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1.
Gastroenterology ; 153(4): 1054-1067, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28642198

RESUMO

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.


Assuntos
Íleo/efeitos dos fármacos , Doenças Inflamatórias Intestinais/prevenção & controle , Obstrução Intestinal/prevenção & controle , Miofibroblastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Transferência Adotiva , Animais , Autofagia/efeitos dos fármacos , Estudos de Casos e Controles , Colágeno/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Ativação Enzimática , Fibrose , Humanos , Íleo/enzimologia , Íleo/imunologia , Íleo/patologia , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-6/metabolismo , Obstrução Intestinal/induzido quimicamente , Obstrução Intestinal/enzimologia , Obstrução Intestinal/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Miofibroblastos/enzimologia , Miofibroblastos/imunologia , Miofibroblastos/patologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/transplante , Fatores de Tempo , Técnicas de Cultura de Tecidos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismo
2.
J Med Chem ; 58(10): 4309-24, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25898023

RESUMO

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Células CACO-2/efeitos dos fármacos , Técnicas de Química Sintética , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Humanos , Masculino , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Coelhos , Relação Estrutura-Atividade , Quinases Associadas a rho/química
3.
Invest Ophthalmol Vis Sci ; 56(2): 1335-48, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25626969

RESUMO

PURPOSE: Rho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD. METHODS: The effect of AMA0428 (0.5-5.0 µM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I). RESULTS: The AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis. CONCLUSIONS: The results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Degeneração Macular/tratamento farmacológico , Neovascularização Retiniana/complicações , Quinases Associadas a rho/antagonistas & inibidores , Animais , Apoptose , Linhagem Celular , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Degeneração Macular/etiologia , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Bioorg Med Chem Lett ; 24(18): 4594-4597, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25149511

RESUMO

PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-associated side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors containing a γ-lactone structure. Such molecules are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors were highly active on both PDE4B1 and PDE4D2 and underwent rapid degradation in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.


Assuntos
Aminopiridinas/farmacologia , Benzamidas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Aminopiridinas/sangue , Aminopiridinas/química , Benzamidas/sangue , Benzamidas/química , Ciclopropanos/sangue , Ciclopropanos/química , Ciclopropanos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hidrólise , Estrutura Molecular , Inibidores da Fosfodiesterase 4/sangue , Inibidores da Fosfodiesterase 4/química , Relação Estrutura-Atividade
5.
Invest Ophthalmol Vis Sci ; 55(2): 1006-16, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24474276

RESUMO

PURPOSE: To determine whether ROCK inhibition for the treatment of glaucoma can be improved by using novel, locally acting Rho kinase (ROCK) inhibitors, such as AMA0076, that lower IOP without inducing hyperemia. METHODS: On-target potency of AMA0076 was compared with other ROCK inhibitors (Y-27632 and Y-39983) and conversion of AMA0076 into its functionally inactive metabolite was evaluated in rabbit eye tissues. Human trabecular meshwork (HTM) cell morphology, actin filaments, and focal adhesion were studied in vitro after exposure to AMA0076. The effect of AMA0076 on IOP was investigated in normotensive rabbits and a new, acute hypertensive rabbit model. Intraocular pressure lowering efficacy of AMA0076 was compared with pharmacologic treatments. Hyperemia after single topical dosing of AMA0076 and Y-39983 was scored. RESULTS: AMA0076 and Y-39983 showed similar on-target potency. AMA0076 was most stable in aqueous humor and converted into its metabolite in other eye tissues. Exposure of HTM cells to AMA0076 led to significant and reversible changes in cell shape and a decrease in actin stress fibers and focal adhesions. Both AMA0076 and Y-39983 provided an equivalent IOP control. Compared with latanoprost and bimatoprost, AMA0076 was more potent in preventing the IOP elevation in the acute hypertensive rabbit model. The degree of hyperemia was significantly lower in rabbits treated with AMA0076 then with Y-39983. CONCLUSIONS: AMA0076 is a locally acting ROCK inhibitor that is able to induce altered cellular behavior of HTM cells. Administration of AMA0076 effectively reduces IOP in ocular normotensive and acute hypertensive rabbits without causing distinct hyperemia.


Assuntos
Anti-Hipertensivos/uso terapêutico , Benzoatos/uso terapêutico , Túnica Conjuntiva/irrigação sanguínea , Hiperemia/induzido quimicamente , Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Actinas/metabolismo , Amidas/farmacologia , Animais , Benzoatos/efeitos adversos , Modelos Animais de Doenças , Adesões Focais/metabolismo , Masculino , Hipertensão Ocular/metabolismo , Piridinas/farmacologia , Coelhos , Tonometria Ocular , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Vinculina/metabolismo
6.
Bioorg Med Chem Lett ; 23(23): 6442-6, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24119557

RESUMO

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC50=2.5 nM) with rapid inactivation in plasma (t1/2 <5'). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.


Assuntos
Benzoatos/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Amidas/química , Amidas/farmacologia , Animais , Benzoatos/química , Modelos Moleculares , Fosforilação , Piridinas/química , Piridinas/farmacologia , Coelhos
7.
J Comput Chem ; 32(11): 2441-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21598279

RESUMO

The biosynthesis of the mineralocorticoid hormone aldosterone involves a multistep hydroxylation of 11-deoxycorticosterone at the 11- and 18-positions, resulting in the formation of corticosterone and 18-hydroxycorticosterone, the final precursor of aldosterone. Two members of the cytochrome P450 11B family, CYP11B1 and CYP11B2, are known to catalyze these 11- and 18-hydroxylations, however, only CYP11B2 can oxidize 18-hydroxycorticosterone to aldosterone. It is unknown what sequence of hydroxylations leads to the formation of 18-hydroxycorticosterone. In this study we have investigated which of the possible conversion paths towards formation of 18-hydroxycorticosterone and aldosterone are most likely from the ligand perspective. Therefore, we combined quantum mechanical investigations on the steroid conformations of 11-deoxycorticosterone and its ensuing reaction intermediates with Fukui indices calculations to predict the reactivity of their carbon atoms for an attack by the iron-oxygen species. Both F(-) and F(0) were calculated to account for different mechanisms of substrate conversion. We show which particular initial conformations of 11-deoxycorticosterone and which conversion paths are likely to result in the successful synthesis of aldosterone, and thereby may be representative for the mechanism of aldosterone biosynthesis by CYP11B2. Moreover, we found that the most likely path for aldosterone synthesis coincides with the substrate conformation proposed in an earlier publication. To summarize, we show that on a theoretical and strictly ligand-directed basis only a limited number of reaction paths in the conversion of 11-deoxycorticosterone to aldosterone is possible. Despite its theoretical nature, this knowledge may help to understand the catalytic function of CYP11B1 and CYP11B2.


Assuntos
Aldosterona/biossíntese , Aldosterona/química , Citocromo P-450 CYP11B2/química , Ligantes , Teoria Quântica , Ferro/química , Estrutura Molecular , Oxigênio/química
8.
J Med Chem ; 53(4): 1712-25, 2010 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-20121113

RESUMO

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, this is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, we used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-yl)methyl)benzonitrile) displaying an IC(50) for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC(50) for CYP11B2 6.0 nM, selectivity 19.8). Molecular docking of the inhibitors in the models enabled us to generate posthoc hypotheses on their binding modes, providing a valuable basis for future studies and further design of CYP11B2 inhibitors.


Assuntos
Compostos de Benzil/síntese química , Citocromo P-450 CYP11B2/antagonistas & inibidores , Imidazóis/síntese química , Modelos Moleculares , 18-Hidroxicorticosterona/química , Animais , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Domínio Catalítico , Linhagem Celular , Cricetinae , Cricetulus , Citocromo P-450 CYP11B2/química , Fadrozol/química , Humanos , Imidazóis/química , Imidazóis/farmacologia , Simulação de Dinâmica Molecular , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade
9.
J Comput Aided Mol Des ; 21(8): 455-71, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17646925

RESUMO

Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11beta-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1.


Assuntos
Simulação por Computador , Citocromo P-450 CYP11B2/química , Esteroide 11-beta-Hidroxilase/química , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Citocromo P-450 CYP11B2/antagonistas & inibidores , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Esteroide 11-beta-Hidroxilase/antagonistas & inibidores , Esteroide 11-beta-Hidroxilase/genética , Esteroide 11-beta-Hidroxilase/metabolismo , Termodinâmica
10.
Steroids ; 68(3): 213-20, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12628684

RESUMO

A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.


Assuntos
Microssomos Hepáticos/metabolismo , Nandrolona/análogos & derivados , Nandrolona/química , Nandrolona/metabolismo , Ésteres/química , Ésteres/metabolismo , Humanos , Hidrólise , Masculino , Modelos Moleculares , Relação Estrutura-Atividade
11.
Steroids ; 68(3): 235-43, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12628686

RESUMO

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.


Assuntos
Aromatase/metabolismo , Etinilestradiol/análogos & derivados , Etinilestradiol/metabolismo , Norpregnenos/metabolismo , Androgênios/metabolismo , Animais , Inibidores da Aromatase , Células CHO , Linhagem Celular , Cromatografia Líquida/métodos , Cricetinae , Humanos , Imidazóis/farmacologia , Espectrometria de Massas/métodos , Fenalenos/farmacologia , Especificidade por Substrato , Fatores de Tempo
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