RESUMEN

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.

Asunto(s)

Vesículas Extracelulares , Neuronas , Hidrolasas Diéster Fosfóricas , Animales , Humanos , Ratones , Vesículas Extracelulares/metabolismo , Neuronas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Proteómica/métodos

RESUMEN

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.

Asunto(s)

Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Hidrolasas Diéster Fosfóricas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/metabolismo , ADN de Cadena Simple/metabolismo , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Reparación del ADN

RESUMEN

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 N6-methyladenosine (m6A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m6A 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 m6A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues. CONCLUSIONS: We describe a novel mechanism in which WTAP catalyzes m6A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis.

Asunto(s)

Adenosina , Proliferación Celular , Metabolismo de los Lípidos , Cirrosis Hepática , Oxidación-Reducción , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , ARN Mensajero , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Animales , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proliferación Celular/genética , Metabolismo de los Lípidos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Movimiento Celular/genética , Ratones Endogámicos C57BL , Masculino , Epigénesis Genética , Fibroblastos/metabolismo , Fibroblastos/patología , Metilación , Factores de Empalme de ARN , Proteínas de Ciclo Celular

RESUMEN

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.

Asunto(s)

Hidroxicloroquina , Ratones Noqueados , Prurito , Receptores del Ácido Lisofosfatídico , Animales , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Prurito/inducido químicamente , Prurito/metabolismo , Prurito/genética , Prurito/tratamiento farmacológico , Ratones , Hidroxicloroquina/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Masculino , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Lisofosfolípidos/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Bibliotecas de Moléculas Pequeñas , Animales , Humanos , Estructura Molecular , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad , AMP Cíclico/química , AMP Cíclico/metabolismo , AMP Cíclico/farmacología

RESUMEN

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Asunto(s)

Fosfolipasa D , Proteínas Recombinantes , Venenos de Araña , Animales , Fosfolipasa D/inmunología , Fosfolipasa D/genética , Venenos de Araña/inmunología , Ratones , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Picaduras de Arañas/inmunología , Araña Reclusa Parda/inmunología , Femenino , Antígenos/inmunología , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/inmunología , Anticuerpos Neutralizantes , Antivenenos/inmunología , Antivenenos/administración & dosificación , Modelos Animales de Enfermedad , Inmunización , Hidrolasas Diéster Fosfóricas

RESUMEN

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 Ca2+-activated lysophospholipase D activity of glycerophosphodiesterase 7.

Asunto(s)

Lisofosfatidilcolinas , Lisofosfolípidos , Mucosa Bucal , Hidrolasas Diéster Fosfóricas , Saliva , Humanos , Lisofosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Lisofosfatidilcolinas/metabolismo , Saliva/metabolismo , Saliva/enzimología , Mucosa Bucal/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/enzimología , Masculino , Femenino , Adulto

RESUMEN

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 Cß1 (PLCß1) 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.

Asunto(s)

Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Lisofosfolípidos , Transducción de Señal , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Lisofosfolípidos/metabolismo , Humanos , Ácidos Araquidónicos/metabolismo , Animales , Hidrolasas Diéster Fosfóricas/metabolismo

RESUMEN

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.

Asunto(s)

Proteína Quinasa CDC2 , Reparación del ADN , ADN-Topoisomerasas de Tipo I , Proteínas de Unión al ADN , Endonucleasas , Mitosis , Hidrolasas Diéster Fosfóricas , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/genética , Fosforilación , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Endonucleasas/metabolismo , Endonucleasas/genética , ADN/metabolismo , Células HeLa , Daño del ADN

RESUMEN

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 IC50 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.

Asunto(s)

Relación Dosis-Respuesta a Droga , Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Triterpenos , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Relación Estructura-Actividad , Triterpenos/farmacología , Triterpenos/química , Triterpenos/síntesis química , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/química , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Furanos/farmacología , Furanos/química , Furanos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Modelos Moleculares , Línea Celular Tumoral , Ácido Betulínico

RESUMEN

Lysophosphatidic acid (LPA)-mediated activation of LPA receptor 1 (LPAR1) contributes to the pathophysiology of fibrotic diseases such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). These diseases are associated with high morbidity and mortality despite current treatment options. The LPA-producing enzyme autotaxin (ATX) and LPAR1 activation contribute to inflammation and mechanisms underlying fibrosis in preclinical fibrotic models. Additionally, elevated levels of LPA have been detected in bronchoalveolar lavage fluid from patients with IPF and in serum from patients with SSc. Thus, ATX and LPAR1 have gained considerable interest as pharmaceutical targets to combat fibrotic disease and inhibitors of these targets have been investigated in clinical trials for IPF and SSc. The goals of this review are to summarise the current literature on ATX and LPAR1 signalling in pulmonary fibrosis and to help differentiate the novel inhibitors in development. The mechanisms of action of ATX and LPAR1 inhibitors are described and preclinical studies and clinical trials of these agents are outlined. Because of their contribution to numerous physiologic events underlying fibrotic disease, ATX and LPAR1 inhibition presents a promising therapeutic strategy for IPF, SSc and other fibrotic diseases that may fulfil unmet needs of the current standard of care.

Asunto(s)

Fibrosis Pulmonar Idiopática , Hidrolasas Diéster Fosfóricas , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/metabolismo , Animales , Transducción de Señal/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Terapia Molecular Dirigida , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Pulmón/metabolismo , Antifibróticos/uso terapéutico , Lisofosfolípidos/metabolismo , Resultado del Tratamiento , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/fisiopatología , Inhibidores de Fosfodiesterasa/uso terapéutico

RESUMEN

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.

Asunto(s)

Pentoxifilina , Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Espermatozoides , Teofilina , Pentoxifilina/farmacología , Pentoxifilina/química , Pentoxifilina/metabolismo , Teofilina/farmacología , Teofilina/química , Teofilina/metabolismo , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Masculino , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/metabolismo , Humanos , Sitios de Unión , Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular

RESUMEN

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.

Asunto(s)

Inmunidad Innata , Hidrolasas Diéster Fosfóricas , Infecciones por Yersinia pseudotuberculosis , Yersinia pseudotuberculosis , Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/genética , Animales , Ratones , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Nucleótidos Cíclicos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Fosfatos de Dinucleósidos/metabolismo , Femenino , GMP Cíclico/análogos & derivados

RESUMEN

Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.

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Title: Les phosphodiestérases des nucléotides cycliques - Cibles thérapeutiques dans l'hypertrophie et l'insuffisance cardiaques. Abstract: Les phosphodiestérases des nucléotides cycliques (PDE) modulent la régulation neuro-hormonale de la fonction cardiaque en dégradant l'AMPc et le GMPc. Dans les cardiomyocytes, de multiples isoformes de PDE, aux propriétés enzymatiques et aux localisations subcellulaires différentes, régulent localement les niveaux de nucléotides cycliques et les fonctions cellulaires associées. Cette organisation est fortement perturbée au cours de l'hypertrophie et de l'insuffisance cardiaque à fraction d'éjection réduite (IC), ce qui peut contribuer à la progression de la maladie. Sur le plan clinique, l'inhibition des PDE a été considérée comme une approche prometteuse pour compenser la désensibilisation aux catécholamines qui accompagne l'IC. Bien que des inhibiteurs de la PDE3, tels que la milrinone ou l'énoximone, puissent être utilisés cliniquement pour améliorer la fonction systolique et soulager les symptômes de l'IC aiguë, leur utilisation chronique s'est avérée préjudiciable. D'autres PDE, telles que les PDE1, PDE2, PDE4, PDE5, PDE9 et PDE10, sont apparues comme de nouvelles cibles potentielles pour le traitement de l'IC, chacune ayant un rôle unique dans les voies de signalisation locales des nucléotides cycliques. Dans cette revue, nous décrivons la signalisation de l'AMPc et du GMPc dans les cardiomyocytes et présentons les différentes familles de PDE exprimées dans le cœur ainsi que leurs modifications dans l'hypertrophie cardiaque pathologique et dans l'IC. Nous évaluons également les résultats issus de modèles précliniques ainsi que les données cliniques indiquant l'utilisation d'inhibiteurs ou d'activateurs de PDE spécifiques qui pourraient avoir un potentiel thérapeutique dans l'IC.

Asunto(s)

Cardiomegalia , Insuficiencia Cardíaca , Inhibidores de Fosfodiesterasa , Humanos , Cardiomegalia/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Animales , Inhibidores de Fosfodiesterasa/uso terapéutico , Inhibidores de Fosfodiesterasa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Terapia Molecular Dirigida/métodos , GMP Cíclico/metabolismo , GMP Cíclico/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/fisiología

RESUMEN

Topotecan administered intraperitoneally at single doses of 0.25, 0.5, and 1 mg/kg induced chromosomal aberrations in bone marrow cells of F1(CBA×C57BL/6) hybrid mice in a dose-dependent manner. A tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitor, an usnic acid derivative OL9-116 was inactive in a dose range of 20-240 mg/kg, but enhanced the cytogenetic effect of topotecan (0.25 mg/kg) at a dose of 40 mg/kg (per os). The TDP1 inhibitor, a coumarin derivative TX-2552 (at doses of 20, 40, 80, and 160 mg/kg per os), increased the level of aberrant metaphases induced by topotecan (0.25 mg/kg) by 2.1-2.6 times, but was inactive at a dose of 10 mg/kg. The results indicate that TDP1 inhibitors enhance the clastogenic activity of topotecan in mouse bone marrow cells in vivo and are characterized by different dose profiles of the co-mutagenic effects.

Asunto(s)

Células de la Médula Ósea , Hidrolasas Diéster Fosfóricas , Topotecan , Animales , Topotecan/farmacología , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Masculino , Aberraciones Cromosómicas/efectos de los fármacos , Aberraciones Cromosómicas/inducido químicamente , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Topoisomerasa I/farmacología , Ratones Endogámicos C57BL , Mutágenos/toxicidad

RESUMEN

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.

Asunto(s)

Hidrolasas Diéster Fosfóricas , Síndrome de Tourette , Adulto , Animales , Perros , Femenino , Humanos , Masculino , Ratas , Trastornos del Movimiento/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/metabolismo , Tomografía de Emisión de Positrones , Ratas Sprague-Dawley , Síndrome de Tourette/tratamiento farmacológico , Haplorrinos

RESUMEN

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

Asunto(s)

5'-Nucleotidasa , Adenosina , Proliferación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Transducción de Señal , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Animales , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Adenosina/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Ratones , Humanos , Adenosina Monofosfato/metabolismo , Ratones Endogámicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/genética

RESUMEN

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.

Asunto(s)

Lisofosfolípidos , Neoplasias , Hidrolasas Diéster Fosfóricas , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Lisofosfolípidos/metabolismo , Animales , Transducción de Señal , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo

RESUMEN

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 LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 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 LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 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.

Asunto(s)

Anafilaxia , Fibroblastos , Lisofosfolípidos , Mastocitos , Ratones Noqueados , Comunicación Paracrina , Hidrolasas Diéster Fosfóricas , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Animales , Mastocitos/inmunología , Mastocitos/metabolismo , Anafilaxia/inmunología , Anafilaxia/metabolismo , Ratones , Fibroblastos/metabolismo , Lisofosfolípidos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Prostaglandina D2/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Oxidorreductasas Intramoleculares/genética , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/genética , Diferenciación Celular , Ratones Endogámicos C57BL , Proteína 1 Similar al Receptor de Interleucina-1 , Lipocalinas

RESUMEN

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.

Asunto(s)

Antipsicóticos , Hidrolasas Diéster Fosfóricas , Pirimidinas , Risperidona , Esquizofrenia , Compuestos de Azufre , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Enfermedad Aguda , Antipsicóticos/farmacología , Antipsicóticos/efectos adversos , Antipsicóticos/administración & dosificación , Método Doble Ciego , Evaluación de Resultado en la Atención de Salud , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/metabolismo , Prueba de Estudio Conceptual , Pirimidinas/efectos adversos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Risperidona/farmacología , Risperidona/efectos adversos , Risperidona/administración & dosificación , Esquizofrenia/tratamiento farmacológico , Compuestos de Azufre/efectos adversos , Compuestos de Azufre/farmacología , Compuestos de Azufre/uso terapéutico