RESUMO
Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.
Assuntos
Adenocarcinoma de Pulmão , Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Invasividade Neoplásica , Receptor Notch3 , Análise de Célula Única , Células Estromais , Microambiente Tumoral , Humanos , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
One of the central challenges for cancer therapy is the identification of factors in the tumor microenvironment that increase tumor progression and immune tolerance. In breast cancer, fibrosis is a histopathologic criterion for invasive cancer and poor survival that results from inflammatory factors and remodeling of the extracellular matrix to produce an immune tolerant microenvironment. To determine whether tolerance is associated with the immune checkpoint, Programmed Cell Death 1 (PD-1), NeuT/ATTAC mice, a conditional model of mammary fibrosis that we recently developed, were administered a murine-specific anti-PD-1 mAb related to pembrolizumab, and drug response was monitored by tumor development, imaging mass cytometry, immunohistochemistry and tumor gene expression by RNAseq. Tumor progression in NeuT/ATTAC mice was unaffected by weekly injection of anti-PD-1 over four months. Insensitivity to anti-PD-1 was associated with several processes, including increased tumor-associated macrophages (TAM), epithelial to mesenchymal transition (EMT), fibroblast proliferation, an enhanced extracellular matrix and the Wnt signaling pathway, including increased expression of Fzd5, Wnt5a, Vimentin, Mmp3, Col2a1 and Tgfß1. These results suggest potential therapeutic avenues that may enhance PD-1 immune checkpoint sensitivity, including the use of tumor microenvironment targeted agents and Wnt pathway inhibitors.
Assuntos
Antineoplásicos , Neoplasias , Camundongos , Animais , Via de Sinalização Wnt , Transição Epitelial-Mesenquimal , Antineoplásicos/farmacologia , Macrófagos , Microambiente Tumoral , Linhagem Celular TumoralRESUMO
Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. IMPLICATIONS: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.
Assuntos
Melanoma/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Receptores Imunológicos/imunologia , Animais , Feminino , Xenoenxertos , Humanos , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Monócitos/metabolismo , Células Supressoras Mieloides/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Imidazóis/química , Inibidores de Proteínas Quinases/química , Pirazinas/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Sítios de Ligação , Compostos Bicíclicos com Pontes/química , Cristalografia por Raios X , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Meia-Vida , Humanos , Imidazóis/metabolismo , Imidazóis/uso terapêutico , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/metabolismo , Pirazinas/uso terapêutico , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.
Assuntos
Fibroblastos Associados a Câncer/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias Pulmonares/imunologia , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Espécies Reativas de Oxigênio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Terapia de Imunossupressão , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , NADPH Oxidase 2/imunologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/imunologia , NADPH Oxidase 4/metabolismo , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Glucocorticoides/administração & dosagem , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoconjugados/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Linfócitos B/efeitos dos fármacos , Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , Fluticasona/administração & dosagem , Humanos , Camundongos , Camundongos Transgênicos , Receptores de Glucocorticoides/agonistasRESUMO
8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Descoberta de Drogas , Imidazóis/farmacologia , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Artrite Reumatoide/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imidazóis/síntese química , Imidazóis/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/metabolismoRESUMO
We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.
Assuntos
Ácidos Carboxílicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Humanos , RatosRESUMO
In an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel phosphate bridged Cathepsin B sensitive linker was developed to enable the targeted delivery of glucocorticoids. Phosphate bridging of the Cathepsin B sensitive linkers allows for payload attachment at an aliphatic alcohol. As small molecule drug-linkers, these aqueous soluble phosphate containing drug-linkers were found to have robust plasma stability coupled with rapid release of payload in a lysosomal environment. Site-specific ADCs were successfully made between these drug-linkers and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. These ADCs demonstrated in vitro targeted delivery of glucocorticoids to a representative cell line as measured by changes in glucocorticoid receptor (GR) mediated gene mRNA levels. This novel linker expands the scope of potential ADC payloads by allowing an aliphatic alcohol to be a stable, yet cleavable attachment site. This phosphate linker may have broad utility for internalizing ADCs as well as other targeted delivery platforms.
Assuntos
Catepsina B/metabolismo , Imunoconjugados/química , Imunoconjugados/metabolismo , Fosfatos/química , Água/química , Álcoois/química , Carbonatos/química , Estabilidade de Medicamentos , Humanos , Lisossomos/metabolismo , SolubilidadeRESUMO
Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.
RESUMO
As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.
Assuntos
Difosfatos/química , Glucocorticoides/química , Imunoconjugados/química , ÉsteresRESUMO
Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked ß-N-acetylglucosamine transferase (OGT), which adds an O-linked ß-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.
Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Piranos/farmacologia , Receptores de Glucocorticoides/metabolismo , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , Prednisolona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Benzamidas/uso terapêutico , Dermatite de Contato/tratamento farmacológico , Edema/tratamento farmacológico , Indazóis/uso terapêutico , Receptores de Glucocorticoides/metabolismo , Animais , Anti-Inflamatórios/sangue , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Benzamidas/sangue , Benzamidas/farmacocinética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Colágeno , Citocinas/sangue , Cães , Feminino , Células HeLa , Humanos , Indazóis/sangue , Indazóis/farmacocinética , Indazóis/farmacologia , Insulina , Lipopolissacarídeos , Masculino , Metilprednisolona/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Reatores Biológicos , Receptores de Glucocorticoides/agonistas , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Linhagem Celular , Citocromo P-450 CYP3A/metabolismo , Remoção de Radical Alquila , Dermatite/tratamento farmacológico , Feminino , Glucocorticoides/metabolismo , Humanos , Insetos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismoRESUMO
The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.
Assuntos
Coração/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Eplerenona , Coração/fisiopatologia , Hipertensão/sangue , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Losartan/farmacologia , Losartan/uso terapêutico , Masculino , Ratos , Ratos Endogâmicos SHR , Cloreto de Sódio na Dieta/farmacologia , Cloreto de Sódio na Dieta/uso terapêutico , Espironolactona/análogos & derivadosRESUMO
INTRODUCTION: Mineralocorticoid receptor (MR) antagonists are useful for the treatment of hypertension and heart failure. In the present work we sought to develop a simplified protocol for measuring the acute activity of MR antagonists on urinary excretion of sodium and potassium in rats based on the original studies of mineralocorticoids in adrenalectomized rats reported by Kagawa et al. (Kagawa, C. M., & Jacobs Jr., R. S. (1960) Mineralocorticoid effects of 9 alphafluorodeoxycorticosterone in adrenalectomized rats. Proceedings of the Society for Experimental Biology and Medicine, 104, 60-62). METHODS: Rats with intact adrenal glands were treated with test compounds and challenged with a bolus oral dose of saline. Urine was collected over 4 h in metabolism cages and urinary sodium and potassium concentrations were measured. RESULTS: Aldosterone had no significant effect on sodium or potassium excretion and MR antagonists dose-dependently increased the ratio of sodium to potassium. Diuretics with distinct mechanisms of action were differentiated via their relative effects on sodium, potassium and urine volumes and the new assay protocol was used to characterize a novel MR antagonist. DISCUSSION: A facile and robust protocol for the measurement of antagonism of renal MRs was established. This protocol used fewer animals than previously described methods and did not require preparative surgery, factors which contributed favorably to cost, experimental throughput and animal use.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Antagonistas de Receptores de Mineralocorticoides , Aldosterona/farmacologia , Amilorida/farmacologia , Análise de Variância , Animais , Creatinina/urina , Diuréticos/farmacologia , Relação Dose-Resposta a Droga , Eplerenona , Hidroclorotiazida/farmacologia , Imidazóis/farmacologia , Masculino , Potássio/urina , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/metabolismo , Sódio/urina , Espironolactona/análogos & derivados , Espironolactona/farmacologiaRESUMO
Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.
Assuntos
D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Análise Serial de Tecidos/métodos , Animais , Automação , Células CHO , Cricetinae , Humanos , Modelos Biológicos , TransfecçãoRESUMO
Lithium inhibits inositol monophosphatase at therapeutically effective concentrations, and it has been hypothesized that depletion of brain inositol levels is an important chemical alteration for lithium's therapeutic efficacy in bipolar disorder. We have employed adult rat cortical slices as a model to investigate the gene regulatory consequences of inositol depletion effected by lithium using cytidine diphosphoryl-diacylglycerol as a functionally relevant biochemical marker to define treatment conditions. Genes coding for the neuropeptide hormone pituitary adenylate cyclase activating polypeptide (PACAP) and the enzyme that processes PACAP's precursor to the mature form, peptidylglycine alpha-amidating monooxygenase, were upregulated by inositol depletion. Previous work has shown that PACAP can increase tyrosine hydroxylase (TH) activity and dopamine release, and we found that the gene for GTP cyclohydrolase, which effectively regulates TH through synthesis of tetrahydrobiopterin, was also upregulated by inositol depletion. We propose that modulation of brain PACAP signaling might represent a new opportunity in the treatment of bipolar disorder.
Assuntos
Antimaníacos/farmacologia , Biopterinas/análogos & derivados , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inositol/metabolismo , Cloreto de Lítio/farmacologia , Animais , Biomarcadores/metabolismo , Biopterinas/metabolismo , Transtorno Bipolar/metabolismo , Córtex Cerebral/fisiopatologia , Diglicerídeos de Citidina Difosfato/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Masculino , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Fatores de Crescimento Neural/biossíntese , Neuropeptídeos/biossíntese , Neurotransmissores/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/biossíntese , Regulação para Cima/genéticaRESUMO
Inositol monophosphatase is a potential drug target for developing lithium-mimetic agents for the treatment of bipolar disorder. Enzyme-based assays have been traditionally used in compound screening to identify inositol monophosphatase inhibitors. A cell-based screening assay in which the compound needs to cross the cell membrane before reaching the target enzyme offers a new approach for discovering novel structure leads of the inositol monophosphatase inhibitor. The authors have recently reported a high-throughput measurement of G-protein-coupled receptor activation by determining inositol phosphates in cell extracts using scintillation proximity assay. This cell-based assay has been modified to allow the determination of inositol monophosphatase activity instead of G-protein-coupled receptors. The enzyme is also assayed in its native form and physiological environment. The authors have applied this cell-based assay to the high-throughput screening of a large compound collection and identified several novel inositol monophosphatase inhibitors.
Assuntos
Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Atropina/farmacologia , Bioensaio/métodos , Carbacol/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Modelos Biológicos , Contagem de CintilaçãoRESUMO
Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.