RESUMEN
Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Cadenas Pesadas de Inmunoglobulina/administración & dosificación , Región Variable de Inmunoglobulina/administración & dosificación , Biblioteca de Péptidos , Neumonía Viral/tratamiento farmacológico , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/ultraestructura , Afinidad de Anticuerpos , COVID-19 , Cricetinae , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/ultraestructura , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/ultraestructura , Ratones , Ratones Endogámicos BALB C , Mutación , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Tratamiento Farmacológico de COVID-19RESUMEN
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/inmunología , Bacteriófagos/genética , Bacteriófagos/inmunología , Proteínas Asociadas a CRISPR/inmunología , Proteínas Asociadas a CRISPR/ultraestructura , ADN Viral/química , Modelos Químicos , Modelos Moleculares , Complejos Multiproteicos/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestructuraRESUMEN
Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ â¼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
Asunto(s)
Descubrimiento de Drogas , Glutamato Deshidrogenasa/ultraestructura , Isocitrato Deshidrogenasa/ultraestructura , L-Lactato Deshidrogenasa/ultraestructura , Aminoquinolinas/química , Aminoquinolinas/farmacología , Animales , Bovinos , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/química , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Modelos Moleculares , Conformación Proteica , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD â¼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (â¼ 10-25 Å) by hinge-like motions as large as â¼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Proteínas de Transporte de Catión/ultraestructura , Magnesio/metabolismo , Thermotoga maritima/química , Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Microscopía por Crioelectrón , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.
Asunto(s)
Diagnóstico por Imagen , Humanos , Microscopía por CrioelectrónRESUMEN
Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus-host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Humanos , Péptido Hidrolasas , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
The recently reported "UK variant" (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , COVID-19/virología , Microscopía por Crioelectrón , Epítopos , Humanos , Modelos Moleculares , Mutación , Pruebas de Neutralización , Unión Proteica , Conformación Proteica , Dominios Proteicos , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The global spread of SARS-CoV-2 has proceeded at an unprecedented rate. Remarkably, characterization of the virus using modern tools in structural biology has also progressed at exceptional speed. Advances in electron-based imaging techniques, combined with decades of foundational studies on related viruses, have enabled the research community to rapidly investigate structural aspects of the novel coronavirus from the level of individual viral proteins to imaging the whole virus in a native context. Here, we provide a detailed review of the structural biology and pathobiology of SARS-CoV-2 as it relates to all facets of the viral life cycle, including cell entry, replication, and three-dimensional (3D) packaging based on insights obtained from X-ray crystallography, cryo-electron tomography, and single-particle cryo-electron microscopy. The structural comparison between SARS-CoV-2 and the related earlier viruses SARS-CoV and MERS-CoV is a common thread throughout this review. We conclude by highlighting some of the outstanding unanswered structural questions and underscore areas that are under rapid current development such as the design of effective therapeutics that block viral infection.
Asunto(s)
COVID-19 , SARS-CoV-2 , Microscopía por Crioelectrón , Humanos , Imagenología Tridimensional , Estructuras ViralesRESUMEN
In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.
RESUMEN
G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the ß2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Rodopsina/metabolismo , Rodopsina/ultraestructura , Arrestina/química , Arrestina/metabolismo , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Rodopsina/química , Transducción de Señal , Especificidad por SustratoRESUMEN
Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.
Asunto(s)
Ehrlichia chaffeensis/metabolismo , Ehrlichiosis/patología , Cuerpos de Inclusión/metabolismo , Fosfatidilcolinas/metabolismo , Vacuolas/microbiología , Animales , Autofagosomas/metabolismo , Membrana Celular/metabolismo , Perros , Ehrlichia chaffeensis/citología , Ehrlichia chaffeensis/patogenicidad , Ehrlichiosis/sangre , Ehrlichiosis/microbiología , Endosomas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Cuerpos de Inclusión/ultraestructura , Microscopía Intravital , Microscopía Electrónica de Rastreo , Células THP-1 , Imagen de Lapso de Tiempo , Vacuolas/ultraestructuraRESUMEN
The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function.
Asunto(s)
Mutación Missense , Proteína que Contiene Valosina/química , Regulación Alostérica , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this 'desensitization ring' is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.
Asunto(s)
Microscopía por Crioelectrón , Receptores de Ácido Kaínico/metabolismo , Receptores de Ácido Kaínico/ultraestructura , Animales , Sitios de Unión , Regulación hacia Abajo , Activación del Canal Iónico , Ligandos , Modelos Moleculares , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Receptores de Ácido Kaínico/química , Receptor de Ácido Kaínico GluK2RESUMEN
Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
Asunto(s)
Neurofibromina 1/química , Neurofibromina 1/metabolismo , Multimerización de Proteína , Células HEK293 , Humanos , Neurofibromina 1/ultraestructura , Dominios Proteicos , Relación Estructura-Actividad , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion; however, genetic evidence suggests that facilitated diffusion is not critical for normal function. We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.
Asunto(s)
Metabolismo Energético , Mitocondrias Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Difusión , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Fuerza Protón-MotrizRESUMEN
ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at â¼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/ultraestructura , Nucleosomas/ultraestructura , Factores de Transcripción/ultraestructura , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/ultraestructura , Animales , Chaetomium/genética , Chaetomium/ultraestructura , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Escherichia coli/genética , Histonas/química , Histonas/ultraestructura , Nucleosomas/genética , Unión Proteica/genética , Dominios Proteicos/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
On May 24, 2019, the Food and Drug Administration approved ruxolitinib for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in adult and pediatric patients 12 years and older. Approval was based on Study INCB 18424-271 (REACH-1; NCT02953678), an open-label, single-arm, multicenter trial that included 49 patients with grades 2-4 SR-aGVHD occurring after allogeneic hematopoietic stem cell transplantation. Ruxolitinib was administered at 5 mg twice daily, with dose increases to 10 mg twice daily permitted after 3 days in the absence of toxicity. The Day-28 overall response rate was 57.1% (95% confidence interval [CI]: 42.2-71.2). The median duration of response was 0.5 months (95% CI: 0.3-2.7), and the median time from Day-28 response to either death or need for new therapy for acute GVHD was 5.7 months (95% CI: 2.2 to not estimable). Common adverse reactions included anemia, thrombocytopenia, neutropenia, infections, edema, bleeding, and elevated transaminases. Ruxolitinib is the first drug approved for treatment of SR-aGVHD. IMPLICATIONS FOR PRACTICE: Ruxolitinib is the first Food and Drug Administration-approved treatment for steroid-refractory acute graft-versus-host disease in adult and pediatric patients 12 years and older. Its approval provides a treatment option for the 60% of those patients who do not respond to steroid therapy.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Adulto , Niño , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Nitrilos , Pirazoles/efectos adversos , Pirimidinas , Esteroides/uso terapéuticoAsunto(s)
Algoritmos , Biología Computacional/métodos , Bases de Datos de Proteínas , Modelos Moleculares , Proteínas/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopía por Crioelectrón , Bases de Datos de Proteínas/estadística & datos numéricos , Humanos , Ligandos , Conformación Proteica , Mapas de Interacción de Proteínas , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of â¼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein-coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.