RESUMO
BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.
Assuntos
Anticolesterolemiantes , Inibidores de Hidroximetilglutaril-CoA Redutases , Hipercolesterolemia , Adulto , Humanos , Anticolesterolemiantes/efeitos adversos , Colesterol , LDL-Colesterol , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Peptídeos/uso terapêutico , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
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
Following the discovery of a metabolic 'soft-spot' on a bicyclo[2.2.2]octyltriazole lead, an extensive effort was undertaken to block the oxidative metabolism and improve PK of this potent HSD1 lead. In this communication, SAR survey focusing on various alkyl chain replacements will be detailed. This effort culminated in the discovery of a potent ethyl sulfone inhibitor with an improved PK profile across species and improved physical properties.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Compostos Bicíclicos com Pontes/química , Inibidores Enzimáticos/química , Síndrome Metabólica/tratamento farmacológico , Triazóis/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Camundongos , Relação Estrutura-Atividade , Triazóis/farmacocinética , Triazóis/uso terapêuticoRESUMO
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.
Assuntos
Inibidores de PCSK9/farmacologia , Peptídeos Cíclicos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Macaca fascicularis , Estrutura Molecular , Inibidores de PCSK9/química , Inibidores de PCSK9/farmacocinética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores de PCSK9 , Pró-Proteína Convertase 9/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X/métodos , Inibidores Enzimáticos/química , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/química , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography-mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Hipercolesterolemia/tratamento farmacológico , Pró-Proteína Convertase 9/química , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Células Hep G2 , Humanos , Hipercolesterolemia/genética , Cinética , Lactonas/antagonistas & inibidores , Espectrometria de Massas/métodos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/genética , Conformação Proteica/efeitos dos fármacos , Receptores de LDL/genéticaRESUMO
11beta-Hydroxysteroid dehydrogenase type-1 (11beta-HSD1) is a potential target for the treatment of diabetes, obesity, and hyperlipidemia. This enzyme is mainly responsible for reactivating glucocorticoid hormone inside cells such as adipose cells and liver cells by converting the inactive cortisone to active cortisol. Enzyme assays for 11beta-HSD1 involve either a thin-layer chromatography or high-performance liquid chromatography step to separate cortisol from the substrate cortisone. This additional step is labor intensive and increases the assay time, which limits assay throughput. A homogenous scintillation proximity assay-based method has been recently developed that enables high-throughput screening of 11beta-HSD1 inhibitors. We have applied this novel 11beta-HSD1 assay to screening a large-size compound collection and identified several structural classes of lead compounds that selectively inhibit the activity of 11beta-HSD1.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Contagem de Cintilação/métodos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Anticorpos Monoclonais , Células CHO , Técnicas de Química Combinatória , Cricetinae , Cricetulus , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidrocortisona/análise , Hidrocortisona/imunologia , Hidrocortisona/metabolismo , Microssomos/enzimologia , Transfecção , TrítioRESUMO
Replacement of the pentyl chain on our original bicyclo[2.2.2]octyltriazole leads 1 and 2 has led to the discovery that heteroaryl substituted bicyclo[2.2.2]octyltriazoles are potent and selective 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) inhibitors with excellent pharmacokinetic profiles.