RESUMO
Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a Ki of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.
Assuntos
Carnosina/administração & dosagem , Dipeptidases/antagonistas & inibidores , Descoberta de Drogas , Imidazóis/farmacologia , Propionatos/farmacologia , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Adulto , Animais , Carnosina/sangue , Dipeptidases/sangue , Dipeptidases/genética , Feminino , Expressão Gênica , Humanos , Imidazóis/química , Injeções Subcutâneas , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Propionatos/química , Inibidores de Proteases/química , Ligação Proteica , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas/química , TransgenesRESUMO
For fasiglifam (TAK875) and its metabolites the substance-specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole-cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short-term treatment (24 hours or 48 hours) with TC50 values of 56 to 68 µM (adenosine triphosphate endpoint). The long-term treatment for 14 days in the hLiMT resulted in a slight TC50 shift over time of 2.7/3.6 fold lower vs 24-hour treatment indicating possibly a higher risk for cytotoxicity during long-term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.
Assuntos
Benzofuranos/toxicidade , Ácidos Graxos não Esterificados/metabolismo , Fígado/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Sulfonas/toxicidade , Animais , Benzofuranos/metabolismo , Células Cultivadas , Cães , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Sulfonas/metabolismoRESUMO
The discovery of potent benzimidazole stearoyl-CoA desaturase (SCD1) inhibitors by ligand-based virtual screening is described. ROCS 3D-searching gave a favorable chemical motif that was subsequently optimized to arrive at a chemical series of potent and promising SCD1 inhibitors. In particular, compound SAR224 was selected for further pharmacological profiling based on favorable in vitro data. After oral administration to male ZDF rats, this compound significantly decreased the serum fatty acid desaturation index, thus providing conclusive evidence for SCD1 inhibition in vivo by SAR224.
Assuntos
Amidas/química , Benzimidazóis/química , Inibidores Enzimáticos/química , Estearoil-CoA Dessaturase/antagonistas & inibidores , Tiofenos/química , Amidas/metabolismo , Amidas/farmacocinética , Animais , Benzimidazóis/síntese química , Disponibilidade Biológica , Células CACO-2 , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Humanos , Ligantes , Masculino , Camundongos , Ligação Proteica , Ratos , Ratos Zucker , Estearoil-CoA Dessaturase/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese químicaRESUMO
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.
Assuntos
Artrite Experimental , Artrite Reumatoide , Sinoviócitos , Animais , Humanos , Camundongos , Artrite Experimental/tratamento farmacológico , Células Cultivadas , Fibroblastos/patologia , Membrana Sinovial/patologia , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Inibidores do Fator de Necrose Tumoral/metabolismo , Inibidores do Fator de Necrose Tumoral/farmacologia , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/imunologiaRESUMO
Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.
RESUMO
Several polypeptides comprising the carboxy-terminal domain of the 1-amyloid precursor protein (cAPP) were prepared by solid phase peptide synthesis, and employed as antigens for the determination of the epitopes recognised by anti-cAPP antibodies. Selective proteolytic epitope-excision and -extraction on the immobilised immune complexes, in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were used as major methods for epitope identification. The epitope recognised by a polyclonal anti-cAPP antibody (36-BO) was identified as APP(727-737), a sequence close to the APP transmembrane region. In contrast, the epitope recognised by a monoclonal anti-cAPP antibody (Jonas-mAb) was identified at APP(740-747) to be located more remote from the transmembrane region. The two adjacent, yet distinct epitopes recognised by two different antibodies should provide efficient tools for (i), molecular diagnostic applications, and (ii), the study of intracellular processing pathways of APP relevant to Alzheimer's disease, utilising suitable mass spectrometric and molecular imaging approaches.
Assuntos
Doença de Alzheimer/imunologia , Precursor de Proteína beta-Amiloide/imunologia , Anticorpos/imunologia , Epitopos/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Humanos , Dados de Sequência Molecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile.
Assuntos
Carboxipeptidase B2/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Zinco/química , Animais , Carboxipeptidase B2/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Camundongos , Microssomos/química , Microssomos/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Ratos , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.
Assuntos
Precursor de Proteína beta-Amiloide/química , Espectrometria de Massas/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/imunologia , Animais , Ciclotrons , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Análise de Fourier , Humanos , Íons , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
AIM: To compare the therapeutic efficacy of SAR407899 with the current standard treatment for hypertension [an angiotensin converting enzyme (ACE)-inhibitor and a calcium channel blocker] and compare the frequency and severity of the hypertension-related end-organ damage. METHODS: Long-term pharmacological characte-rization of SAR407899 has been performed in two animal models of hypertension, of which one is sensitive to ACE-inhibition (LNAME) and the other is insensitive [deoxycorticosterone acetate (DOCA)]. SAR407899 efficiently lowered high blood pressure and significantly reduced late-stage end organ damage as indicated by improved heart, kidney and endothelial function and reduced heart and kidney fibrosis in both models of chronic hypertension. RESULTS: Long term treatment with SAR407899 has been well tolerated and dose-dependently reduced elevated blood pressure in both models with no signs of tachyphylaxia. Blood pressure lowering effects and protective effects on hypertension related end organ damage of SAR407899 were superior to ramipril and amlodipine in the DOCA rat. Typical end-organ damage was significantly reduced in the SAR407899-treated animals. Chronic administration of SAR407899 significantly reduced albuminuria in both models. The beneficial effect of SAR407899 was associated with a reduction in leukocyte/macrophage tissue infiltration. The overall protective effect of SAR407899 was superior or comparable to that of ACE-inhibition or calcium channel blockade. Chronic application of SAR407899 protects against hypertension and hypertension-induced end organ damage, regardless of the pathophysiological mechanism of hypertension. CONCLUSION: Rho-kinases-inhibition by the SAR407899 represents a new therapeutic option for the treatment of hypertension and its complications.
RESUMO
Anabaenopeptins isolated from cyanobacteria were identified as inhibitors of carboxypeptidase TAFIa. Cocrystal structures of these macrocyclic natural product inhibitors in a modified porcine carboxypeptidase B revealed their binding mode and provided the basis for the rational design of small molecule inhibitors with a previously unknown central urea motif. Optimization based on these design concepts allowed for a rapid evaluation of the SAR and delivered potent small molecule inhibitors of TAFIa with a promising overall profile.
Assuntos
Produtos Biológicos/farmacologia , Carboxipeptidase B2/antagonistas & inibidores , Fibrinólise/efeitos dos fármacos , Microssomos/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Produtos Biológicos/química , Células Cultivadas , Cristalografia por Raios X , Cianobactérias/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Peptídeos Cíclicos/química , Ratos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , SuínosRESUMO
Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.
RESUMO
The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Terapia de Alvo Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Animais , Antituberculosos/química , Antituberculosos/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêutico , Estrutura Secundária de Proteína , Streptomyces/química , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Starting from clinical candidates Firategrast, Valategrast, and AJM-300, a series of novel macrocyclic platelet collagen receptor α2ß1 antagonists were developed. The amino acid derived low molecular weight 14-18-membered macrocycles turned out to be highly active toward integrin α2ß1 with IC50s in the low nanomolar range. The conformation of the macrocycles was found to be highly important for the activity, and an X-ray crystal structure was obtained to clarify this. Subsequent docking into the metal-ion-dependent adhesion site (MIDAS) of a ß1 unit revealed a binding model indicating key binding features. Macrocycle 38 was selected for further in vitro and in vivo profiling.
RESUMO
Stearoyl-CoA desaturase (SCD1) is linked to the pathogenesis of obesity, dyslipidemia and type 2 diabetes. It is the rate-limiting enzyme in the synthesis of monounsaturated 16:1 n-7 and 18:1 n-9 fatty acyl-CoAs and catalyzes an essential part of lipogenesis. Here, we describe the identification, in vitro properties and in vivo efficacy of a novel class of heterocyclic small molecule hexahydro-pyrrolopyrrole SCD1 inhibitors. SAR707, a compound representative for the series, was optimized to high in vitro potency, selectivity and favorable overall properties in enzymatic and cellular assays. In vivo, this compound reduced serum desaturation index, decreased body weight gain and improved lipid parameters and blood glucose levels of obese Zucker diabetic fatty rats treated for 4 weeks in a chronic study. In parallel, fissures of the eye lid, alopecia and inflammation of the skin were observed from day 11 on in all animals treated with the same metabolically active dose. In summary, we described in vitro and in vivo properties of a novel, potent and selective SCD1 inhibitor that improved body weight, blood glucose and triglycerides in an animal model of obesity, type 2 diabetes and dyslipidemia. However, the favorable in vivo properties of systemic SCD1 inhibition shown in our study were accompanied by dose-dependently occurring adverse target-related effects observed in skin. Thus, systemic SCD1 inhibition by small molecules might therefore not represent a feasible approach for the treatment of chronic metabolic diseases.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Piridazinas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/toxicidade , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Dislipidemias/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/toxicidade , Masculino , Obesidade/tratamento farmacológico , Piridazinas/administração & dosagem , Piridazinas/toxicidade , Ratos , Ratos Wistar , Ratos Zucker , Pele/efeitos dos fármacos , Pele/patologia , Triglicerídeos/sangueRESUMO
A series of novel, highly potent P2Y12 antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y12 antagonists displaying not only low nanomolar binding affinity to the P2Y12 receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC50 values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.
Assuntos
Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/síntese química , Piperazinas/síntese química , Antagonistas do Receptor Purinérgico P2Y/síntese química , Pirazóis/síntese química , Receptores Purinérgicos P2Y12/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Simulação de Acoplamento Molecular , Piperazinas/química , Piperazinas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2Y/química , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Relação Quantitativa Estrutura-Atividade , Ensaio Radioligante , EstereoisomerismoRESUMO
Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel ß-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.
Assuntos
Aminoácidos/farmacologia , Catepsina A/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Cristalografia por Raios X , Modelos MolecularesRESUMO
Recent advances in basic and clinical research have identified Rho kinase as an important target potentially implicated in a variety of cardiovascular diseases. Rho kinase is a downstream mediator of RhoA that leads to stress fiber formation, membrane ruffling, smooth muscle contraction, and cell motility. Increased Rho-kinase activity is associated with vasoconstriction and elevated blood pressure. We identified a novel inhibitor of Rho kinase (SAR407899) and characterized its effects in biochemical, cellular, tissue-based, and in vivo assays. SAR407899 is an ATP-competitive Rho-kinase inhibitor, equipotent against human and rat-derived Rho-kinase 2 with inhibition constant values of 36 nM and 41 nM, respectively. It is highly selective in panel of 117 receptor and enzyme targets. SAR407899 is approximately 8-fold more active than fasudil. In vitro, SAR407899 demonstrated concentration-dependent inhibition of Rho-kinase-mediated phosphorylation of myosin phosphatase, thrombin-induced stress fiber formation, platelet-derived growth factor-induced proliferation, and monocyte chemotactic protein-1-stimulated chemotaxis. SAR407899 potently (mean IC(50) values: 122 to 280 nM) and species-independently relaxed precontracted isolated arteries of different species and different vascular beds. In vivo, over the dose range 3 to 30 mg/kg PO, SAR407899 lowered blood pressure in a variety of rodent models of arterial hypertension. The antihypertensive effect of SAR407899 was superior to that of fasudil and Y-27632. In conclusion, SAR407899 is a novel and potent selective Rho-kinase inhibitor with promising antihypertensive activity.
Assuntos
Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Catálise/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Immunoblotting , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Fibras de Estresse/metabolismo , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.