in vitro and in vivo. Nrf2 activation has a therapeutic potential for preventing ectopic calcification. Full article

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PP_i) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5′-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A_2aR and A_2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy. Full article

Pseudoxanthoma Elasticum (PXE) is an inherited disease characterized by elastic fiber calcification in the eyes, the skin and the cardiovascular system. PXE results from mutations in ABCC6 that encodes an ABC transporter primarily expressed in the liver and kidneys. It took nearly 15 years after identifying the gene to better understand the etiology of PXE. ABCC6 function facilitates the efflux of ATP, which is sequentially hydrolyzed by the ectonucleotidases ENPP1 and CD73 into pyrophosphate (PPi) and adenosine, both inhibitors of calcification. PXE, together with General Arterial Calcification of Infancy (GACI caused by ENPP1 mutations) as well as Calcification of Joints and Arteries (CALJA caused by NT5E/CD73 mutations), forms a disease continuum with overlapping phenotypes and shares steps of the same molecular pathway. The explanation of these phenotypes place ABCC6 as an upstream regulator of a purinergic pathway (ABCC6 → ENPP1 → CD73 → TNAP) that notably inhibits mineralization by maintaining a physiological Pi/PPi ratio in connective tissues. Based on a review of the literature and our recent experimental data, we suggest that PXE (and GACI/CALJA) be considered as an authentic “purinergic disease”. In this article, we recapitulate the pathobiology of PXE and review molecular and physiological data showing that, beyond PPi deficiency and ectopic calcification, PXE is associated with wide and complex alterations of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease. Full article

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) regulates skeletal and soft tissue mineralization by hydrolyzing nucleotide triphosphates and cyclic nucleotides, and is involved in the modulation of immune system. In fact, ENPP1 degrades 2′,3′-cyclic GMP-AMP dinucleotide (2′,3′-cGAMP), which is an agonist of surface receptor stimulator of interferon genes (STING), thus downregulating immune response. Consequently, ENPP1 inhibitors are being studied as adjuvant agents in infections and cancer. Pistacia chinensis is a medicinal plant endowed with several biological activities and traditional uses. In the current study, we report the isolation of transilitin (2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-3-methoxychromen-4-one) from the methanolic extract of P. chinensis barks and the investigation of its activity as ENPP1 inhibitor. The compound was tested in vitro against snake venom phosphodiesterase, which is structurally related to ENPP1, and dose-dependently inhibited the enzyme. Moreover, molecular modeling studies were employed to assess the binding motif of the transilitin with the macromolecular target. Our findings support the traditional medical application of P. chinensis and its extracts by shedding new light on the mechanisms underlying their biological action. Full article

Adenosine 5′-triphosphate (ATP) is released in the bladder lumen during filling. Urothelial ATP is presumed to regulate bladder excitability. Urinary ATP is suggested as a urinary biomarker of bladder dysfunctions since ATP is increased in the urine of patients with overactive bladder, interstitial cystitis or bladder pain syndrome. Altered urinary ATP might also be associated with voiding dysfunctions linked to disease states associated with metabolic syndrome. Extracellular ATP levels are determined by ATP release and ATP hydrolysis by membrane-bound and soluble nucleotidases (s-NTDs). It is currently unknown whether s-NTDs regulate urinary ATP. Using etheno-ATP substrate and HPLC-FLD detection techniques, we found that s-NTDs are released in the lumen of ex vivo mouse detrusor-free bladders. Capillary immunoelectrophoresis by ProteinSimple Wes determined that intraluminal solutions (ILS) collected at the end of filling contain ENTPD3 > ENPP1 > ENPP3 ≥ ENTPD2 = NT5E = ALPL/TNAP. Activation of adenylyl cyclase with forskolin increased luminal s-NTDs release whereas the AC inhibitor SQ22536 had no effect. In contrast, forskolin reduced and SQ22536 increased s-NTDs release in the lamina propria. Adenosine enhanced s-NTDs release and accelerated ATP hydrolysis in ILS and lamina propria. Therefore, there is a regulated release of s-NTDs in the bladder lumen during filling. Aberrant release or functions of urothelial s-NTDs might cause elevated urinary ATP in conditions with abnormal bladder excitability. Full article

Cyclic GMP-AMP synthase (cGAS) is an endogenous DNA sensor that synthesizes cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) from ATP and GTP. 2′3′-cGAMP activates the stimulator of interferon genes (STING) pathway, resulting in the production of interferons and pro-inflammatory cytokines. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the phosphodiesterase that negatively regulates the STING pathway by hydrolyzing 2′3′-cGAMP. It has been established that the cGAS–STING pathway plays a major role in inhibiting tumor growth by upregulating T cell response. Herein, we demonstrate that AVA-NP-695, a selective and highly potent ENPP1 inhibitor, apart from the immunomodulatory effect also modulates cancer metastasis by negatively regulating epithelial–mesenchymal transition (EMT). We established that the combined addition of 2′3′-cGAMP and AVA-NP-695 significantly abrogated the transforming growth factor beta (TGF-ꞵ)-induced EMT in MDA-MB-231 cells. Finally, results from the in vivo study showed superior tumor growth inhibition and impact on tumor metastasis of AVA-NP-695 compared to Olaparib and PD-1 in a syngeneic 4T1 breast cancer mouse model. The translation of efficacy from in vitro to in vivo 4T1 tumor model provides a strong rationale for the therapeutic potential of AVA-NP-695 against triple-negative breast cancer (TNBC) as an immunomodulatory and anti-metastatic agent. Full article

It was recently revealed that naturally occurring myricetin can inhibit ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which, in turn, can treat ischemic cardiac injury. However, due to myricetin’s poor druggability, its further developments are relatively limited, which necessitates the discovery of novel ENPP1-inhibiting myricetin analogs as alternatives. In this study, the binding model of myricetin with ENPP1 was elucidated by molecular docking and molecular dynamics studies. Subsequently, virtual screening on the self-developed flavonoid natural product database (FNPD), led to the identification of two flavonoid glycosides (Cas No: 1397173-50-0 and 1169835-58-8), as potential ENPP1 inhibitors. Docking scores and MM/GBSA binding energies predicted that they might have higher inhibitory effects than myricetin. This study provides a strong foundation for the future development of ischemic cardiac injury drugs. Full article

The cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes–TANK-binding kinase 1–interferon regulating factor 3 (cGAS-STING-TBK1-IRF3) axis is now acknowledged as the major signaling pathway in innate immune responses. However, 2′,3′-cGAMP as a STING stimulator is easily recognized and degraded by ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which reduces the effect of tumor immunotherapy and promotes metastatic progression. In this investigation, the structure-based virtual screening strategy was adopted to discover eight candidate compounds containing zinc-binding quinazolin-4(3H)-one scaffold as ENPP1 inhibitors. Subsequently, these novel inhibitors targeting ENPP1 were synthesized and characterized by NMR and high-resolution mass spectra (HRMS). In bioassays, 7-fluoro-2-(((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)methyl)quina-zolin-4(3H)-one(compound 4e) showed excellent activity against the ENPP1 at the molecular and cellular levels, with IC₅₀ values of 0.188 μM and 0.732 μM, respectively. Additionally, compound 4e had superior selectivity towards metastatic breast cancer cells (4T1) than towards normal cells (LO2 and 293T) in comparison with cisplatin, indicating that compound 4e can potentially be used in metastatic breast cancer therapy. On the other hand, compound 4e upgraded the expression levels of IFN-β in vivo by preventing the ENPP1 from hydrolyzing the cGAMP to stimulate a more potent innate immune response. Therefore, this compound might be applied to boost antitumor immunity for cancer immunotherapy. Overall, our work provides a strategy for the development of a promising drug candidate targeting ENPP1 for tumor immunotherapy. Full article

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-β1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5′-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-β1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD. Full article

Autotaxin (ATX) is the only enzyme of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP2) family with lysophospholipase D (lysoPLD) activity, which is mainly responsible for the hydrolysis of extracellular lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA can induce various responses, such as cell proliferation, migration, and cytokine production, through six G protein-coupled receptors (LPA1-6). This signaling pathway is associated with metabolic and inflammatory disorder, and inhibiting this pathway has a positive effect on the treatment of related diseases, while ATX, as an important role in the production of LPA, has been shown to be associated with the occurrence and metastasis of tumors, fibrosis and cardiovascular diseases. From mimics of ATX natural lipid substrates to the rational design of small molecule inhibitors, ATX inhibitors have made rapid progress in structural diversity and design over the past 20 years, and three drugs, GLPG1690, BBT-877, and BLD-0409, have entered clinical trials. In this paper, we will review the structure of ATX inhibitors from the perspective of the transformation of design ideas, discuss the advantages and disadvantages of each inhibitor type, and put forward prospects for the development of ATX inhibitors in the future. Full article

Adult T-cell leukemia/lymphoma (ATL) is an intractable disease affecting nearly 4% of Human T-cell Leukemia Virus Type 1 (HTLV-1) carriers. Acute ATL has a unique interaction with bone characterized by aggressive bone invasion, osteolytic metastasis, and hypercalcemia. We hypothesized that dual tumor and bone-targeted therapies would decrease tumor burden in bone, the incidence of metastasis, and ATL-associated osteolysis. Our goal was to evaluate dual targeting of both ATL bone tumors and the bone microenvironment using an anti-tumor HDACi (AR-42) and an osteoclast inhibitor (zoledronic acid, Zol), alone and in combination. Our results showed that AR-42, Zol, and AR-42/Zol significantly decreased the viability of multiple ATL cancer cell lines in vitro. Zol and AR-42/Zol decreased tumor growth in vivo. Zol ± AR-42 significantly decreased ATL-associated bone resorption and promoted new bone formation. AR-42-treated ATL cells had increased mRNA levels of PTHrP, ENPP2 (autotaxin) and MIP-1α, and TAX viral gene expression. AR-42 alone had no significant effect on tumor growth or osteolysis in mice. These findings indicate that Zol adjuvant therapy has the potential to reduce growth of ATL in bone and its associated osteolysis. Full article