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
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/inmunología , Antivirales/farmacología , Endorribonucleasas/inmunología , Exorribonucleasas/química , Inmunidad Innata , Bibliotecas de Moléculas Pequeñas/farmacología , 2',5'-Oligoadenilato Sintetasa/genética , Nucleótidos de Adenina/inmunología , Nucleótidos de Adenina/metabolismo , Antivirales/síntesis química , Cristalografía por Rayos X , Virus de la Encefalomiocarditis/genética , Virus de la Encefalomiocarditis/metabolismo , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/genética , Exorribonucleasas/inmunología , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Interferón-alfa/farmacología , Modelos Moleculares , Oligorribonucleótidos/inmunología , Oligorribonucleótidos/metabolismo , Poli I-C/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-ActividadRESUMEN
Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.