RESUMEN
MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.
Asunto(s)
Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Regulación de la Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Linfocitos/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/ultraestructura , FN-kappa B/metabolismo , Proteínas de Neoplasias , Transducción de SeñalRESUMEN
Complement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop.
RESUMEN
Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.
Asunto(s)
Inhibidores de Proteasas/farmacología , Dominio Catalítico , Factor D del Complemento/química , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Proteasas/químicaRESUMEN
CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.
Asunto(s)
Complejo del Señalosoma COP9/antagonistas & inhibidores , Indoles/metabolismo , Zinc/metabolismo , Sitios de Unión , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Dominio Catalítico , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Células HCT116 , Humanos , Indoles/química , Indoles/farmacología , Simulación del Acoplamiento Molecular , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Zinc/químicaRESUMEN
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
Asunto(s)
Factor D del Complemento/antagonistas & inhibidores , Vía Alternativa del Complemento/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Factor D del Complemento/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Adenoviral infections are associated with a wide range of acute diseases, among which ocular viral conjunctivitis (EKC) and disseminated disease in immunocompromised patients. To date, no approved specific anti-adenoviral drug is available, but there is a growing need for an effective treatment of such infections. The adenoviral protease, adenain, plays a crucial role for the viral lifecycle and thus represents an attractive therapeutic target. Structure-guided design with the objective to depeptidize tetrapeptide nitrile 1 led to the novel chemotype 2. Optimization of scaffold 2 resulted in picomolar adenain inhibitors 3a and 3b. In addition, a complementary series of irreversible vinyl sulfone containing inhibitors were rationally designed, prepared and evaluated against adenoviral protease. High resolution X-ray co-crystal structures of representatives of each series proves the successful design of these inhibitors and provides an excellent basis for future medicinal chemistry optimization of these compounds.
Asunto(s)
Adenoviridae/enzimología , Antivirales/química , Cisteína Endopeptidasas/química , Diseño de Fármacos , Inhibidores de Proteasas/química , Proteínas Virales/antagonistas & inhibidores , Adenoviridae/efectos de los fármacos , Antivirales/metabolismo , Antivirales/toxicidad , Sitios de Unión , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/toxicidad , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteínas Virales/metabolismoRESUMEN
The cysteine protease adenain is the essential protease of adenovirus and, as such, represents a promising target for the treatment of ocular and other adenoviral infections. Through a concise two-pronged hit discovery approach we identified tetrapeptide nitrile 1 and pyrimidine nitrile 2 as complementary starting points for adenain inhibition. These hits enabled the first high-resolution X-ray cocrystal structures of adenain with inhibitors bound and revealed the binding mode of 1 and 2. The screening hits were optimized by a structure-guided medicinal chemistry strategy into low nanomolar drug-like inhibitors of adenain.
RESUMEN
The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.
Asunto(s)
Virus del Dengue/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Sitios de Unión , Activación Enzimática , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Oligopéptidos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismoRESUMEN
The covalent attachment of heme cofactors to the apo-polypeptides via thioether bonds is unique to the maturation of c-type cytochromes. A number of thiol-disulfide oxidoreductases prepare the apocytochrome for heme insertion in system I and II cytochrome c maturation. Although most thiol-disulfide oxidoreductases are nonspecific, the less common, specific thiol-disulfide oxidoreductases may be key to directing the usage of electrons. Here we demonstrate that unlike other thiol-disulfide oxidoreductases, the protein responsible for reducing oxidized apocytochrome c in Bacillus subtilis, ResA, is specific for cytochrome c550 and utilizes alternate conformations to recognize redox partners. We report solution NMR evidence that ResA undergoes a redox-dependent conformational change between oxidation states, as well as data showing that ResA utilizes a surface cavity present only in the reduced state to recognize a peptide derived from cytochrome c550. Finally, we confirm that ResA is a specific thiol-disulfide oxidoreductase by comparing its reactivity to our mimetic peptide with its reactivity to oxidized glutathione, a nonspecific substrate. This study biochemically demonstrates the specificity of this thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Grupo Citocromo c/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/química , Proteínas Bacterianas/metabolismo , Grupo Citocromo c/química , Transporte de Electrón , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Conformación Proteica , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Soluciones , Especificidad por Sustrato , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B-NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.
Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Virus del Dengue/enzimología , Serina Endopeptidasas/aislamiento & purificación , Proteínas no Estructurales Virales/aislamiento & purificación , Virus del Nilo Occidental/enzimología , Cristalización , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Virus del Dengue/aislamiento & purificación , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Serina Endopeptidasas/química , Proteínas no Estructurales Virales/química , Virus del Nilo Occidental/aislamiento & purificaciónRESUMEN
The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous bHLH-PAS (Per-ARNT-Sim) protein that forms heterodimeric transcriptional regulator complexes with several other bHLH-PAS subunits to control a variety of biological pathways, some of which are centrally involved in disease initiation and/or progression. One of these is the hypoxia response pathway, which allows eukaryotic cells to respond to low oxygen tension via the formation of a heterodimeric complex between ARNT and another bHLH-PAS protein, the hypoxia-inducible factor alpha (HIF-alpha). We have previously shown that the C-terminal PAS domains of an HIF-alpha isoform (HIF-2alpha) and ARNT interact in vitro, and that mutations in the solvent-exposed beta-sheet surface of the HIF-2alpha domain not only disrupt this interaction, but also greatly attenuate the hypoxia response in living cells. Here, we have solved the solution structure of the corresponding PAS domain of ARNT and show that it utilizes a very similar interface for the interaction with the HIF-2alpha PAS domain. We also show that this domain self-associates in a concentration-dependent manner, and that the interface used in this homodimeric complex is very similar to that used in the formation of heterodimer. In addition, using experimentally derived NMR restraints, we used the program HADDOCK to calculate a low-resolution model of the complex formed in solution by these two PAS domains, and confirm the validity of this model using site-directed spin labeling to obtain long-range distance information in solution. With this information, we propose a model for the mode of multi-PAS domain interaction in bHLH-PAS transcriptional activation complexes.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Dimerización , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Electricidad EstáticaRESUMEN
The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O2-dependent regulation of HIF through its O2-dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2alpha PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-alpha subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.
Asunto(s)
Factor 1 Inducible por Hipoxia/química , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Células CHO , Línea Celular , Núcleo Celular/metabolismo , Cricetinae , Citoplasma/metabolismo , Análisis Mutacional de ADN , Dimerización , Eliminación de Gen , Genes Reporteros , Proteínas HSP90 de Choque Térmico/metabolismo , Hipoxia , Factor 1 Inducible por Hipoxia/fisiología , Inmunoprecipitación , Modelos Genéticos , Mutación , Proteínas Nucleares/metabolismo , Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Activación Transcripcional , TransfecciónRESUMEN
We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.
Asunto(s)
Antígenos Bacterianos/química , Espectroscopía de Resonancia Magnética , Polisacáridos Bacterianos/química , Escherichia coli/químicaRESUMEN
Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.
Asunto(s)
Transactivadores/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Hipoxia de la Célula , Clonación Molecular , Dimerización , Secuencias Hélice-Asa-Hélice , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Phosphoglyceride-linked enterobacterial common antigen (ECA(PG)) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECA(PG) consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECA(CYC), purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECA(CYC) molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECA(CYC) was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECA(CYC) and ECA(PG) are assembled by a common biosynthetic pathway.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/química , Escherichia coli/inmunología , Antígenos Bacterianos/aislamiento & purificación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Electroforesis/métodos , Enterobacteriaceae/inmunología , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , Transactivadores/aislamiento & purificación , Transactivadores/metabolismoRESUMEN
Glycosylation of Asn-52 of the alpha-subunit (alphaAsn-52) is required for bioactivity of the alphabeta-dimeric human chorionic gonadotropin (hCG), although at a molecular level the effect of the glycan at alphaAsn-52 is not yet understood. To study the role of this glycan for heterodimer stability, the beta-subunit was recombined in solution with either the alpha-subunit or the alpha-subunit enzymically deglycosylated at alphaAsn-52. Enzymic deglycosylation avoids modification of the glycans at alphaAsn-78 and disturbing the protein folding. The efficiency of recombination after 16 h is 80%, independent of whether alphaAsn-52 is glycosylated or not. The dissociation constant of the hCG complex, with or without the glycan at alphaAsn-52, is less than 1 x 10(-5) s(-1), indicating that the glycan at alphaAsn-52 does not contribute significantly to the stability of the dimer. CD and NMR spectra indicate a local conformational difference between both alphabeta-dimeric hCG variants, most probably involving amino acids of the hCG beta-subunit close to the glycan at alphaAsn-52. These data explain the native-like receptor-binding abilities of hCG lacking the glycan at alphaAsn-52. It is proposed that for bioactivity the glycan at alphaAsn-52 is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of the signal-transduction pathway.