RESUMEN
Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.
Asunto(s)
Arginasa/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/fisiología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Transducción de Señal/inmunología , Animales , Arginasa/metabolismo , Arginina/inmunología , Arginina/metabolismo , Western Blotting , Células Dendríticas/metabolismo , Femenino , Perfilación de la Expresión Génica , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma , Triptófano/inmunología , Triptófano/metabolismoRESUMEN
Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) accounts for the catabolism of the endogenous inhibitors of nitric oxide (NO) synthases, namely, ADMA (Nω,Nω-dimethyl-l-arginine) and NMMA (Nω-monomethyl-l-arginine). Inhibition of DDAH-1 may prove a therapeutic benefit in diseases associated with elevated nitric oxide (NO) levels by providing a tissue-specific increase of ADMA and NMMA. In this work, we have used molecular dynamics to generate a pool of DDAH-1 conformations in the apo and holo forms. Ensemble docking has been instrumental in screening an in-house fragment-based library of 824 compounds. Resulting virtual hits have been validated for their binding activity to recombinant human DDAH-1 using microscale thermophoresis (MST). As a key result, three non-amino acidic ligands of DDAH-1 (VIS212, VIS268, VIS726) are identified with higher binding efficiency index than ADMA. Amid these compounds, purpurogallin (VIS726) proves a potent ligand of DDAH-1, showing a mixed behavior of enzymatic inhibition in a biochemical assay. This finding widens the panel of known molecular targets of purpurogallin and provides clues into the molecular mechanisms of its cellular NO inhibition activity as well as its anti-inflammatory and neuroprotective effects.
RESUMEN
A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.
RESUMEN
Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.
Asunto(s)
Antivirales , Productos Biológicos , Ácido Elágico , SARS-CoV-2 , Replicación Viral , Antivirales/farmacología , Productos Biológicos/farmacología , Ácido Elágico/farmacología , Compuestos Heterocíclicos/farmacología , Ligandos , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.
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Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Biocatálisis , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Quinurenina/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Ratones Noqueados , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Triptófano/metabolismoRESUMEN
PD-1/PD-L1 protein complex is attracting a great deal of interest as a drug target for the design of immune therapies able to block its assembly. Although some biologic drugs have entered clinical use, their poor response rate in patients are demanding further efforts to design small molecule inhibitors of PD-1/PD-L1 complex with higher efficacy and optimal physicochemical properties. Dysregulation of pH in the tumor microenvironment is indeed one of the key mechanisms promoting drug resistance and lack of response in cancer therapy. Integrating computational and biophysical approaches, herein we report a screening campaign that has led to identifying VIS310 as a novel ligand of PD-L1, with physicochemical properties enabling a pH-dependent binding potency. Additional optimization efforts by analogue-based screening have been instrumental to disclosing VIS1201, which exhibits improved binding potency against PD-L1 and is able to inhibit PD-1/PD-L1 complex formation in a ligand binding displacement assay. While providing preliminary structure-activity relationships (SARs) of a novel class of PD-L1 ligands, our results lay the foundation for the discovery of immunoregulatory small molecules resilient to tumor microenvironmental conditions for escaping drug-resistance mechanisms.
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Antígeno B7-H1 , Microambiente Tumoral , Humanos , Antígeno B7-H1/metabolismo , Ligandos , Receptor de Muerte Celular Programada 1/metabolismo , Concentración de Iones de HidrógenoRESUMEN
The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Antivirales/farmacología , Antivirales/química , Simulación del Acoplamiento MolecularRESUMEN
Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.
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Proteínas Portadoras , Imagen por Resonancia Magnética , Humanos , Preparaciones Farmacéuticas , Espectroscopía de Resonancia Magnética , Proteínas Portadoras/química , Cristalografía por Rayos XRESUMEN
Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa , Fosfatidilinositol 3-Quinasas , Células Dendríticas/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inflamación , Fosfatidilinositol 3-Quinasas/genética , Transducción de SeñalRESUMEN
Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.
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Inhibidores Enzimáticos , Indolamina-Pirrol 2,3,-Dioxigenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ligandos , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral-chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.
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Aminoácidos , Antifibrinolíticos , Suplementos Dietéticos , Ácido Acético , Etanol , AguaRESUMEN
SLC26A5 transporter prestin is fundamental for the higher hearing sensitivity and frequency selectivity of mammals. Prestin is a voltage-dependent transporter found in the cochlear outer hair cells responsible for their electromotility. Intracellular chloride binding is considered essential for voltage sensitivity and electromotility. Prestin is composed by a transmembrane domain and by a cytosolic domain called STAS. There is evidence of a calcium/calmodulin regulation of prestin mediated by the STAS domain. Using different biophysical techniques, namely SEC, CD, ITC, MST, NMR and SAXS, here we demonstrate and characterize the direct interaction between calmodulin and prestin STAS. We show that the interaction is calcium-dependent and that involves residues at the N-terminal end of the "variable loop". This is an intrinsically disordered insertion typical of the STAS domains of the SLC26 family of transporters whose function is still unclear. We derive a low-resolution model of the STAS/CaM complex, where only one lobe of calmodulin is engaged in the interaction, and build a model for the entire dimeric prestin in complex with CaM, which can use the unoccupied lobe to interact with other regions of prestin or with other regulatory proteins. We show that also a non-mammalian STAS can interact with calmodulin via the variable loop. These data start to shed light on the regulatory role of the STAS variable loop of prestin.
Asunto(s)
Calmodulina/metabolismo , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Calmodulina/química , Pollos , Cromatografía en Gel , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Conformación Proteica , Dominios Proteicos , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) - the enzyme catalyzing the rate-limiting step of tryptophan catabolism along the kynurenine pathway - belongs to the class of inhibitory immune checkpoint molecules. Such regulators of the immune system are crucial for maintaining self-tolerance and thus, when properly working, preventing autoimmunity. A dysfunctional IDO1 has recently been associated with a specific single nucleotide polymorphism (SNP) and with the occurrence of autoimmune diabetes and multiple sclerosis. Many genetic alterations of IDO1 have been proposed being related with dysimmune disorders. However, the molecular and functional meaning of variations in IDO1 exomes as well as the promoter region remains a poorly explored field. In the present study, we identified a rare missense variant (rs751360195) at the IDO1 gene in a patient affected by coeliac disease, thyroiditis, and selective immunoglobulin A deficiency. Molecular and functional studies demonstrated that the substitution of lysine (K) at position 257 with a glutamic acid (E) results in an altered IDO1 protein that undergoes a rapid protein turnover. This genotype-to-phenotype relation is produced by peripheral blood mononuclear cells (PBMCs) of the patient bearing this variation and is associated with a specific phenotype (i.e., impaired tryptophan catabolism and defective mechanisms of immune tolerance). Thus decoding functional mutations of the IDO1 exome may provide clinically relevant information exploitable to personalize therapeutic interventions.
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Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Síndromes Mielodisplásicos/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Análisis Mutacional de ADN , Exones/genética , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Simulación de Dinámica Molecular , Mutación Missense , Síndromes Mielodisplásicos/inmunología , ProteolisisRESUMEN
Leukotrienes (LTs) are proinflammatory mediators derived from arachidonic acid (AA), which play significant roles in inflammatory diseases. The 5-lipoxygenase-activating protein (FLAP) is an integral membrane protein, which is essential for the initial step in LT biosynthesis. The aim of this study was to discover novel and chemically diverse FLAP inhibitors for treatment of inflammatory diseases requiring anti-LT therapy. Both ligand- and structure-based approaches were applied to explain the activities of known FLAP inhibitors in relation to their predicted binding modes. We gained valuable insights into the binding modes of the inhibitors by molecular modeling and generated a multistep virtual screening (VS) workflow in which 6.2 million compounds were virtually screened, and the molecular hypotheses were validated by testing VS-hit compounds biologically. The most potent hit compounds showed significant inhibition of FLAP-dependent cellular LT biosynthesis with IC50 values in the range from 0.13 to 0.87 µM. Collectively, this study provided novel bioactive chemotypes with potential for further development as effective anti-inflammatory drugs.
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Leucotrienos , Inhibidores de la Lipooxigenasa , Proteínas Activadoras de la 5-Lipooxigenasa , Antiinflamatorios , Inhibidores de la Lipooxigenasa/farmacología , Modelos MolecularesRESUMEN
The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.
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Ácidos Carboxílicos/síntesis química , Diseño de Fármacos , Ácidos Carboxílicos/química , Bases de Datos Factuales , Modelos Moleculares , Estructura MolecularRESUMEN
Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.
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Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Resistencia a la Enfermedad/efectos de los fármacos , Endotoxemia/genética , Endotoxemia/inmunología , Endotoxemia/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inflamación/enzimología , Inflamación/genética , Inflamación/metabolismo , Quinurenina/metabolismo , Lipopolisacáridos/farmacología , Ratones , Fosforilación , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal , Triptófano Oxigenasa/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
In the present work, we illustrate the ability of high-performance liquid chromatography (HPLC) analysis to assist the synthesis of chiral imidazolines within our medicinal chemistry programs. In particular, a Chiralpak® IB® column containing cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto a 5 µm silica gel was used for the enantioselective HPLC analysis of chiral imidazolines synthesized in the frame of hit-to-lead explorations and designed for exploring the effect of diverse amide substitutions. Very profitably, reversed-phase (RP) conditions succeeded in resolving the enantiomers in nine out of the 10 investigated enantiomeric pairs, with α values always higher than 1.10 and RS values up to 2.31. All compounds were analysed with 50% (v) water while varying the content of the two organic modifiers acetonitrile and methanol. All the employed eluent systems were buffered with 40 mM ammonium acetate while the apparent pH was fixed at 7.5. Based on the experimental results, the prominent role of π-π stacking interactions between the substituted electron-rich phenyl groups outside of the polymeric selector and the complementary aromatic region in defining analyte retention and stereodiscrimination was identified. The importance of compound polarity in explaining the retention behaviour with the employed RP system was readily evident when a quantitative structure-property relationship study was performed on the retention factor values (k) of the 10 compounds, as computed with a 30% (v) methanol containing mobile phase. Indeed, good Pearson correlation coefficients of retention factors (r - log k1st = -0.93; r - log k2nd = -0.94) were obtained with a water solubility descriptor (Ali-logS). Interestingly, a n-hexane/chloroform/ethanol (88:10:2, v/v/v)-based non-standard mobile phase allowed the almost base-line enantioseparation (α = 1.06; RS = 1.26) of the unique compound undiscriminated under RP conditions.
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Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Imidazolinas , Celulosa/química , Estructura MolecularRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAFV600E- and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAFV600E-expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors.
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Regulación Enzimológica de la Expresión Génica , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Factor de Transcripción STAT1/metabolismo , Neoplasias de la Tiroides/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Mutación Missense , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Ratas , Factor de Transcripción STAT1/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Microambiente Tumoral/genéticaRESUMEN
The enantiomers of trans-paroxetine (the selectand) were separated on four chiral stationary phases incorporating either quinine [ZWIX(+), ZWIX(+A)] or quinidine [ZWIX(-), ZWIX(-A)] and (R,R)-aminocyclohexanesulfonic acid [in ZWIX(-), and ZWIX(+A)] or (S,S)-aminocyclohexanesulfonic acid [in ZWIX(+), and ZWIX(-A)] chiral selectors. The zwitterion nature of the phases is due to the presence of either (R,R)- or (S,S)-aminocyclohexanesulfonic acid in the selector structure bearing the quinuclidine moiety. ZWIX(+) and ZWIX(-) phases are available on the market with the commercial names CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-), respectively. With the aim of rationalizing the enantiomer elution order with the above chiral stationary phases, a molecular dynamic protocol was applied and two energetic parameters were initially measured: selectand conformational energy and selectand interaction energy. In the search for other descriptors allowing a better fitting with the experimental evidences, in the present work we consider an energetic parameter, defined as the selector conformational energy, which resulted to be relevant in the explanation of the experimental elution order in most of the cases. Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analytes.
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Alcaloides de Cinchona/química , Paroxetina/aislamiento & purificación , Modelos Moleculares , Conformación Molecular , Paroxetina/química , EstereoisomerismoRESUMEN
The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment.