Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway.

In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)-2'3'-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which thecGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling.

The metazoan innate immune second messenger 2'3'-cGAMP is present both inside and outside cells. However, only extracellular cGAMP can be negatively regulated by the extracellular hydrolase ENPP1. Here, we determine whether ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating stimulator of interferon genes (STING) signaling. We identified ENPP1H362A, a point mutation that cannot degrade the 2'-5' linkage in cGAMP while maintaining otherwise normal function. The selectivity of this histidine is conserved down to bacterial nucleotide pyrophosphatase/phosphodiesterase (NPP), allowing structural analysis and suggesting an unexplored ancient history of 2'-5' cyclic dinucleotides. Enpp1H362A mice demonstrated that extracellular cGAMP is not responsible for the devastating phenotype in ENPP1-null humans and mice but is responsible for antiviral immunity and systemic inflammation. Our data define extracellular cGAMP as a pivotal STING activator, identify an evolutionarily critical role for ENPP1 in regulating inflammation, and suggest a therapeutic strategy for viral and inflammatory conditions by manipulating ENPP1 activity.

Natural history of ENPP1 deficiency: Nationwide Turkish Cohort Study of autosomal-recessive hypophosphataemic rickets type 2.

OBJECTIVE: Autosomal-recessive hypophosphataemic rickets type 2 (ARHR2) is a rare disease that is reported in survivors of generalized arterial calcification of infancy (GACI). DESIGN, PATIENTS AND MEASUREMENT: The objective of this study was to characterize a multicenter paediatric cohort with ARHR2 due to ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) deficiency and with a diagnosis of GACI or GACI-related findings. The clinical, biochemical and genetic characteristics of the patients were retrospectively retrieved. RESULTS: We identified 18 patients from 13 families diagnosed with ARHR2. Fifteen of the patients had an ENPP1 variation confirmed with genetic analyses, and three were siblings of one of these patients, who had clinically diagnosed hypophosphataemic rickets (HRs) with the same presentation. From nine centres, 18 patients, of whom 12 (66.7%) were females, were included in the study. The mean age at diagnosis was 4.2 ± 2.2 (1.6-9) years. The most frequently reported clinical findings on admission were limb deformities (66.6%) and short stature (44.4%). At diagnosis, the mean height SD was -2.2 ± 1.3. Five of the patients were diagnosed with GACI in the neonatal period and treated with bisphosphonates. Other patients were initially diagnosed with ARHR2, but after the detection of a biallelic variant in the ENPP1 gene, it was understood that they previously had clinical findings associated with GACI. Three patients had hearing loss, and two had cervical fusion. After the treatment of HRs, one patient developed calcification, and one developed intimal proliferation. CONCLUSION: ARHR2 represents one manifestation of ENPP1 deficiency that usually manifests later in life than GACI. The history of calcifications or comorbidities that might be associated with GACI will facilitate the diagnosis in patients with ARHR2, and patients receiving calcitriol and phosphate medication should be carefully monitored for signs of calcification or intimal proliferation.

[1,2,4]Triazolo[1,5-a]pyrimidine derivatives: Structure-activity relationship study leading to highly selective ENPP1 inhibitors.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.

Second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP): the cell autonomous and non-autonomous roles in cancer progression.

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy.

ENPP1 in the Regulation of Mineralization and Beyond.

ENPP1 is well known for its role in regulating skeletal and soft tissue mineralization. It primarily exerts its function through the generation of pyrophosphate, a key inhibitor of hydroxyapatite formation. Several previous studies have suggested that ENPP1 also contributes to a range of human diseases including diabetes, cancer, cardiovascular disease, and osteoarthritis. In this review, we summarize the pathological roles of ENPP1 in mineralization and these soft tissue disorders. We also discuss the underlying mechanisms through which ENPP1 exerts its pathological effects. A fuller understanding of the pathways through which ENPP1 acts may help to develop novel therapeutic strategies for these commonly diagnosed morbidities.

Genetics of Diffuse Idiopathic Skeletal Hyperostosis and Ossification of the Spinal Ligaments.

PURPOSE OF REVIEW: The study aims to provide updated information on the genetic factors associated with the diagnoses 'Diffuse Idiopathic Skeletal Hyperostosis' (DISH), 'Ossification of the Posterior Longitudinal Ligament' (OPLL), and in patients with spinal ligament ossification. RECENT FINDINGS: Recent studies have advanced our knowledge of genetic factors associated with DISH, OPLL, and other spinal ossification (ossification of the anterior longitudinal ligament [OALL] and the yellow ligament [OYL]). Several case studies of individuals afflicted with monogenic disorders, such as X-linked hypophosphatemia (XLH), demonstrate the strong association of fibroblast growth factor 23-related hypophosphatemia with OPLL, suggesting that pathogenic variants in PHEX, ENPP1, and DMP1 are associated with FGF23-phosphate wasting phenotype and strong genetic factors placing patients at risk for OPLL. Moreover, emerging evidence demonstrates that heterozygous and compound heterozygous ENPP1 pathogenic variants inducing 'Autosomal Recessive Hypophosphatemic Rickets Type 2' (ARHR2) also place patients at risk for DISH and OPLL, possibly due to the loss of inhibitory plasma pyrophosphate (PPi) which suppresses ectopic calcification and enthesis mineralization. Our findings emphasize the importance of genetic and plasma biomarker screening in the clinical evaluation of DISH and OPLL patients, with plasma PPi constituting an important new biomarker for the identification of DISH and OPLL patients whose disease course may be responsive to ENPP1 enzyme therapy, now in clinical trials for rare calcification disorders.

Role of ENPP1 Gene Variants in the Susceptibility to Diabetic Nephropathy in Patients with type 2 Diabetes Mellitus.

Genetic factors are known to play a significant role in the susceptibility of diabetic patients to severe complications such as diabetic nephropathy (DN). This study aimed to evaluate the association between polymorphism of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) variants (rs997509, K121Q, rs1799774, and rs7754561) and DN in patients with type 2 diabetes mellitus (T2DM). A total number of 492 patients with T2DM with and without DN were categorized into case and control groups. The extracted DNA samples were genotyped using TaqMan allelic discrimination assay amplified by polymerase chain reaction (PCR). The haplotype analysis among the case and control groups was performed using an expectation-maximization algorithm by the maximum-likelihood method. The analysis of laboratory findings demonstrated significant differences in fasting blood sugar (FBS) and hemoglobin A1c (HbA1c) between the case and control groups (P < 0.05). The results showed that K121Q was significantly related to DN under a recessive model of inheritance (P = 0.006); however, rs1799774 and rs7754561 both were protective for DN under a dominant model of inheritance (P = 0.034 and P = 0.010, respectively) among four studied variants. Two haplotypes, including C-C-delT-G with a frequency < 0.02 and T-A-delT-G with a frequency < 0.01, were associated with the increased risk of DN (P < 0.05). The present study demonstrated that K121Q was associated with the susceptibility of DN; however, rs1799774 and rs7754561 were protecrtive variants for DN in patients with T2DM.

Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice.

The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 µg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.

ENPP1 deficiency: A clinical update on the relevance of individual variants using a locus-specific patient database.

Loss-of-function variants in the ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) cause ENPP1 Deficiency, a rare disorder characterized by pathological calcification, neointimal proliferation, and impaired bone mineralization. The consequence of ENPP1 Deficiency is a broad range of age dependent symptoms and morbidities including cardiovascular complications and 50% mortality in infants, autosomal recessive hypophosphatemic rickets type 2 (ARHR2) in children, and joint pain, osteomalacia and enthesopathies in adults. Recent research continues to add to the growing clinical presentation profile as well as expanding the role of ENPP1 itself. Here we review the current knowledge on the spectrum of clinical and genetic findings of ENPP1 Deficiency reported in patients diagnosed with GACI or ARHR2 phenotypes using a comprehensive database of known ENPP1 variants with associated clinical data. A total of 108 genotypes were identified from 154 patients. Of the 109 ENPP1 variants reviewed, 72.5% were demonstrably disease-causing, a threefold increase in pathogenic/likely pathogenic variants over other databases. There is substantial heterogeneity in disease severity, even among patients with the same variant. The approach to creating a continuously curated database of ENPP1 variants accessible to clinicians is necessary to increase the diagnostic yield of clinical genetic testing and accelerate diagnosis of ENPP1 Deficiency.

Mutation update: Variants of the ENPP1 gene in pathologic calcification, hypophosphatemic rickets, and cutaneous hypopigmentation with punctate keratoderma.

ENPP1 encodes ENPP1, an ectonucleotidase catalyzing hydrolysis of ATP to AMP and inorganic pyrophosphate (PPi), and an endogenous plasma protein physiologically preventing ectopic calcification of connective tissues. Mutations in ENPP1 have been reported in association with a range of human genetic diseases. In this mutation update, we provide a comprehensive review of all the pathogenic variants, likely pathogenic variants, and variants of unknown significance in ENPP1 associated with three autosomal recessive disorders-generalized arterial calcification of infancy (GACI), autosomal recessive hypophosphatemic rickets type 2 (ARHR2), and pseudoxanthoma elasticum (PXE), as well as with a predominantly autosomal dominant disorder-Cole disease. The classification of all variants is determined using the latest ACMG guidelines. A total of 140 ENPP1 variants were curated consisting of 133 previously reported variants and seven novel variants, with missense variants being the most prevalent (70.0%, 98/140). While the pathogenic variants are widely distributed in the ENPP1 gene of patientsgen without apparent genotype-phenotype correlation, eight out of nine variants associated with Cole disease are confined to the somatomedin-B-like (SMB) domains critical for homo-dimerization of the ENPP1 protein.

Hyperpigmentation in a Chinese family with autosomal dominant Cole disease.

Cole disease (OMIM 615522), caused by mutations in ENPP1, is a rare autosomal dominant or recessive genodermatosis characterized by guttate hypopigmentation and punctate palmoplantar keratoderma. To date, a few cases with autosomal recessive inheritance had been reported with hyperpigmentation. The aim of this case report was to investigate the molecular basis of individuals with hyperpigmentation, hypopigmentation and punctate keratoderma in a Chinese family. A Chinese pedigree of suspected Cole disease with hyperpigmentation was subjected to mutation detection in the ENPP1 gene. All exons of the ENPP1 gene and adjacent exon-intron border sequences were amplified using polymerase chain reaction and directly sequenced. Three-dimensional (3D) models of the wild-type and mutated ENPP1 proteins were predicted by PyMOL viewer. Both of the proband and his affected father carried a heterozygous missense mutation p.C176R in ENPP1. In silico modelling of the ENPP1 wild-type and ENPP1 with the p.C176R mutation showed the residue Arg-176 disturbed the fold of the loop conformation. Based on clinical and genetic findings, a diagnosis of Cole disease was made. We identified a heterozygous mutation, p.C176R, in the ENPP1 gene in a Chinese family with Cole disease. This study clearly showed that hyperpigmentation could also occur in Cole disease in cases with autosomal dominant inheritance. Our data expand the phenotypic spectrum of ENPP1 mutations underlying Cole disease.

An Unusual Case of Transient Cardiac Calcification Identified on Antenatal Echocardiography: A Generalized Arterial Calcification of Infancy (GACI) Like Presentation.

Generalised arterial calcification of infancy (GACI) is an ultra-rare life-threatening genetic disorder. Arterial calcification is identified during foetal ultrasound scan (USS) as increased cardiac and/or vascular echogenicity. Inorganic pyrophosphate (PPi) is the main inhibitor of arterial calcification. Pathogenic variants in ENPP1, ABCC6 and NT5E causing low PPi lead to ectopic calcifications. Rheumatoid arthritis (RA) is an acquired condition that can also lead to arterial calcification in adults. We present an extremely rare case of a transient GACI-like condition identified during foetal echocardiogram of an infant born to a mother diagnosed with RA, which spontaneously resolved postnatally. This case highlights that foetal ultrasound scans of pregnant women with RA should be carefully evaluated for cardiovascular calcifications.

Discovery of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one and 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one derivatives as novel ENPP1 inhibitors.

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) negatively regulates the anti-cancer Stimulator of Interferon Genes (STING) pathway. We discovered that 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one and 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one derivatives possessed inhibitory activities on ENPP1. A structure-activity relationship (SAR) study led to the identification of 46 and 23 as potent ENPP1 inhibitors. Also, compounds 46 and 23 possessed high microsomal stabilities in human, rat, and mouse liver microsome. Additionally, CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) were not inhibited by 46 and 23. Molecular dynamics simulations provided an insight of binding modes between ENPP1 and compounds (46 and 23).

Design, synthesis and biological evaluation studies of novel small molecule ENPP1 inhibitors for cancer immunotherapy.

Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents.

Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors.

In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found 18p possessed high potency (IC50 = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN-ß and IP-10 were induced by 18p in a concentration dependent manner. Finally, we discovered that 18p causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.

Histone Deacetylase Inhibitors Downregulate Calcium Pyrophosphate Crystal Formation in Human Articular Chondrocytes.

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)-ß1-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-ß1. 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.

Development of Novel Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Tumor Immunotherapy.

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-ß 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.

From Myricetin to the Discovery of Novel Natural Human ENPP1 Inhibitors: A Virtual Screening, Molecular Docking, Molecular Dynamics Simulation, and MM/GBSA Study.

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.