RESUMEN
Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.
Asunto(s)
Neuronas , Canales de Potasio con Entrada de Voltaje , Humanos , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio/metabolismo , Potenciales de Acción/fisiología , Proteínas de la Membrana/metabolismoRESUMEN
The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S2. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported, but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α-subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the binding interactions at S1 and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative subdomain orientations and triggers downstream signal propagation.
Asunto(s)
Microscopía por Crioelectrón , Insulina/química , Insulina/metabolismo , Multimerización de Proteína , Receptor de Insulina/química , Receptor de Insulina/ultraestructura , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Receptor de Insulina/metabolismo , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
We present here a new CryoEM grid boxes storage system designed to simplify sample labeling, tracking and retrieval. The system is based on the crystal pucks widely used by the X-ray crystallographic community for storage and shipping of crystals. This system is suitable for any cryoEM laboratory, but especially for large facilities that will need accurate tracking of large numbers of samples coming from different sources.
Asunto(s)
Microscopía por Crioelectrón/métodos , Manejo de Especímenes/métodos , Automatización de Laboratorios , Preservación Biológica/métodos , Proyectos de Investigación , Manejo de Especímenes/normasRESUMEN
Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53-hDM2 inhibitors was conducted. A structure-activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.
Asunto(s)
Leucina/química , Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Clostridioides difficile/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Clostridioides difficile/química , Clostridioides difficile/genética , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/química , Epítopos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Iminas/farmacología , Pirimidinonas/farmacología , Renina/antagonistas & inhibidores , Administración Oral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Iminas/síntesis química , Iminas/química , Modelos Moleculares , Estructura Molecular , Pirimidinonas/síntesis química , Pirimidinonas/química , Renina/metabolismo , Relación Estructura-ActividadRESUMEN
Tertiary hydroxyl class of C-imidazole bridgehead azaheptapyridine FPT inhibitors were prepared in an attempt to block in vivo oxidation of secondary hydroxyl series. One representative compound 5a exhibited potent enzyme (IC50=1.4 nM) and cellular activities (soft agar IC50=1.3 nM) with excellent oral pharmacokinetic profiles in rats, mice, monkeys and dogs. The in vivo study in wap-ras TG mouse models showed dose dependent tumor growth inhibition and regression.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/farmacología , Compuestos Aza/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Piridinas/farmacología , Transferasas Alquil y Aril/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Compuestos Aza/síntesis química , Compuestos Aza/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Haplorrinos , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/patología , Piridinas/síntesis química , Piridinas/química , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aß40 levels upon subcutaneous and oral administration to rats.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Ácido Aspártico Endopeptidasas/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Resonancia Magnética Nuclear Biomolecular , Bibliotecas de Moléculas Pequeñas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
From an initial lead 1, a structure-based design approach led to identification of a novel, high-affinity iminohydantoin BACE1 inhibitor that lowers CNS-derived Aß following oral administration to rats. Herein we report SAR development in the S3 and F' subsites of BACE1 for this series, the synthetic approaches employed in this effort, and in vivo data for the optimized compound.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Anticonvulsivantes/síntesis química , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Hidantoínas/síntesis química , Administración Oral , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticonvulsivantes/farmacología , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Hidantoínas/farmacología , Modelos Moleculares , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Recent evidence suggests that deletion of STUB1âa pivotal negative regulator of interferon-γ sensingâmay potentially clear malignant cells. However, current studies rely primarily on genetic approaches, as pharmacological inhibitors of STUB1 are lacking. Identifying a tool compound will be a step toward validating the target in a broader therapeutic sense. Herein, screening more than a billion macrocyclic peptides resulted in STUB1 binders, which were further optimized by a structure-enabled in silico design. The strategy to replace the macrocyclic peptides' hydrophilic and solvent-exposed region with a hydrophobic scaffold improved cellular permeability while maintaining the binding conformation. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to a certain extent while improving permeability, suggesting a path forward. Although not optimal for cellular study, the current lead provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.
RESUMEN
TNF-α converting enzyme (TACE) inhibitors are promising agents to treat inflammatory disorders and cancer. We have investigated novel tartrate diamide TACE inhibitors where the tartrate core binds to zinc in a unique tridentate fashion. Incorporating (R)-2-(2-N-alkylaminothiazol-4-yl)pyrrolidines into the left hand side amide of the tartrate scaffold led to the discovery of potent and selective TACE inhibitors, some of which exhibited good rat oral bioavailability.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Pirrolidinas/química , Tartratos/química , Proteína ADAM17 , Amidas/síntesis química , Amidas/química , Animales , Disponibilidad Biológica , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , RatasRESUMEN
Arginase (EC 3.5.3.1) is an aminohydrolase that acts on L-arginine to produce urea and ornithine. Two isotypes of the enzyme are found in humans. Type I is predominantly produced in the liver and is a homotrimer of 35â kDa subunits. Human arginase (hArginase) I is seen to be up-regulated in many diseases and is a potential therapeutic target for many diverse indications. Previous reports of crystallization and structure determination of hArginase have always included inhibitors of the enzyme: here, the first case of a true apo crystal form of the enzyme which is suitable for small-molecule soaking is reported. The crystals belonged to space group P2(1)2(1)2(1) and have approximate unit-cell parameters a=53, b=67.5, c=250â Å. The crystals showed slightly anisotropic diffraction to beyond 2.0â Å resolution.
Asunto(s)
Arginasa/química , Isoenzimas/química , Animales , Arginasa/antagonistas & inhibidores , Cristalización , Humanos , Isoenzimas/antagonistas & inhibidores , Datos de Secuencia MolecularRESUMEN
Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low µM range, typical of many specific and biologically relevant protein-protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab'2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab'2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development.
RESUMEN
Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.
Asunto(s)
Aminopiridinas/química , Azepinas/química , Antagonistas de los Receptores de Orexina/química , Receptores de Orexina/química , Péptidos/química , Fármacos Inductores del Sueño/química , Sulfonamidas/química , Triazoles/química , Aminopiridinas/metabolismo , Azepinas/metabolismo , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Antagonistas de los Receptores de Orexina/metabolismo , Receptores de Orexina/agonistas , Receptores de Orexina/metabolismo , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fármacos Inductores del Sueño/metabolismo , Sulfonamidas/metabolismo , Triazoles/metabolismoRESUMEN
Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.
Asunto(s)
Arginasa/genética , Arginasa/metabolismo , Arginina/química , Sitios de Unión , Microscopía por Crioelectrón , Ornitina/química , Unión Proteica , Especificidad por SustratoRESUMEN
By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.
RESUMEN
The discovery of C-linked imidazole azaheptapyridine bridgehead FPT inhibitors is described. This novel class of compounds are sub nM FPT enzyme inhibitors with potent cellular inhibitory activities. This series also has reduced hERG activity versus previous N-linked imidazole series. X-ray of compound 10a bound to FTase revealed strong interaction between bridgehead imidazole 3N with catalytic zinc atom.
Asunto(s)
Descubrimiento de Drogas/métodos , Farnesiltransferasa/antagonistas & inhibidores , Imidazoles/química , Piridinas/química , Línea Celular Tumoral , Cristalografía por Rayos X , Farnesiltransferasa/metabolismo , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Piridinas/metabolismo , Piridinas/farmacologíaRESUMEN
With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2' sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Abeta(40) in transgenic mice with a single subcutaneous dose.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Piperazinas/química , Inhibidores de Proteasas/síntesis química , Sulfonamidas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Ratones , Ratones Transgénicos , Modelos Moleculares , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Piperazina , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/uso terapéuticoRESUMEN
A novel series of TNF-alpha convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of L-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP's. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.