RESUMO
The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability.
Assuntos
Desenvolvimento de Medicamentos/métodos , Indazóis/química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura MolecularRESUMO
Na(+)/H(+) exchanger 1 (NHE-1) inhibition attenuates the hypertrophic response and heart failure in various experimental models. As the hypertrophic program is rapidly initiated following insult, we investigated whether early and transient administration of a NHE-1 inhibitor will exert salutary effects on cardiomyocyte hypertrophy or heart failure using both in vitro and in vivo approaches. Neonatal cardiomyocytes were treated with the novel, potent, and highly specific NHE-1 inhibitor BIX (N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine; 100 nM) for 1 hour in the presence of 10 µM phenylephrine, after which the cells were maintained for a further 23 hours in the absence of NHE-1 inhibition. One-hour treatment with the NHE-1 inhibitor prevented phenylephrine-induced hypertrophy, which was associated with prevention of activation of calcineurin, a key component of the hypertrophic process. Experiments were then performed in rats subjected to coronary artery ligation, in which the NHE-1 inhibitor was administered immediately after infarction for a 1-week period followed by a further 5 weeks of sustained coronary artery occlusion in the absence of drug treatment. This approach significantly attenuated left ventricular hypertrophy and improved both left ventricular systolic and diastolic dysfunction, which was also associated with inhibition of calcineurin activation. Our findings indicate that early and transient administration of an NHE-1 inhibitor bestows subsequent inhibition of cardiomyocyte hypertrophy in culture as well as cardiac hypertrophy and heart failure in vivo, suggesting a critical early NHE-1-dependent initiation of the hypertrophic program. The study also suggests a preconditioning-like phenomenon in preventing hypertrophy and heart failure by early and transient NHE-1 inhibition.
Assuntos
Cardiomegalia/prevenção & controle , Vasos Coronários/efeitos dos fármacos , Insuficiência Cardíaca/prevenção & controle , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ligadura/efeitos adversos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Piperidinas/química , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de TempoRESUMO
We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC(50) = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (-93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to -39 ± 4 mm Hg on day 4 at T(max)); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic α(1A) and α(2A) was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0-92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC(50) <0.14 nM) without relevant α(1A) and α(2A) inhibition and no adverse cardiovascular effects in vivo.
Assuntos
Azepinas/farmacologia , Benzimidazóis/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Descoberta de Drogas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subsequent kinase selectivity optimization.
Assuntos
Azepinas/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/química , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Amidas/química , Azepinas/farmacologia , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Indóis/síntese química , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , Nitrogênio/química , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: Blood C-reactive protein (CRP) is routinely measured to gauge inflammation. In rheumatoid arthritis (RA), a heightened CRP level is predictive of a poor outcome, while a lowered CRP level is indicative of a positive response to therapy. CRP interacts with the innate and adaptive immune systems in ways that suggest it may be causal in RA and, although this is not proven, it is widely assumed that CRP makes a detrimental contribution to the disease process. Paradoxically, results from animal studies have indicated that CRP might be beneficial in RA. This study was undertaken to study the role of CRP in a mouse model of RA, the collagen-induced arthritis (CIA) model. METHODS: We compared the impact of CRP deficiency with that of transgenic overexpression of CRP on inflammatory and immune responses in mice, using CRP-deficient (Crp-/-) and human CRP-transgenic (CRP-Tg) mice, respectively. Susceptibility to CIA, a disease that resembles RA in humans, was compared between wild-type, Crp-/-, and CRP-Tg mice. RESULTS: CRP deficiency significantly altered the inflammatory cytokine response evoked by challenge with endotoxin or anti-CD3 antibody, and heightened some immune responses. Compared to that in wild-type mice, CIA in Crp-/- mice progressed more rapidly and was more severe, whereas CIA in CRP-Tg mice was dramatically attenuated. Despite these disparate clinical outcomes, anticollagen autoantibody responses during CIA did not differ among the genotypes. CONCLUSION: CRP exerts an early and beneficial effect in mice with CIA. The mechanism of this effect remains unknown but does not involve improvement of the autoantibody profile. In humans, the presumed detrimental role of a heightened blood CRP level during active RA might be balanced by a beneficial effect of the baseline CRP (i.e., levels manifest during the preclinical stages of disease).
Assuntos
Artrite Experimental/genética , Proteína C-Reativa/genética , Citocinas/imunologia , Inflamação/imunologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Índice de Gravidade de DoençaRESUMO
Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKß, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/ß activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKß) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.
Assuntos
Proteínas I-kappa B/metabolismo , Imunidade Inata/fisiologia , Linhagem Celular , Ciclobutanos/química , Ciclobutanos/farmacologia , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Interleucina-1/genética , Interleucina-1/metabolismo , Estrutura Molecular , Morfolinas/química , Morfolinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Transdução de Sinais , Sulfonamidas/química , Sulfonamidas/farmacologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.
Assuntos
2-Naftilamina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Ureia/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , 2-Naftilamina/química , Animais , Química Orgânica/métodos , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Concentração Inibidora 50 , Lipopolissacarídeos/metabolismo , Camundongos , Modelos Químicos , Estrutura Molecular , Fator de Necrose Tumoral alfa/metabolismo , Ureia/químicaRESUMO
Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.
Assuntos
Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Sulfonamidas/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química , Animais , Sítios de Ligação , Disponibilidade Biológica , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Niacinamida/análogos & derivados , Niacinamida/síntese química , Niacinamida/química , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
INTRODUCTION: Mouse transverse aortic constriction (TAC) is a widely-used model of pressure overload-induced heart failure. An intrinsic limitation of the model is variability in the response to pressure overload even when employing a standard severity of stenosis. Few literature studies have explicitly reported the use of entry criteria or early predictors to mitigate variability and enrich outcomes in this model. METHODS: Eleven-week-old male C57BL/6J mice underwent TAC or sham surgery. Left ventricular (LV) function and dimensions were assessed by M-mode echocardiography at baseline (pre) and 3, 9 and 12weeks post-procedure (end-study). At 24h post-procedure, transverse aortic flow velocities were obtained for estimating trans-TAC pressure gradients. Invasive LV hemodynamic assessments were performed and terminal heart and lung weights obtained at end-study. RESULTS: TAC mice displayed early development of LV hypertrophy and wall thickening followed by the later development of LV chamber dilation, and progressive development of LV systolic and diastolic dysfunction. The use of a pre-defined trans-TAC pressure gradient criterion of 45-60mmHg did not affect end-study organ weight, echocardiographic and invasive hemodynamic outcomes. A post-hoc receiver operator characteristic (ROC) analysis identified early 3week echocardiographic measures of LVmass(echo) and ejection fraction, with threshold changes of ~+30% and -10% normalized to baseline respectively, as good predictors for multiple end-study organ weight, echocardiographic and invasive hemodynamic outcomes. DISCUSSION: This ROC analysis has identified early predictive threshold changes which may serve, alone or in combination, as entry criteria to enrich outcome in this model.
Assuntos
Modelos Animais de Doenças , Ecocardiografia/métodos , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Animais , Constrição , Insuficiência Cardíaca/etiologia , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Distribuição Aleatória , Função Ventricular Esquerda/fisiologiaRESUMO
INTRODUCTION: Despite the widespread use of the mouse transverse aortic constriction heart failure model, there are no reports on the characterization of the standard-of-care agent carvedilol in this model. METHODS: Left ventricular pressure overload was produced in mice by transverse aortic constriction between the innominate and left common carotid arteries. Carvedilol was administered at multiple dose levels (3, 10 and 30 mg/kg/day per os; yielding end-study mean plasma concentrations of 0.002, 0.015 and 0.044 µM, respectively) in a therapeutic design protocol with treatment initiated after the manifestation of left ventricular remodeling at 3 weeks post transverse aortic constriction and continued for 10 weeks. RESULTS: Carvedilol treatment in transverse aortic constriction mice significantly decreased heart rate and left ventricular dP/dt (max) at all dose levels consistent with ß-adrenoceptor blockade. The middle dose of carvedilol significantly decreased left ventricular weight, whereas the higher dose decreased total heart, left and right ventricular weight and wet lung weight compared to untreated transverse aortic constriction mice. The higher dose of carvedilol significantly increased cardiac performance as measured by ejection fraction and fractional shortening and decreased left ventricular end systolic volume consistent with the beneficial effect on cardiac function. End-study plasma sST-2 and Gal-3 levels did not differ among sham, transverse aortic constriction control and transverse aortic constriction carvedilol groups. Plasma brain natriuretic peptide concentrations were elevated significantly in transverse aortic constriction control animals (~150%) compared to shams in association with changes in ejection fraction and heart weight and tended to decrease (~30%, p = 0.10-0.12) with the mid- and high-dose carvedilol treatment. CONCLUSION: A comparison of carvedilol hemodynamic and structural effects in the mouse transverse aortic constriction model versus clinical use indicates a strong agreement in effect profiles preclinical versus clinical, providing important translational validation for this widely used animal model. The present plasma brain natriuretic peptide biomarker findings support the measurement of plasma natriuretic peptides in the mouse transverse aortic constriction model to extend the translational utility of the model.
Assuntos
Descoberta de Drogas/métodos , Insuficiência Cardíaca/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto/métodos , Descoberta de Drogas/organização & administração , Avaliação Pré-Clínica de Medicamentos , Insuficiência Cardíaca/metabolismo , Humanos , Seleção de Pacientes , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/organização & administração , Resultado do TratamentoRESUMO
BACKGROUND: Mouse transgenesis has provided the unique opportunity to investigate mechanisms underlying sodium kidney reabsorption as well as end organ damage. However, understanding mouse background and the experimental conditions effects on phenotypic readouts of engineered mouse lines such as blood pressure presents a challenge. Despite the ability to generate high sodium and chloride plasma levels during high-salt diet, observed changes in blood pressure are not consistent between wild-type background strains and studies. METHODS: The present work was designed in an attempt to determine guidelines in the field of salt-induced hypertension by recording continuously blood pressure by telemetry in mice submitted to different sodium and potassium loaded diets and changing experimental conditions in both C57BL/6N and C57BL/6J mice strain (Normal salt vs. Low salt vs. High-salt/normal potassium vs. High salt/low potassium, standard vs. modified light cycle, Non-invasive tail cuff blood pressure vs. telemetry). RESULTS: In this study, we have shown that, despite a strong blood pressure (BP) basal difference between C57BL/6N and C57BL/6J mice, High salt/normal potassium diet increases BP and heart rate during the active phase only (dark period) in the same extent in both strains. On the other hand, while potassium level has no effect on salt-induced hypertension in C57BL/6N mice, high-salt/low potassium diet amplifies the effect of the high-salt challenge only in C57BL/6J mice. Indeed, in this condition, salt-induced hypertension can also be detected during light period even though this BP increase is lower compared to the one occurring during the dark period. Finally, from a methodological perspective, light cycle inversion has no effect on this circadian BP phenotype and tail-cuff method is less sensitive than telemetry to detect BP phenotypes due to salt challenges. CONCLUSIONS: Therefore, to carry investigations on salt-induced hypertension in mice, chronic telemetry and studies in the active phase are essential prerequisites.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Ritmo Circadiano/fisiologia , Hipertensão/fisiopatologia , Cloreto de Sódio na Dieta/farmacologia , Animais , Determinação da Pressão Arterial/métodos , Dieta Hipossódica , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipertensão/induzido quimicamente , Masculino , Camundongos Endogâmicos C57BL/genética , Potássio/sangue , Potássio/farmacologia , Potássio/urina , Sódio/sangue , Sódio/urina , Cloreto de Sódio na Dieta/efeitos adversos , Sódio na Dieta/efeitos adversos , Sódio na Dieta/farmacologia , TelemetriaRESUMO
We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Inibidores Enzimáticos/síntese química , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftalenos/síntese química , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Fluorescência , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Naftalenos/química , Naftalenos/farmacologia , Ligação Proteica , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese , Ureia/química , Ureia/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38alpha MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38alpha inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important pi-CH(2) interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38alpha by 16-17 degrees C translating into K(d) values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-alpha production when dosed orally in mice.
Assuntos
Inibidores Enzimáticos/síntese química , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftalenos/síntese química , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Animais , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ensaio de Imunoadsorção Enzimática , Calefação , Humanos , Técnicas In Vitro , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Proteína Quinase 14 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/química , Naftalenos/química , Ligação Proteica , Desnaturação Proteica , Pirazóis/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Ureia/químicaRESUMO
Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.
Assuntos
Benzamidas/síntese química , Guanidinas/síntese química , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Benzamidas/química , Benzamidas/farmacologia , Disponibilidade Biológica , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular , Tamanho Celular , Inibidores das Enzimas do Citocromo P-450 , Cães , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Guanidinas/química , Guanidinas/farmacologia , Humanos , Masculino , Membranas Artificiais , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Permeabilidade , Isoformas de Proteínas/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Trocador 1 de Sódio-Hidrogênio , Relação Estrutura-AtividadeRESUMO
Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKß), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFß-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC(50) of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFß-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers.
RESUMO
We investigated whether or not p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). dTGR, with or without p38 inhibitor (BIRB796; 30 mg/kg per day in the diet), and nontransgenic Sprague-Dawley rats were studied in 2 protocols. In protocol 1 (week 7), systolic blood pressure of untreated dTGRs was 204+/-4 mm Hg, but partially reduced after BIRB796 treatment (166+/-7 mm Hg), whereas Sprague-Dawley rats were normotensive. The cardiac hypertrophy index was unchanged in untreated and BIRB796-treated dTGRs. The beta-myosin heavy chain expression of BIRB796-treated hearts was significantly lower in BIRB796 compared with dTGRs, indicating a delayed switch to the fetal isoform. BIRB796 treatment significantly reduced cardiac fibrosis, connective tissue growth factor, tumor necrosis factor-alpha, interleukin-6, and macrophage infiltration. Albuminuria was not reduced in BIRB796-treated dTGRs. Tubular and glomerular damage with tumor necrosis factor-alpha expression was unaltered, although serum creatinine and cystatin C were normalized. Renal macrophage infiltration, fibrosis, and vessel damage were reduced. In protocol 2 (week 8), we focused on mortality and arrhythmogenic electrical remodeling. Mortality of untreated dTGRs was 100% but was reduced to 10% in the BIRB796 group. Cardiac magnetic field mapping showed prolongation of depolarization and repolarization in untreated dTGRs compared with Sprague-Dawley rats with a partial reduction by BIRB796. Programmed electrical stimulation elicited ventricular tachycardias in 81% of untreated dTGRs but only in 48% of BIRB796-treated dTGRs. In conclusion, BIRB796 improved survival, target organ damage, and arrhythmogenic potential in angiotensin II-induced target organ damage.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Nefropatias/prevenção & controle , Naftalenos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Angiotensina II/efeitos adversos , Angiotensinogênio/genética , Animais , Animais Geneticamente Modificados , Doenças Cardiovasculares/etiologia , Modelos Animais de Doenças , Nefropatias/etiologia , Masculino , Ratos , Ratos Sprague-Dawley , Renina/genéticaRESUMO
Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.
Assuntos
Adenina/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Sítios de Ligação , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-alpha, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-alpha(1)-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.