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Myocardial infarction (MI) results in aberrant cardiac metabolism, but no therapeutics have been designed to target cardiac metabolism to enhance heart repair. We engineer a humanized monoclonal antibody against the ectonucleotidase ENPP1 (hENPP1mAb) that targets metabolic crosstalk in the infarcted heart. In mice expressing human ENPP1, systemic administration of hENPP1mAb metabolically reprograms myocytes and non-myocytes and leads to a significant rescue of post-MI heart dysfunction. Using metabolomics, single-nuclear transcriptomics, and cellular respiration studies, we show that the administration of the hENPP1mAb induces organ-wide metabolic and transcriptional reprogramming of the heart that enhances myocyte cellular respiration and decreases cell death and fibrosis in the infarcted heart. Biodistribution and safety studies showed specific organ-wide distribution with the antibody being well tolerated. In humanized animals, with drug clearance kinetics similar to humans, we demonstrate that a single "shot" of the hENPP1mAb after MI is sufficient to rescue cardiac dysfunction., Competing Interests: Declaration of interests The intellectual property associated with hENPP1mAb is held by the Regents, University of California., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid ( Euprymna scolopes ) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri . The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V . fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions., Competing Interests: The authors declare no conflict of interest.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Vascular calcification is a major contributor to cardiovascular disease, especially diabetes, where it exacerbates morbidity and mortality. Although pyrophosphate is a recognized natural inhibitor of vascular calcification, there have been no prior studies examining its specific deficiency in diabetic conditions. This study is the first to analyze the direct link between elevated glucose levels and disruptions in extracellular pyrophosphate metabolism., Methods: Rat aortic smooth muscle cells, streptozotocin (STZ)-induced diabetic rats, and diabetic human aortic smooth muscle cells were used to assess the effects of elevated glucose levels on pyrophosphate metabolism and vascular calcification. The techniques used include extracellular pyrophosphate metabolism assays, thin-layer chromatography, phosphate-induced calcification assays, BrdU incorporation for DNA synthesis, aortic smooth muscle cell viability and proliferation assays, and quantitative PCR for enzyme expression analysis. Additionally, extracellular pyrophosphate metabolism was examined through the use of radiolabeled isotopes to track ATP and pyrophosphate transformations., Results: Elevated glucose led to a significant reduction in extracellular pyrophosphate across all diabetic models. This metabolic disruption was marked by notable downregulation of both the expression and activity of ectonucleotide pyrophosphatase/phosphodiesterase 1, a key enzyme that converts ATP to pyrophosphate. We also observed an upregulation of ectonucleoside triphosphate diphosphohydrolase 1, which preferentially hydrolyzes ATP to inorganic phosphate rather than pyrophosphate. Moreover, tissue-nonspecific alkaline phosphatase activity was markedly elevated across all diabetic models. This shift in enzyme activity significantly reduced the pyrophosphate/phosphate ratio. In addition, we noted a marked downregulation of matrix Gla protein, another inhibitor of vascular calcification. The impaired pyrophosphate metabolism was further corroborated by calcification experiments across all three diabetic models, which demonstrated an increased propensity for vascular calcification., Conclusions: This study demonstrated that diabetes-induced high glucose disrupts extracellular pyrophosphate metabolism, compromising its protective role against vascular calcification. These findings identify pyrophosphate deficiency as a potential mechanism in diabetic vascular calcification, highlighting a new therapeutic target. Strategies aimed at restoring or enhancing pyrophosphate levels may offer significant potential in mitigating cardiovascular complications in diabetic patients, meriting further investigation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O 2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determine six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme containing domain and phosphodiesterase separated by nearly 180 Å. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels., (© 2024. The Author(s).)

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of a potent endogenous calcification inhibitor, plasma inorganic pyrophosphate (PPi); the deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1 that generates PPi and is now in clinical trials, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of Abcc6 -/- mice. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6 -/- mice. When Abcc6 -/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 h. When Abcc6 -/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising second generation enzyme therapy for PXE, the first generation of which is currently in clinical testing., Competing Interests: Declaration of interests D.T.B. and P.R.S. are inventors on patents owned by Yale University for therapeutics treating ENPP1 deficiency. D.T.B. is an equity holder and receives research and consulting support from Inozyme Pharma, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Phosphodiesterases (PDEs) constitute a family of enzymes that play a pivotal role in the regulation of intracellular levels of cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Dysregulation of PDE activity has been implicated in diverse pathological conditions encompassing cardiovascular disorders, pulmonary diseases, and neurological disorders. Small-molecule inhibitors targeting PDEs have emerged as promising therapeutic agents for the treatment of these ailments, some of which have been approved for their clinical use. Despite their success, challenges such as resistance mechanisms and off-target effects persist, urging continuous research for the development of next-generation PDE inhibitors. The objective of this review is to provide an overview of the synthesis and clinical application of representative approved small-molecule PDE inhibitors, with the aim of offering guidance for further advancements in the development of novel PDE inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Excitation/inhibition (E/I) balance plays important roles in mental disorders. Bioactive phospholipids like lysophosphatidic acid (LPA) are synthesized by the enzyme autotaxin (ATX) at cortical synapses and modulate glutamatergic transmission, and eventually alter E/I balance of cortical networks. Here, we analyzed functional consequences of altered E/I balance in 25 human subjects induced by genetic disruption of the synaptic lipid signaling modifier PRG-1, which were compared to 25 age and sex matched control subjects. Furthermore, we tested therapeutic options targeting ATX in a related mouse line. Using EEG combined with TMS in an instructed fear paradigm, neuropsychological analysis and an fMRI based episodic memory task, we found intermediate phenotypes of mental disorders in human carriers of a loss-of-function single nucleotide polymorphism of PRG-1 (PRG-1 R345T/WT ). Prg-1 R346T/WT animals phenocopied human carriers showing increased anxiety, a depressive phenotype and lower stress resilience. Network analysis revealed that coherence and phase-amplitude coupling were altered by PRG-1 deficiency in memory related circuits in humans and mice alike. Brain oscillation phenotypes were restored by inhibtion of ATX in Prg-1 deficient mice indicating an interventional potential for mental disorders., (© 2024. The Author(s).)

DNA-protein crosslinks (DPC) are common DNA lesions induced by various external and endogenous agents. One of the sources of DPC is the apurinic/apyrimidinic site (AP site) and proteins interacting with it. Some proteins possessing AP lyase activity form covalent complexes with AP site-containing DNA without borohydride reduction (suicidal crosslinks). We have shown earlier that tyrosyl-DNA phosphodiesterase 1 (TDP1) but not AP endonuclease 1 (APE1) is able to remove intact OGG1 from protein-DNA adducts, whereas APE1 is able to prevent the formation of DPC by hydrolyzing the AP site. Here we demonstrate that TDP1 can remove intact PARP2 but not XRCC1 from covalent enzyme-DNA adducts with AP-DNA formed in the absence of APE1. We also analyzed an impact of APE1 and TDP1 on the efficiency of DPC formation in APE1 -/- or TDP1 -/- cell extracts. Our data revealed that APE1 depletion leads to increased levels of PARP1-DNA crosslinks, whereas TDP1 deficiency has little effect on DPC formation., (© 2024 International Union of Biochemistry and Molecular Biology.)

Replication stresses are the major source of break-induced replication (BIR). Here, we show that in alternative lengthening of telomeres (ALT) cells, replication stress-induced polyubiquitinated proliferating cell nuclear antigen (PCNA) (polyUb-PCNA) triggers BIR at telomeres and the common fragile site (CFS). Consistently, depleting RAD18, a PCNA ubiquitinating enzyme, reduces the occurrence of ALT-associated promyelocytic leukemia (PML) bodies (APBs) and mitotic DNA synthesis at telomeres and CFS, both of which are mediated by BIR. In contrast, inhibiting ubiquitin-specific protease 1 (USP1), an Ub-PCNA deubiquitinating enzyme, results in an increase in the above phenotypes in a RAD18- and UBE2N (the PCNA polyubiquitinating enzyme)-dependent manner. Furthermore, deficiency of ATAD5, which facilitates USP1 activity and unloads PCNAs, augments recombination-associated phenotypes. Mechanistically, telomeric polyUb-PCNA accumulates SLX4, a nuclease scaffold, at telomeres through its ubiquitin-binding domain and increases telomere damage. Consistently, APB increase induced by Ub-PCNA depends on SLX4 and structure-specific endonucleases. Taken together, our results identified the polyUb-PCNA-SLX4 axis as a trigger for directing BIR., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ENPP1 acts as a negative regulator of the cGAS-STING pathway through the hydrolysis of 2'3'-cGAMP. Inhibitors of ENPP1 are regarded as promising agents for stimulating the immune response in cancer immunotherapy. This study describes the identification and optimization of imidazo[1,2- a ]pyrazine derivative 7 as a highly potent and selective ENPP1 inhibitor. Compound 7 demonstrated substantial inhibitory activity against ENPP1 with an IC 50 value of 5.70 or 9.68 nM while showing weak inhibition against ENPP2 and ENPP3. Furthermore, compound 7 was shown to enhance the mRNA expression of cGAMP-induced STING pathway downstream target genes, such as IFNB1 , CXCL10 , and IL 6. In vivo pharmacokinetic and antitumor studies showed promising results, with 7 not only exhibiting efficient pharmacokinetic properties but also enhancing the antitumor efficacy of the anti-PD-1 antibody. Treatment with 7 (80 mg/kg) combined with anti-PD-1 antibody achieved a tumor growth inhibition rate of 77.7% and improved survival in a murine model.

The emergence of perturbation transcriptomics provides a new perspective for drug discovery, but existing analysis methods suffer from inadequate performance and limited applicability. In this work, we present PertKGE, a method designed to deconvolute compound-protein interactions from perturbation transcriptomics with knowledge graph embedding. By considering multi-level regulatory events within biological systems that share the same semantic context, PertKGE significantly improves deconvoluting accuracy in two critical "cold-start" settings: inferring targets for new compounds and conducting virtual screening for new targets. We further demonstrate the pivotal role of incorporating multi-level regulatory events in alleviating representational biases. Notably, it enables the identification of ectonucleotide pyrophosphatase/phosphodiesterase-1 as the target responsible for the unique anti-tumor immunotherapy effect of tankyrase inhibitor K-756 and the discovery of five novel hits targeting the emerging cancer therapeutic target aldehyde dehydrogenase 1B1 with a remarkable hit rate of 10.2%. These findings highlight the potential of PertKGE to accelerate drug discovery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by unpredictable progression and limited therapeutic options. Current diagnosis relies on high resolution computed tomography (HRCT), which may not adequately capture early signs of deterioration. The enzyme autotaxin (ATX) emerges as a prominently expressed extracellular secretory enzyme in the lungs of IPF patients. The objective of this study was to evaluate the effectiveness of 18 F-labeled ATX-targeted tracer [ 18 F]ATX-1905, in comparison with [ 18 F]FDG, for early fibrosis diagnosis, disease evolution monitoring, and treatment efficacy assessment in bleomycin-induced pulmonary fibrosis (BPF) models. To assess treatment efficacy, mice were treated with two commonly used drugs for IPF, pirfenidone or nintedanib, from Day 9 to Day 23 postbleomycin administration. Lung tissue assessments encompassed inflammation severity via H&E staining, and Ashcroft scoring via Masson staining, alongside quantification of ATX expression through ELISA. Positron emission tomography (PET) imaging employing [ 18 F]FDG and [ 18 F]ATX-1905 tracked disease progression pre- and post-treatment. The extent of pulmonary fibrosis corresponded to changes in ATX expression levels in the BPF mouse model. Notably, [ 18 F]ATX-1905 exhibited elevated uptake in BPF lungs during the progression of the disease, particularly evident at the early stage (Day 9). This uptake was inhibited by an ATX inhibitor, PF-8380, underscoring the specificity of the radiotracer. Conversely, [ 18 F]FDG uptake, peaking at Day 15, decreased subsequently, likely reflective of diminished inflammation. A 2-week treatment regimen using either pirfenidone or nintedanib resulted in notable reductions of ATX expression levels and fibrosis degrees within lung tissues, based on ELISA and Masson staining, as evidenced by PET imaging with [ 18 F]ATX-1905. [ 18 F]FDG uptake also decreased following the treatment period. Additionally, PET/CT imaging extended to a nonhuman primate (NHP) BPF model. The uptake of [ 18 F]ATX-1905 (SUV max = 2.2) was significantly higher than that of [ 18 F]FDG (SUV max = 0.7) in fibrotic lung tissue. Using our novel ATX-specific radiotracer [ 18 F]ATX-1905 and PET/CT imaging, we demonstrated excellent ability in early fibrosis detection, disease monitoring, and treatment assessment within lungs of the BPF mouse models. [ 18 F]ATX-1905 displayed remarkable specificity for ATX expression and high sensitivity for ATX alterations, suggesting its potential for monitoring varying ATX expression in lungs of IPF patients.

For the first time, a synthetic route for preparing lupane and oleanane derivatives with a hydrogenated furan ring as a cycle A of triterpene scaffold is described. Most of the synthesized compounds, furanoterpenoids and their synthetic intermediates, were non-toxic against the tested cancer and non-cancerous cell lines, and evinced significant inhibitory activity with IC 50 1.0-9.0 μM in the tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibition test. Lupane derivatives - 1-oxime 7, 1,10-seco-hydroxynitrile 11 and furanoterpenoid 14 - were selected as those expected to be the most promising compounds. The results of molecular modeling evinced the strongest binding of compound 11 to the active site of Tdp1 compared to the reference drug. Simultaneously, only compound 11 at subtoxic concentration (10 μM) produced a synergetic effect on the topotecan activity against HeLa-V cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Phosphodiesterase 10 A (PDE10A), a pivotal element of the second messenger signaling downstream of the dopamine receptor stimulation, is conceived to be crucially involved in the mood instability of bipolar I disorder (BD-I) as a primary causal factor or in response to dysregulated dopaminergic tone. We aimed to determine whether striatal PDE10A availability is altered in patients with BD-I and assessed its relationship with the clinical characteristics of BD-I. This case-control study used positron emission tomography (PET) with 2-(2-(3-(4-(2-[ 18 F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([ 18 F]MNI-659), a radioligand that binds to PDE10A, to examine the alterations of the striatal PDE10A availability in the living brains of individuals with BD-I and their association with the clinical characteristics of BD-I. [ 18 F]MNI-659 PET data were acquired from 25 patients with BD-I and 27 age- and sex-matched healthy controls. Patients with BD-I had significantly lower PDE10A availability than controls in the executive (F = 8.86; P = 0.005) and sensorimotor (F = 6.13; P = 0.017) subregions of the striatum. Lower PDE10A availability in the executive subregion was significantly associated with a higher frequency of mood episodes in patients with BD-I (r = -0.546; P = 0.007). This study provides the first evidence of altered PDE10A availability in patients with BD-I. Lower PDE10A availability in the executive subregion of the striatum is associated with an increased recurrence risk, suggesting that PDE10A may prevent BD-I relapse. Further studies are required to elucidate the role of PDE10A in BD-I pathophysiology and explore its potential as a treatment target., (© 2024. The Author(s).)

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

We reported that lysophosphatidic acid (LPA) is present at 0.8 μM in mixed human saliva (MS). In this study, we examined the distribution, origin, and enzymatic generation pathways of LPA in MS. LPA was distributed in the medium and cell pellet fraction; a true level of soluble LPA in MS was about 150 nM. The soluble LPA was assumed to be generated by ecto-type lysophospholipase D on exfoliated cells in MS from LPC that originated mainly from the major salivary gland saliva. Our results with the albumin-back extraction procedures suggest that a significant pool of LPA is kept in the outer layer of the plasma membranes of detached oral mucosal cells. Such pool of LPA may contribute to wound healing in upper digestive organs including oral cavity. We obtained evidence that the choline-producing activity in MS was mainly due to Ca 2+ -activated lysophospholipase D activity of glycerophosphodiesterase 7., Competing Interests: Declaration of Competing Interest All authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fertility is a result of a synergy among the sperm's various functions including capacitation, motility, chemotaxis, acrosome reaction, and, finally, the fertilization of the oocyte. Subpar motility is the most common cause of infertility in males. Cyclic adenosine monophosphate (cAMP) signalling underlies motility and is depleted by the phosphodiesterases (PDEs) in sperm, such as PDE10A, PDE1, and PDE4. Therefore, the PDE inhibitor (PDEI) category of fertility drugs aim to enhance motility in assisted reproduction technologies (ARTs) through inhibition of PDEs, though they might have adverse effects on other physiological variables. For example, the popular drug pentoxifylline (PTX), widely used in ARTs, improves motility but causes premature acrosome reaction and exerts toxicity on the fertilized oocyte. Another xanthine-derived drug, theophylline (TP), has been repurposed for treating infertility, but its mechanism of PDE inhibition remains unexplored. Here, using biophysical and computational approaches, we identified that TP binds to the same binding pocket as PTX with higher affinity than PTX. We also found that PTX and TP co-bind to the same binding pocket, but at different sites., Competing Interests: Declaration of competing interest The Authors declare that there are no competing interests associated with the manuscript., (Copyright © 2024. Published by Elsevier B.V.)

Myocardial infarction (MI) triggers a complex inflammatory response that is essential for cardiac repair but can also lead to adverse outcomes if left uncontrolled. Recent studies have highlighted the importance of epigenetic modifications in regulating post-MI inflammation. This study investigated the role of the autotaxin (ATX)/lysophosphatidic acid (LPA) signaling axis in modulating myocardial inflammation through epigenetic pathways in a mouse model of MI. C57BL/6 J mice underwent left anterior descending coronary artery ligation to induce MI and were treated with the ATX inhibitor, PF-8380, or vehicle. Cardiac tissue from the border zone was collected at 6 h, 1, 3, and 7 days post-MI for epigenetic gene profiling using RT 2 Profiler PCR Arrays. The results revealed distinct gene expression patterns across sham, MI + Vehicle, and MI + PF-8380 groups. PF-8380 treatment significantly altered the expression of genes involved in inflammation, stress response, and epigenetic regulation compared to the vehicle group. Notably, PF-8380 downregulated Hdac5, Prmt5, and Prmt6, which are linked to exacerbated inflammatory responses, as early as 6 h post-MI. Furthermore, PF-8380 attenuated the reduction of Smyd1, a gene important in myogenic differentiation, at 7 days post-MI. This study demonstrates that the ATX/LPA signaling axis plays a pivotal role in modulating post-MI inflammation via epigenetic pathways. Targeting ATX/LPA signaling may represent a novel therapeutic strategy to control inflammation and improve outcomes after MI. Further research is needed to validate these findings in preclinical and clinical settings and to elucidate the complex interplay between epigenetic mechanisms and ATX/LPA signaling in the context of MI., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host's innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3'3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3'3'-cGAMP and host-derived 2'3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2'3'-cGAMP. In addition, CpdB has 2'3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The development of drug resistance in cancer cells poses a significant challenge for treatment, with nearly 90% of cancer-related deaths attributed to it. Over 50% of ovarian cancer patients and 30-40% of breast cancer patients exhibit resistance to therapies such as Taxol. Previous literature has shown that cytotoxic cancer therapies and ionizing radiation damage tumors, prompting cancer cells to exploit the autotaxin (ATX)-lysophosphatidic acid (LPA)-lysophosphatidic acid receptor (LPAR) signaling axis to enhance survival pathways, thus reducing treatment efficacy. Therefore, targeting this signaling axis has become a crucial strategy to overcome some forms of cancer resistance. Addressing this challenge, we identified and assessed ATX-1d, a novel compound targeting ATX, through computational methods and in vitro assays. ATX-1d exhibited an IC 50 of 1.8 ± 0.3 μM for ATX inhibition and demonstrated a significant binding affinity for ATX, as confirmed by MM-GBSA, QM/MM-GBSA, and SAPT in silico methods. ATX-1d significantly amplified the potency of paclitaxel, increasing its effectiveness tenfold in 4T1 murine breast carcinoma cells and fourfold in A375 human melanoma cells without inducing cytotoxic effects as a single agent.

Background: Dysregulated lipid oxidation occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the molecular mechanism of lipid oxidation is not well appreciated in liver fibrosis, which is accompanied by enhanced fibroblast proliferation and migration., Methods: We investigated the causes and consequences of lipid oxidation in liver fibrosis using cultured cells, animal models, and clinical samples., Results: Increased ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) expression caused increased lipid oxidation, resulting in the proliferation and migration of hepatic stellate cells (HSCs) that lead to liver fibrosis, whereas fibroblast-specific ENPP1 knockout reversing these results. Elevated ENPP1 and N 6 -methyladenosine (m 6 A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m 6 A methylation of the 3'UTR of ENPP1 mRNA and induces its translation dependent of YTH domain family proteins 1 (YTHDF1). Additionally, ENPP1 could interact with hypoxia inducible lipid droplet associated (HILPDA) directly; overexpression of ENPP1 further recruits HILPDA-mediated lipid oxidation, thereby promotes HSCs proliferation and migration, while inhibition of ENPP1 expression produced the opposite effect. Clinically, increased expression of WTAP, YTHDF1, ENPP1, and HILPDA, and increased m 6 A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues., Conclusions: We describe a novel mechanism in which WTAP catalyzes m 6 A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis., (© 2024. The Author(s).)

Oxygen sensing is of paramount importance for maintaining cellular and systemic homeostasis. In response to diminished oxygen levels, the hypoxia-inducible factors (HIFs) orchestrate various biological processes. These pivotal transcription factors have been identified as key regulators of several biological events. Notably, extensive research from our group and others has demonstrated that HIF1α exerts an inverse regulatory effect on steroidogenesis, leading to the suppression of crucial steroidogenic enzyme expression and a subsequent decrease in steroid levels. These steroid hormones occupy pivotal roles in governing a myriad of physiological processes. Substantial or prolonged fluctuations in steroid levels carry detrimental consequences across multiple organ systems and underlie various pathological conditions, including metabolic and immune disorders. MicroRNAs serve as potent mediators of multifaceted gene regulatory mechanisms, acting as influential epigenetic regulators that modulate a broad spectrum of gene expressions. Concomitantly, phosphodiesterases (PDEs) play a crucial role in governing signal transduction. PDEs meticulously manage intracellular levels of both cAMP and cGMP, along with their respective signaling pathways and downstream targets. Intriguingly, an intricate interplay seems to exist between hypoxia signaling, microRNAs, and PDEs in the regulation of steroidogenesis. This review highlights recent advances in our understanding of the role of microRNAs during hypoxia-driven processes, including steroidogenesis, as well as the possibilities that exist in the application of HIF prolyl hydroxylase (PHD) inhibitors for the modulation of steroidogenesis., (© 2024. The Author(s).)

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value., (© 2024 Federation of European Biochemical Societies.)

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence 1 . Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database 2,3 . Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus-host interactions that are common across the virome., (© 2024. The Author(s).)

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress., (© 2024. The Author(s).)

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present., Competing Interests: Declaration of competing interest DTB is an inventor on patents owned by Yale University for therapeutics treating ENPP1 deficiency and is an equity holder and receives research and consulting support from Inozyme Pharma, Inc. TOC is an advisor and has received consulting support from Inozyme Pharma. Y.S. is an employee of Inozyme Pharma., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cyclic dinucleotides (CDNs) are a significant and expanding class of secondary messengers that influence several vital bacterial physiological functions. Therefore, an understanding of the process by which CDNs are degraded by their cognate PDEs is crucial for comprehending a variety of cellular processes, such as the formation and dissemination of biofilms. As an alternative, it might be beneficial to create and/or identify non-hydrolyzable CDN derivatives to employ them as chemical probes of cyclic-di-GMP (c-di-GMP) signaling. Cyclic-di-inosine monophosphate, or c-di-IMP, is not a naturally occurring signaling molecule in biological systems, but it has strong adjuvant effects on metazoans and functions as an immunological modulator and stimulant. Here we report the first structural details of c-di-IMP and EAL interaction through high-resolution (2.2 Å) crystal structure of VcEAL in complex with c-di-IMP + Ca 2+ . Comparison of the VcEAL structures bound with cyclic-di-GMP (c-di-GMP), 3',5'-cyclic-AMP-GMP (cGAMP) and c-di-IMP and the structural variations at the chemical level between these CDNs provides their structural basis of recognition and rate of hydrolysis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is the dominant hydrolase of 2',3'-cyclic GMP-AMP (cGAMP). Inhibition of ENPP1 contributes to increased cGAMP concentration and stimulator of interferon gene (STING) activation, with the potential to boost immune response against cancer. ENPP1 is a promising therapeutic target in tumor immunotherapy. To date, orally bioavailable ENPP1 inhibitors with highly potent activity under physiological conditions have been rarely reported. Herein, we report our effort in the design and synthesis of two different series of ENPP1 inhibitors, and in the identification of a highly potent ENPP1 inhibitor 27 (IC 50  = 1.2 nM at pH 7.5), which significantly enhanced the cGAMP-mediated STING activity in THP-1 cells. Phosphonate compound 27 has good preclinical pharmacokinetic profiles with low plasma clearance rate in mouse, rat, and dog. It has been developed as bis-POM prodrug 36 which successfully improves the oral bioavailability of 27. In the Pan02 syngeneic mouse model of pancreatic cancer, orally administered 36 showed synergistic effect in combination with radiotherapy., Competing Interests: Declaration of competing interest The authors are all employees of Haihe Biopharma. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Osteoarthritis (OA) is the most common joint disease that usually starts from joint cartilage injury. Notch2, a versatile signaling in human development and diseases, was recently uncovered to be an important regulator in chondrocyte damage. However, in OA chondrocytes, how Notch2 activation is dysregulated is largely unknown. Here, integrated bioinformatic analysis was performed on GEO datasets (GSE199193 and GSE224255) to search potential extracellular vesicles (EVs) derived regulators of Notch2 in OA chondrocytes. Ectonucleoside triphosphate diphosphohydrolase 3 (Entpd3), a most differentially expressed gene both in LPS-induced macrophage EV and Notch2 mutant chondrocytes, was screened as the candidate regulator of Notch2 in OA chondrocytes. Gain-of-function experiments in cultured human chondrocytes revealed that recombinant Entpd3 protein and macrophage EV both had a protective effect on LPS-induced inflammation, oxidative stress, apoptosis, and collagen loss in chondrocytes. In terms of mechanism, Entpd3 directly interacted with ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and suppressed AKT/Notch2-mediated mitochondrial dysfunction. Finally, we verified that either macrophage EV administration or Entpd3 overexpression was able to alleviate osteoarthritis in mice in vivo. In conclusion, Entpd3 is identified as a new regulator in OA, which alleviates mitochondrial dysfunction induced chondrocyte damage via ENPP1-induced suppression of the AKT/Notch2 pathway., (© 2024 Wiley Periodicals LLC.)

Autotaxin (ATX) has emerged as a promising therapeutic target for liver diseases. In this study, we identified potential drug candidates through in silico high-throughput screening. Subsequently, we synthesized a series of small molecules, specifically KR-40795 (2c), a pyrrolidine-2,5-dione-based analogue that binds to the allosteric tunnel and hydrophobic pocket of ATX. This compound was designed to inhibit the enzymatic activity of ATX for the treatment of liver diseases. The inhibitory potency of KR-40795 was evaluated using a biochemical assay that measured the hydrolysis of a specific substrate (FS-3). Notably, KR-40795 demonstrated significant inhibition of both collagen formation and lipid accumulation in liver cells, suggesting its potential as a therapeutic agent for liver diseases, particularly fibrosis and steatosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kwang-seok Oh acknowledges financial support from the National Research Foundation of Korea and the Korea Research Institute of Chemical Technology. Any additional authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Genotoxic stress activates the DNA-damage response (DDR) signalling cascades responsible for maintaining genome integrity. Downstream DNA repair pathways include the tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme that hydrolyses the phosphodiester bond between the tyrosine of topoisomerase I (TopI) and 3'-phosphate of DNA. The plant TDP1 subfamily contains the canonical TDP1α gene and the TDP1β gene whose functions are not fully elucidated. The current study proposes to investigate the involvement of TDP1 genes in DDR-related processes by using Arabidopsis thaliana mutants treated with genotoxic agents. The phenotypic and molecular characterization of tdp1α, tdp1β and tdp1α/β mutants treated with cisplatin (CIS), curcumin (CUR), NSC120686 (NSC), zeocin (ZEO), and camptothecin (CPT), evidenced that while tdp1β was highly sensitive to CIS and CPT, tdp1α was more sensitive to NSC. Gene expression analyses showing upregulation of the TDP2 gene in the double mutant indicate the presence of compensatory mechanisms. The downregulation of POL2A gene in the tdp1β mutant along with the upregulation of the TDP1β gene in pol2a mutants, together with its sensitivity to replication inhibitors (CIS, CTP), point towards a function of this gene in the response to replication stress. Therefore, this study brings novel information relative to the activity of TDP1 genes in plants., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Extracellular vesicles (EVs) are implicated in a multitude of physiological and pathophysiological processes in the nervous system; however, their biogenesis and cargoes are not well defined. Glycerophosphodiester Phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that cleaves the Glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane and has important roles in neurodevelopment and disease-relevant pathways of neuronal survival. We show here that GDE2 regulates the number of small EVs (sEVs) released from the cell surface of neurons via its GPI-anchor cleavage activity and contributes to the loading of protein cargo through enzymatic and non-enzymatic mechanisms. Proteomic profiling reveals that GDE2 releases at least two distinct EV populations, one containing GDE2 itself and the other harboring the putative ectosomal markers CD9 and BSG. sEVs released by GDE2 are enriched in cytoskeletal and actin-remodeling proteins, suggesting a potential mechanism for GDE2-dependent EV release. Further, sEV populations released by GDE2 are enriched in proteins responsible for modulating synaptic activity and proteins that are critical for cellular redox homeostasis. These studies identify GDE2 as a novel regulator of molecularly distinct sEV populations from neurons with potential roles in the synaptic and redox pathways required for neuronal function and survival.

Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA 1 , like that of the phospholipase PLA2G3, the prostaglandin D 2 (PGD 2 ) synthase L-PGDS, or the PGD 2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA 1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD 2 generation, and feedforward ATX-LPA 1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA 1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation., Competing Interests: Declaration of interests J.C. has an employment relationship with Neurocrine Biosciences, Inc. as a Distinguished Scholar, unrelated to the current manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease., (© 2024. The Author(s).)

Radiation enteritis is the most common complication of pelvic radiotherapy, but there is no effective prevention or treatment drug. Apoptotic T cells and their products play an important role in regulating inflammation and maintaining physiological immune homeostasis. Here it is shown that systemically infused T cell-derived apoptotic extracellular vesicles (ApoEVs) can target mice irradiated intestines and alleviate radiation enteritis. Mechanistically, radiation elevates the synthesis of intestinal 2'3' cyclic GMP-AMP (cGAMP) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) proinflammatory pathway. After systemic infusion of ApoEVs, the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) enriches on the surface of ApoEVs hydrolyze extracellular cGAMP, resulting in inhibition of the cGAS-STING pathway activated by irradiation. Furthermore, after ApoEVs are phagocytosed by phagocytes, ENPP1 on ApoEVs hydrolyzed intracellular cGAMP, which serves as an intracellular cGAMP hydrolyzation mode, thereby alleviating radiation enteritis. The findings shed light on the intracellular and extracellular hydrolysis capacity of ApoEVs and their role in inflammation regulation., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Background: PDE10A inhibition represents a potential mechanism for treating schizophrenia. PDE10A inhibitors increase cyclic nucleotides in striatal neurons, thereby mimicking the effects of dopamine receptor D2 antagonists and D1 agonists. We evaluated the PDE10A inhibitor MK-8189 for treating schizophrenia., Methods: Randomized, double-blind, placebo and active-controlled, phase 2a, multicenter, inpatient trial in adults experiencing an acute episode of schizophrenia. Participants were randomized 2:2:1 to once-daily MK-8189 12 mg, placebo, or risperidone 6 mg (active control) for 4-weeks. The primary outcome was change-from-baseline in total score on the Positive and Negative Syndrome Scale (PANSS) at 4 weeks., Results: The number of treated participants was 90 for MK-8189, 89 for placebo, and 45 for risperidone. MK-8189 demonstrated a trend towards improvement versus placebo for change-from-baseline in PANSS total score after 4 weeks (difference = -4.7 [95 % CI: -9.8,0.5], P = 0.074). The active control risperidone was superior to placebo on PANNS total score (difference = -7.3 [95 % CI: -14.0,-0.6], P = 0.033), demonstrating assay sensitivity, while MK-8189 and risperidone did not significantly differ (difference = 2.6 [95 % CI: -4.0,9.2], P = 0.440). MK-8189 had a nominally significant effect on PANSS positive subscale score compared to placebo (difference = -2.2 [95 % CI: -3.8,-0.5], P = 0.011). Discontinuation of MK-8189 treatment due to an adverse event was low (<10 %). Extrapyramidal symptoms occurred with MK-8189 but were mostly mild and transient. Compared with placebo, MK-8189 reduced body weight while risperidone increased weight., Conclusions: These findings suggest that PDE10A inhibition may produce antipsychotic effects and associated weight loss and that further trials with PDE10A inhibitors are warranted., Trial Registration: Clinicaltrials.gov identifier: NCT03055338., Competing Interests: Declaration of competing interest Mukai, Lupinacci, Snow-adami, Voss, Smith, and Egan are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co. Inc., Rahway, NJ, USA (MSD) and own stock/stock options in Merck & Co., Inc., Rahway, NJ, USA. Marder has acted as a consultant for MSD., (Copyright © 2024. Published by Elsevier B.V.)

The complex mechanism of neuropathic pain involves various aspects of both central and peripheral pain conduction pathways. An effective cure for neuropathic pain therefore remains elusive. We found that deficiency of the gene Gdpd3, encoding a lysophospholipase D enzyme, alleviates the inflammatory responses in dorsal root ganglia (DRG) of mice under neuropathic pain and reduces PE (20:4) and PGE2 in DRG. Gdpd3 deficiency had a stronger analgesic effect on neuropathic pain than Celecoxib, a nonsteroidal anti-inflammatory drug. Gdpd3 deficiency also interferes with the polarization of macrophages, switching from M1 towards M2 phenotype. The PPARγ/ FABP4 pathway was screened by RNA sequencing as functional related with Gdpd3 deficient BMDMs stimulated with LPS. Both protein and mRNA levels of PPARγ in GDPD3 deficient BMDMs were higher than those of the litter control mice. However, GW9962 (inhibitor of PPARγ) could reverse the reprogramming polarization of macrophages caused by GDPD3 deficiency. Therefore, our study suggests that GDPD3 deficiency exerts a relieving effect on neuropathic pain and alleviates neuroinflammation in DRG by switching the phenotype of macrophages from M1 to M2, which was mediated through PGE2 and PPARγ/ FABP4 pathway., (© 2024. The Author(s).)

Competing Interests: Conflict of interest: None declared.

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.

Background: Tourette syndrome is a neurodevelopmental movement disorder involving basal ganglia dysfunction. PDE10A inhibitors modulate signaling in the striatal basal ganglia nuclei and are thus of interest as potential therapeutics in treating Tourette syndrome and other movement disorders., Methods: The preclinical pharmacology and toxicology, human safety and tolerability, and human PET striatal enzyme occupancy data for the PDE10A inhibitor EM-221 are presented., Results: EM-221 inhibited PDE10A with an in vitro IC50 of 9 pM and was >100,000 selective vs. other PDEs and other CNS receptors and enzymes. In rats, at doses of 0.05-0.50 mg/kg, EM-221 reduced hyperlocomotion and the disruption of prepulse inhibition induced by MK-801, attenuated conditioned avoidance, and facilitated novel object recognition, consistent with PDE10A's inhibition. EM-221 displayed no genotoxicity and was well tolerated up to 300 mg/kg in rats and 100 mg/kg in dogs. In single- and multiple-day ascending dose studies in healthy human volunteers, EM-221 was well tolerated up to 10 mg, with a maximum tolerated dose of 15 mg. PET imaging indicated that a PDE10A enzyme occupancy of up to 92.8% was achieved with a ~24 h half-life., Conclusions: The preclinical and clinical data presented here support the study of EM-221 in phase 2 trials of Tourette syndrome and other movement disorders.

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase ( ENPP ) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1-6). The ATX-LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX-LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPβS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

Neurodegenerative diseases (NDs) cause progressive loss of neuron structure and ultimately lead to neuronal cell death. Since the available drugs show only limited symptomatic relief, NDs are currently considered as incurable. This review will illustrate the principal roles of the signaling systems of cyclic adenosine and guanosine 3',5'-monophosphates (cAMP and cGMP) in the neuronal functions, and summarize expression/activity changes of the associated enzymes in the ND patients, including cyclases, protein kinases, and phosphodiesterases (PDEs). As the sole enzymes hydrolyzing cAMP and cGMP, PDEs are logical targets for modification of neurodegeneration. We will focus on PDE inhibitors and their potentials as disease-modifying therapeutics for the treatment of Alzheimer's disease, Parkinson's disease, and Huntington's disease. For the overlapped but distinct contributions of cAMP and cGMP to NDs, we hypothesize that dual PDE inhibitors, which simultaneously regulate both cAMP and cGMP signaling pathways, may have complementary and synergistic effects on modifying neurodegeneration and thus represent a new direction on the discovery of ND drugs., (© 2024 Wiley Periodicals LLC.)

Background: The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport., Methods: To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer., Findings: Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer., Interpretation: We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer., Funding: Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute., Competing Interests: Declaration of interests OW: Personal fees from Amgen, Bayer, BMS, Celgene, Daiicgi Sankyo, Eisai, Incyte, Ipsen, Merck, MSD, Novartis, Pierre Fabre, Roche, Servier; honoraria for lectures and/or presentations from Amgen, AstraZeneca, Bayer, BMS, Eisai, Ipsen, MSD, Novartis, Roche, Zentiva; support for attending meetings and/or travel: Abbvie, AstraZeneca, Bayer, BMS, Gilead, Ipsen, Medac, Merck, Pierre Fabre, Roche. SZ: Consultancy and/or speaker’s bureau: Abbvie, BioMarin, Boehringer Ingelheim, Gilead, GSK, Ipsen, Madrigal, Merck/MSD, NovoNordisk, SoBi. JUM: Grants or contracts from any entity, AstraZeneca, consulting fees: AstraZeneca, Roche, Ipsen, Eisai; Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: AstraZeneca, Roche, Ipsen, Eisai. The other authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)