RESUMEN
Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
Asunto(s)
Apoproteínas/química , Bioquímica/métodos , Furina/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Glicosilación , Células HEK293 , Humanos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Bone Morphogenetic Protein 1 (BMP1) inhibition is a potential method for treating fibrosis because BMP1, a member of the zinc metalloprotease family, is required to convert pro-collagen to collagen. A novel class of reverse hydroxamate BMP1 inhibitors was discovered, and cocrystal structures with BMP1 were obtained. The observed binding mode is unique in that the small molecule occupies the nonprime side of the metalloprotease pocket providing an opportunity to build in metalloprotease selectivity. Structure-guided modification of the initial hit led to the identification of an oral in vivo tool compound with selectivity over other metalloproteases. Due to irreversible inhibition of cytochrome P450 3A4 for this chemical class, the risk of potential drug-drug interactions was managed by optimizing the series for subcutaneous injection.
RESUMEN
Hypothalamic CAMKK2 represents a potential mechanism for chemically affecting satiety and promoting weight loss in clinically obese patients. Single-digit nanomolar inhibitors of CAMKK2 were identified in three related ATP-competitive series. Limited optimization of kinase selectivity, solubility, and pharmacokinetic properties were undertaken on all three series, as SAR was often transferrable. Ultimately, a 2,4-diaryl 7-azaindole was optimized to afford a tool molecule that potently inhibits AMPK phosphorylation in a hypothalamus-derived cell line, is orally bioavailable, and crosses the blood-brain barrier. When dosed orally in rodents, compound 4â¯t limited ghrelin-induced food intake.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Administración Oral , Animales , Encéfalo/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Ghrelina/farmacología , Enlace de Hidrógeno , Indoles/química , Indoles/metabolismo , Concentración 50 Inhibidora , Ratones , Mutagénesis , Inhibidores de Proteínas Quinasas/metabolismoRESUMEN
AIM: To evaluate the clinical outcomes of Descemet's membrane endothelial keratoplasty (DMEK) in the treatment of patients with Fuchs' endothelial dystrophy. METHODS: This prospective study involved 16 consecutive patients who had DMEK done and who were evaluated over a year. Measurements included best corrected visual acuity (BCVA), endothelial cell count, and central corneal thickness. RESULTS: Two patients had failed grafts. Of the remaining 14 patients with successful grafts, after 12â months, 79% had BCVA of 6/6 or better, and all patients had a BCVA of 6/9 or better. Median endothelial cell count was 1567 cells per mm(2) (range=900-2359) representing a 40% reduction compared with preoperative counts. Median central corneal thickness was 498â µm (range 445-567â µm) compared with a median of 649â µm (range 548-740â µm) preoperatively. All patients attained total visual rehabilitation without further surgical intervention. CONCLUSIONS: In our experience, DMEK has the potential to become the primary procedure for treating Fuchs' endothelial dystrophy and endothelial disease as it produces rapid total visual rehabilitation with few complications, and an easy follow-up and management regimen. Donor preparation and graft insertion, however, remain important challenges.
Asunto(s)
Queratoplastia Endotelial de la Lámina Limitante Posterior/métodos , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/cirugía , Supervivencia de Injerto/fisiología , Agudeza Visual/fisiología , Adulto , Anciano , Recuento de Células , Edema Corneal/etiología , Femenino , Estudios de Seguimiento , Distrofia Endotelial de Fuchs/fisiopatología , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Estudios Prospectivos , Donantes de Tejidos , Resultado del Tratamiento , Reino UnidoRESUMEN
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
Asunto(s)
Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Zinc/química , Línea Celular Tumoral , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Histona Desacetilasas/genética , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Oxadiazoles/química , Relación Estructura-Actividad , Zinc/metabolismoRESUMEN
We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.
Asunto(s)
Piridonas/síntesis química , Piridonas/farmacología , Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Serina Endopeptidasas/efectos de los fármacos , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Conformación Molecular , Estructura Molecular , Prolil Oligopeptidasas , Piridonas/sangre , Piridonas/química , Pirrolidinas/sangre , Pirrolidinas/química , Inhibidores de Serina Proteinasa/sangre , Inhibidores de Serina Proteinasa/químicaRESUMEN
The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. We show that heme binds reversibly to the orphan nuclear receptor Rev-erbalpha, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbalpha-mediated gene repression. Thus, Rev-erbalpha serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism.