RESUMO
Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.
Assuntos
Anticorpos/metabolismo , Fibroblastos/metabolismo , Integrina alfaV/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Anticorpos/imunologia , Bleomicina , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/citologia , Humanos , Integrina alfaV/imunologia , Masculino , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Propionatos/farmacologia , Ligação Proteica , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controleRESUMO
Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK-0429, a compound that was originally developed as an αvß3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK-0429 is an equipotent pan-inhibitor of multiple av integrins. MK-0429 dose-dependently inhibited podocyte motility and also suppressed TGF-ß-induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK-0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Marcadores Genéticos/efeitos dos fármacos , Integrina alfaV/metabolismo , Rim/fisiopatologia , Naftiridinas/administração & dosagem , Propionatos/administração & dosagem , Animais , Linhagem Celular , Colágeno/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Testes de Função Renal , Masculino , Naftiridinas/farmacologia , Propionatos/farmacologia , RatosRESUMO
The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Assuntos
Modelos Moleculares , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/química , Bloqueadores do Receptor Tipo 2 de Angiotensina II/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Desenho de Fármacos , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/genética , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato/genética , beta-Arrestinas/metabolismoRESUMO
Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.
Assuntos
Descoberta de Drogas , Indóis/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores de Mineralocorticoides/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
This review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds. For seconds to hours timescale kinetics we discuss surface plasmon resonance-based biosensor, global progress curve analysis for high throughput kinetic profiling of enzyme inhibitors and activators, and filtration plate-based radioligand or fluorescent binding assays for receptor binding kinetics. Jump dilution after pre-incubation is the preferred method for very slow kinetics lasting for days. The basic principles, best practices and simulated data for these technologies are described. Finally, the application of a universal label-free technology, liquid chromatography coupled tandem mass spectrometry (LC/MS/MS), is briefly reviewed. Select literature references are highlighted for in-depth understanding. A new reality is dawning wherein binding kinetics is an integral and routine part of mechanism of action elucidation and translational, quantitative pharmacology for drug discovery.
Assuntos
Descoberta de Drogas/métodos , Ativadores de Enzimas/farmacocinética , Inibidores Enzimáticos/farmacocinética , Ensaios de Triagem em Larga Escala/métodos , Técnicas Biossensoriais/métodos , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Farmacocinética , Ressonância de Plasmônio de Superfície/métodos , Espectrometria de Massas em TandemRESUMO
Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Diacilglicerol O-Aciltransferase/química , Ensaios Enzimáticos/métodos , Humanos , Espectrometria de Massas/métodos , Células Sf9 , Triglicerídeos/químicaRESUMO
DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacologia , Triglicerídeos/metabolismo , Animais , Diacilglicerol O-Aciltransferase/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triglicerídeos/sangueRESUMO
Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.
RESUMO
Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Animais , Carboxipeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Humanos , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure-activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.
Assuntos
Amidas/química , Compostos Heterocíclicos/química , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Oxazóis/farmacologia , Receptores de Mineralocorticoides/metabolismo , Animais , Relação Dose-Resposta a Droga , Humanos , Microssomos Hepáticos , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Ratos , Relação Estrutura-AtividadeRESUMO
The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Cicloexanos/química , Cicloexanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Animais , Cicloexanos/farmacocinética , Descoberta de Drogas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.
Assuntos
Antagonistas de Receptores de Mineralocorticoides/química , Oxazóis/química , Receptores de Mineralocorticoides/metabolismo , Animais , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Microssomos/metabolismo , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/farmacocinética , Simulação de Acoplamento Molecular , Oxazóis/síntese química , Oxazóis/farmacocinética , Estrutura Terciária de Proteína , Ratos , Receptores de Mineralocorticoides/química , Relação Estrutura-AtividadeRESUMO
In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11ß-HSD1 inhibitors, we examined a set of 11ß-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11ß-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11ß-HSD1 independent pathway.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hipertensão/tratamento farmacológico , Hipertensão/enzimologia , Triazóis/farmacologia , Animais , Humanos , Camundongos , Camundongos Knockout , Ratos , Ratos Endogâmicos SHRRESUMO
A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos , Animais , Butanóis/síntese química , Butanóis/química , Butanóis/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Pirrolidinas/síntese química , Pirrolidinas/química , Pirrolidinas/farmacologiaRESUMO
A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.
Assuntos
Aminas/farmacologia , Carboxipeptidases/antagonistas & inibidores , Ciclopentanos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos , Aminas/síntese química , Aminas/química , Animais , Ciclização , Ciclopentanos/síntese química , Ciclopentanos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológicoRESUMO
Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).
Assuntos
Aminas/síntese química , Encéfalo/efeitos dos fármacos , Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , Amidas/química , Aminas/química , Aminas/farmacologia , Animais , Encéfalo/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/química , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.
Assuntos
Alanina/química , Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Alanina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Furanos/química , Furanos/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Estabilidade de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Furanos/síntese química , Humanos , Camundongos , Camundongos Obesos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.
Assuntos
Benzimidazóis/farmacologia , Encéfalo/metabolismo , Carboxipeptidases/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Amidas/química , Animais , Transporte Biológico , Peso Corporal , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Obesidade/tratamento farmacológico , Pirrolidinas/química , Fatores de TempoRESUMO
The generation of peptides presented by MHC class I molecules requires the proteolytic activity of the proteasome and/or other peptidases. The processing of a short vaccinia virus-encoded antigen can take place by a proteasome-independent pathway involving initiator caspase-5 and -10, which generate antigenic peptides recognized by CD8(+) T lymphocytes. In the present study, comparing single versus double enzyme digestions by mass spectrometry analysis, both qualitative and quantitative differences in the products obtained were identified. These in vitro data suggest that each enzyme can use the degradation products of the other as substrate for new cleavages, indicating concerted endoproteolytic activity of caspase-5 and -10.