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1.
Immunity ; 55(6): 1032-1050.e14, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35704993

RESUMO

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase , Cinurenina , Animais , Células Dendríticas , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Camundongos , Transdução de Sinais , Triptofano/metabolismo
2.
Nature ; 563(7731): 354-359, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30356218

RESUMO

Nicotinamide adenine dinucleotide (NAD+) is a co-substrate for several enzymes, including the sirtuin family of NAD+-dependent protein deacylases. Beneficial effects of increased NAD+ levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-ß-carboxymuconate-ε-semialdehyde in the de novo NAD+ synthesis pathway, controls cellular NAD+ levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD+ synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD+ levels, sirtuin activity and mitochondrial homeostasis in kidney and liver.


Assuntos
Carboxiliases/metabolismo , Sequência Conservada , Evolução Molecular , Saúde , Mitocôndrias/fisiologia , NAD/biossíntese , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Carboxiliases/deficiência , Linhagem Celular , Colina , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Masculino , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Ratos , Sirtuínas/metabolismo
3.
Int J Mol Sci ; 24(3)2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36768761

RESUMO

The mineralocorticoid receptor (MR) belongs to the steroid receptor subfamily of nuclear receptors. MR is a transcription factor key in regulating blood pressure and mineral homeostasis. In addition, it plays an important role in a broad range of biological and pathological conditions, greatly expanding its interest as a pharmacological target. Non-steroidal MR antagonists (MRAs) are of particular interest to avoid side effects and achieve tissue-specific modulation of the receptor. The 1,4-dihydropyridine (1,4-DHP) ring has been identified as an appropriate scaffold to develop non-steroidal MRAs. We report the identification of a novel series of 1,4-DHP that has been guided by structure-based drug design, focusing on the less explored DHP position 2. Interestingly, substituents at this position might interfere with MR helix H12 disposition, which is essential for the recruitment of co-regulators. Several of the newly synthesized 1,4-DHPs show interesting properties as MRAs and have a good selectivity profile. These 1,4-DHPs promote MR nuclear translocation with less efficiency than the natural agonist aldosterone, which explains, at least in part, its antagonist character. Molecular dynamic studies are suggestive of several derivatives interfering with the disposition of H12 in the agonist-associated conformation, and thus, they might stabilize an MR conformation unable to recruit co-activators.


Assuntos
Di-Hidropiridinas , Antagonistas de Receptores de Mineralocorticoides , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Receptores de Mineralocorticoides , Di-Hidropiridinas/farmacologia , Di-Hidropiridinas/química , Aldosterona/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico
4.
Gastroenterology ; 159(3): 956-968.e8, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485177

RESUMO

BACKGROUND & AIMS: Renewal and patterning of the intestinal epithelium is coordinated by intestinal stem cells (ISCs); dietary and metabolic factors provide signals to the niche that control ISC activity. Bile acids (BAs), metabolites in the gut, signal nutrient availability by activating the G protein-coupled bile acid receptor 1 (GPBAR1, also called TGR5). TGR5 is expressed in the intestinal epithelium, but it is not clear how its activation affects ISCs and regeneration of the intestinal epithelium. We studied the role of BAs and TGR5 in intestinal renewal, and regulation of ISC function in mice and intestinal organoids. METHODS: We derived intestinal organoids from wild-type mice and Tgr5-/- mice, incubated them with BAs or the TGR5 agonist INT-777, and monitored ISC function by morphologic analyses and colony-forming assays. We disrupted Tgr5 specifically in Lgr5-positive ISCs in mice (Tgr5ISC-/- mice) and analyzed ISC number, proliferation, and differentiation by flow cytometry, immunofluorescence, and organoid assays. Tgr5ISC-/- mice were given cholecystokinin; we measured the effects of BA release into the intestinal lumen and on cell renewal. We induced colitis in Tgr5ISC-/- mice by administration of dextran sulfate sodium; disease severity was determined based on body weight, colon length, and histopathology analysis of colon biopsies. RESULTS: BAs and TGR5 agonists promoted growth of intestinal organoids. Administration of cholecystokinin to mice resulted in acute release of BAs into the intestinal lumen and increased proliferation of the intestinal epithelium. BAs and Tgr5 expression in ISCs were required for homeostatic intestinal epithelial renewal and fate specification, and for regeneration after colitis induction. Tgr5ISC-/- mice developed more severe colitis than mice without Tgr5 disruption in ISCs. ISCs incubated with INT-777 increased activation of yes-associated protein 1 (YAP1) and of its upstream regulator SRC. Inhibitors of YAP1 and SRC prevented organoid growth induced by TGR5 activation. CONCLUSIONS: BAs promote regeneration of the intestinal epithelium via activation of TGR5 in ISCs, resulting in activation of SRC and YAP and activation of their target genes. Release of endogenous BAs in the intestinal lumen is sufficient to promote ISC renewal and drives regeneration in response to injury.


Assuntos
Células-Tronco Adultas/fisiologia , Ácidos e Sais Biliares/metabolismo , Colite/patologia , Mucosa Intestinal/patologia , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/fisiologia , Células Cultivadas , Ácidos Cólicos/farmacologia , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Organoides , Cultura Primária de Células , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas de Sinalização YAP , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
5.
Org Biomol Chem ; 19(24): 5403-5412, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34056641

RESUMO

The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxycholic acid and employed as chemical tools to exploit the importance of the orientation and spatial disposition of the carboxyl tail and the C25-methyl group for the binding at the DAF-12 receptor. The synthesis route was based on (a) Walden inversion and stereoselective PtO2-hydrogenation to convert the L-shaped 5ß-cholanoid scaffold into the planar 5α-sterol intermediate; (b) two-carbon homologation of the side chain by Wittig and cyclopropanation reaction; and (c) formation of the 3-keto group and Δ7 double bond. The synthesized isomers were isolated and tested for their activity as DAF-12 ligands by AlphaScreen assays. Results showed a significant loss of potency and efficacy for all the four stereoisomers when compared to the parent endogenous ligand. Computational analysis has evidenced the configurational and conformational arrangement of both the carboxylic and the C25-methyl group of dafachronic acids as key structural determinants for DAF-12 binding and activation.

6.
Mol Pharmacol ; 98(4): 343-349, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32764096

RESUMO

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.


Assuntos
Bactérias/química , Produtos Biológicos/química , Indóis/farmacologia , Descoberta de Drogas , Humanos , Indóis/química , Ligantes , Mimetismo Molecular
7.
Biochim Biophys Acta ; 1844(10): 1765-72, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25062913

RESUMO

The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

8.
Clin Chem ; 61(7): 955-63, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25931455

RESUMO

BACKGROUND: 3ß-Hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3ß-hydroxy-Δ(5)-bile acids. These atypical bile acids are highly labile and difficult to accurately measure, yet a method for accurate determination of 3ß-hydroxy-Δ(5)-bile acid sulfates is critical for dose titration and monitoring response to therapy. METHODS: We describe a electrospray ionization LC-MS/MS method for the direct measurement of atypical 3ß-hydroxy-Δ(5)-bile acid sulfates in urine from patients with HSD3B7 deficiency that overcomes the deficiencies of previously used GC-MS methods. RESULTS: Separation of sulfated 3ß-hydroxy-Δ(5)-bile acids was achieved by reversed-phase HPLC in a 12-min analytical run. The mean (SE) urinary concentration of the total 3ß-sulfated-Δ(5)-cholenoic acids in patients with HSD3B7 deficiency was 4650 (1711) µmol/L, approximately 1000-fold higher than in noncholestatic and cholestatic patients with intact primary bile acid synthesis. GC-MS was not reliable for measuring 3ß-hydroxy-Δ(5)-bile acid sulfates; however, direct analysis of urine by fast atom bombardment mass spectrometry yielded meaningful semiquantitative assessment of urinary excretion. CONCLUSIONS: The tandem mass spectrometry method described here for the measurement of 3ß-hydroxy-Δ(5)-bile acid sulfates in urine can be applied to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients.


Assuntos
3-Hidroxiesteroide Desidrogenases/deficiência , Ácidos e Sais Biliares/urina , Espectrometria de Massas em Tandem/métodos , Urinálise/métodos , 3-Hidroxiesteroide Desidrogenases/genética , Ácidos e Sais Biliares/metabolismo , Criança , Pré-Escolar , Colestase/urina , Ácido Cólico/uso terapêutico , Ácidos Cólicos/urina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Recém-Nascido , Limite de Detecção , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/urina , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Sulfatos/química
9.
J Cell Mol Med ; 18(3): 468-79, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24444146

RESUMO

Activation of poly(ADP-ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper-reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma-like events to airway damage. Ovalbumin-sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma-like signs, bronchial hyper-reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1-10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen-induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ-treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8-hydroxy-2'-deoxyguanosine and the lung content of pro-inflammatory cytokines (TNF-α, interleukin (IL)-1ß, IL-5, IL-6 and IL-18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ-treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Alérgenos/imunologia , Asma/tratamento farmacológico , Asma/fisiopatologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Isoquinolinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases , Tiofenos/uso terapêutico , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Cobaias , Liberação de Histamina , Inflamação/patologia , Isoquinolinas/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ovalbumina/imunologia , Estresse Oxidativo/efeitos dos fármacos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Tiofenos/farmacologia
10.
J Pharmacol Exp Ther ; 350(1): 56-68, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24784847

RESUMO

We report on the relationship between the structure-pharmacokinetics, metabolism, and therapeutic activity of semisynthetic bile acid analogs, including 6α-ethyl-3α,7α-dihydroxy-5ß-cholan-24-oic acid (a selective farnesoid X receptor [FXR] receptor agonist), 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid (a specific Takeda G protein-coupled receptor 5 [TGR5] receptor agonist), and 6α-ethyl-3α,7α-dihydroxy-24-nor-5ß-cholan-23-sulfate (a dual FXR/TGR5 agonist). We measured the main physicochemical properties of these molecules, including ionization constants, water solubility, lipophilicity, detergency, and protein binding. Biliary secretion and metabolism and plasma and hepatic concentrations were evaluated by high-pressure liquid chromatography-electrospray-mass spectrometry/mass spectrometry in bile fistula rat and compared with natural analogs chenodeoxycholic, cholic acid, and taurochenodexycholic acid and intestinal bacteria metabolism was evaluated in terms of 7α-dehydroxylase substrate-specificity in anaerobic human stool culture. The semisynthetic derivatives detergency, measured in terms of their critical micellar concentration, was quite similar to the natural analogs. They were slightly more lipophilic than the corresponding natural analogs, evaluated by their 1-octanol water partition coefficient (log P), because of the ethyl group in 6 position, which makes these molecules very stable toward bacterial 7-dehydroxylation. The hepatic metabolism and biliary secretion were different: 6α-ethyl-3α,7α-dihydroxy-5ß-cholan-24-oic acid, as chenodeoxycholic acid, was efficiently conjugated with taurine in the liver and, only in this form, promptly and efficiently secreted in bile. 6α-Ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid was poorly conjugated with taurine because of the steric hindrance of the methyl at C23(S) position metabolized to the C23(R) isomer and partly conjugated with taurine. Conversely, 6α-ethyl-3α,7α-dihydroxy-24-nor-5ß-cholan-23-sulfate was secreted in bile unmodified and as 3-glucuronide. Therefore, minor structural modifications profoundly influence the metabolism and biodistribution in the target organs where these analogs exert therapeutic effects by interacting with FXR and/or TGR5 receptors.


Assuntos
Ácidos e Sais Biliares/farmacocinética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Acoplados a Proteínas G/agonistas , Animais , Bactérias/metabolismo , Bile/química , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/sangue , Fenômenos Químicos , Humanos , Fígado/metabolismo , Ratos
11.
Org Biomol Chem ; 12(47): 9592-600, 2014 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-25338931

RESUMO

An efficient method for the C3-glucuronidation of bile acids is developed under flow conditions. A modular mesoreactor assisted flow set-up was combined with statistical design of experiments to speed up the optimization of the Koenigs-Knorr reaction in terms of yield, regioselectivity, costs, as well as technical and practical standpoints. Using the optimal conditions, selective glucuronidation of naturally occurring bile acids was successfully achieved offering a new, valuable route to C3-glucuronidated bile acids useful for biological, diagnostic and PK/ADMET investigations.


Assuntos
Ácidos e Sais Biliares/química , Glucuronídeos/química , Ácidos e Sais Biliares/síntese química , Técnicas de Química Sintética/instrumentação , Desenho de Equipamento , Glucuronídeos/síntese química , Estereoisomerismo
12.
J Med Chem ; 67(5): 4150-4169, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38417155

RESUMO

The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure-activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3 as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.


Assuntos
Strongyloides stercoralis , Estrongiloidíase , Animais , Humanos , Estrongiloidíase/tratamento farmacológico , Estrongiloidíase/parasitologia , Strongyloides stercoralis/metabolismo , Esteroides/uso terapêutico , Estágios do Ciclo de Vida , Relação Estrutura-Atividade
13.
J Physiol ; 591(9): 2307-18, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23507881

RESUMO

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 µm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.


Assuntos
Antidiarreicos/farmacologia , Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Ácido Ursodesoxicólico/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ácidos e Sais Biliares/metabolismo , Colo/citologia , Colo/fisiologia , Células Epiteliais/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Bloqueadores dos Canais de Potássio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
14.
Bioorg Med Chem ; 21(13): 3780-9, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23684233

RESUMO

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.


Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Tiazepinas/química , Tiazepinas/farmacologia , Humanos , Ligação Proteica , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Estereoisomerismo , Tiazepinas/síntese química
15.
Molecules ; 18(9): 10497-513, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23999724

RESUMO

Bile acids have emerged as versatile signalling compounds of a complex network of nuclear and membrane receptors regulating various endocrine and paracrine functions. The elucidation of the interconnection between the biological pathways under the bile acid control and manifestations of hepatic and metabolic diseases have extended the scope of this class of steroids for in vivo investigations. In this framework, the design and synthesis of novel biliary derivatives able to modulate a specific receptor requires a deep understanding of both structure-activity and structure-property relationships of bile acids. In this paper, we report the preparation and the critical micellization concentration evaluation of a series of hyodeoxycholic acid derivatives characterized by a diverse side chain length and by the presence of a methyl group at the alpha position with respect to the terminal carboxylic acid moiety. The data collected are instrumental to extend on a quantitative basis, the knowledge of the current structure-property relationships of bile acids and will be fruitful, in combination with models of receptor activity, to design and prioritize the synthesis of novel pharmacokinetically suitable ligands useful in the validation of bile acid-responsive receptors as therapeutic targets.


Assuntos
Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/síntese química , Acetilação , Ácidos e Sais Biliares/síntese química , Micelas , Modelos Moleculares , Estrutura Molecular , Relação Quantitativa Estrutura-Atividade
16.
Eur J Med Chem ; 261: 115851, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-37813065

RESUMO

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.


Assuntos
Ácidos e Sais Biliares , Receptores Acoplados a Proteínas G , Ácido Cólico/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade , Ácidos e Sais Biliares/farmacologia , Estrutura Molecular
17.
Eur J Med Chem ; 247: 115022, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36549114

RESUMO

After over 30 years of research, the development of HDAC inhibitors led to five FDA/Chinese FDA-approved drugs and many others under clinical or preclinical investigation to treat cancer and non-cancer diseases. Herein, based on our recent development of pyridine-based isomers as HDAC inhibitors, we report a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2'-aminoanilides 5-8 as anticancer agents. The hydroxamate 5d proved to be quite HDAC3/6-selective exhibiting IC50 values of 80 and 11 nM, respectively, whereas the congener 5e behaved as inhibitor of HDAC1-3, -6, -8, and -10 (class I/IIb-selective inhibitor) at nanomolar level. Compound 5e provided a huge antiproliferative activity (nanomolar IC50 values) against both haematological and solid cancer cell lines. In leukaemia U937 cells, the hydroxamate 5d and the 2'-aminoanilide 8f induced remarkable cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, showing a stronger effect with respect to SAHA and MS-275 used as reference compounds. In U937 cells, the highest dose- and time-dependent cytodifferentiation was obtained by the 2'-aminoanilide 8d (up to 35% of CD11c positive/propidium iodide negative cells at 5 µM for 48 h). The same 8d and the hydroxamates 5d and 5e were the most effective in inducing p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f increased mRNA expression of p21, BAX and BAK, downregulated cyclin D1 and BCL-2 and modulated pro- and anti-apoptotic microRNAs towards apoptosis induction. Finally, 5e strongly arrested proliferation in nine different haematological cancer cell lines, with dual-digit nanomolar potency towards MV4-11, Kasumi-1, and NB4, being more potent than mocetinostat, used as reference drug.


Assuntos
Antineoplásicos , MicroRNAs , Neoplasias , Humanos , Inibidores de Histona Desacetilases/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Antineoplásicos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Apoptose , Piridinas/farmacologia , Histona Desacetilase 1
18.
Org Biomol Chem ; 10(20): 4109-15, 2012 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-22517166

RESUMO

A multi-gram scale protocol for the N-acyl amidation of bile acids with glycine and taurine has been successfully developed under continuous flow processing conditions. Selecting ursodeoxycholic acid (UDCA) as the model compound and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as the condensing agent, a modular mesoreactor assisted flow set-up was employed to significantly speed up the optimization of the reaction conditions and the flow scale-up synthesis. The results in terms of yield, in line purification, analysis, and implemented flow set-up for the reaction optimization and large scale production are reported and discussed.


Assuntos
Ácidos e Sais Biliares/química , Glicina/síntese química , Taurina/síntese química , Aminação , Humanos , Estrutura Molecular
19.
Bioorg Med Chem ; 20(11): 3429-45, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22564381

RESUMO

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 µM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.


Assuntos
Pirazóis/química , Receptores Citoplasmáticos e Nucleares/agonistas , Relação Estrutura-Atividade , Tiazepinas/síntese química , Tiazepinas/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Interface Usuário-Computador
20.
Front Mol Biosci ; 9: 834700, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35463964

RESUMO

Human α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) stands at a branch point of the de novo NAD+ synthesis pathway and plays an important role in maintaining NAD+ homeostasis. It has been recently identified as a novel therapeutic target for a wide range of diseases, including inflammatory, metabolic disorders, and aging. So far, in absence of potent and selective enzyme inhibitors, only a crystal structure of the complex of human dimeric ACMSD with pseudo-substrate dipicolinic acid has been resolved. In this study, we report the crystal structure of the complex of human dimeric ACMSD with TES-1025, the first nanomolar inhibitor of this target, which shows a binding conformation different from the previously published predicted binding mode obtained by docking experiments. The inhibitor has a K i value of 0.85 ± 0.22 nM and binds in the catalytic site, interacting with the Zn2+ metal ion and with residues belonging to both chains of the dimer. The results provide new structural information about the mechanism of inhibition exerted by a novel class of compounds on the ACMSD enzyme, a novel therapeutic target for liver and kidney diseases.

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