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1.
Nat Commun ; 13(1): 5282, 2022 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-36075902

RESUMEN

Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. Host-directed therapies present an enticing option for this diverse genus of viruses. We have previously identified the actin histidine methyltransferase SETD3 as a critical host factor physically interacting with the viral protease 2A. Here, we report the 3.5 Å cryo-EM structure of SETD3 interacting with coxsackievirus B3 2A at two distinct interfaces, including the substrate-binding surface within the SET domain. Structure-function analysis revealed that mutations of key residues in the SET domain resulted in severely reduced binding to 2A and complete protection from enteroviral infection. Our findings provide insight into the molecular basis of the SETD3-2A interaction and a framework for the rational design of host-directed therapeutics against enteroviruses.


Asunto(s)
Infecciones por Enterovirus , Enterovirus , Antígenos Virales/metabolismo , Endopeptidasas/metabolismo , Enterovirus/genética , Histona Metiltransferasas/metabolismo , Humanos , Péptido Hidrolasas/metabolismo
2.
Science ; 378(6615): eabn5648, 2022 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-36074821

RESUMEN

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.


Asunto(s)
COVID-19 , Lisosomas , Mucolipidosis , Proteínas , Animales , COVID-19/genética , Catepsinas/metabolismo , Humanos , Lisosomas/metabolismo , Manosa/metabolismo , Ratones , Ratones Noqueados , Mucolipidosis/genética , Mucolipidosis/metabolismo , Proteínas/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)
3.
Viruses ; 13(2)2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33499355

RESUMEN

Enteroviruses are among the most common human infectious agents. While infections are often mild, the severe neuropathogenesis associated with recent outbreaks of emerging non-polio enteroviruses, such as EV-A71 and EV-D68, highlights their continuing threat to public health. In recent years, our understanding of how non-polio enteroviruses co-opt cellular pathways has greatly increased, revealing intricate host-virus relationships. In this review, we focus on newly identified mechanisms by which enteroviruses hijack the cellular machinery to promote their replication and spread, and address their potential for the development of host-directed therapeutics. Specifically, we discuss newly identified cellular receptors and their contribution to neurotropism and spread, host factors required for viral entry and replication, and recent insights into lipid acquisition and replication organelle biogenesis. The comprehensive knowledge of common cellular pathways required by enteroviruses could expose vulnerabilities amenable for host-directed therapeutics against a broad spectrum of enteroviruses. Since this will likely include newly arising strains, it will better prepare us for future epidemics. Moreover, identifying host proteins specific to neurovirulent strains may allow us to better understand factors contributing to the neurotropism of these viruses.


Asunto(s)
Enfermedades Virales del Sistema Nervioso Central/virología , Sistema Nervioso Central/virología , Infecciones por Enterovirus/virología , Enterovirus/patogenicidad , Tropismo Viral , Animales , Autofagia , Enterovirus/genética , Enterovirus/fisiología , Genoma Viral , Interacciones Huésped-Patógeno , Humanos , Sitios Internos de Entrada al Ribosoma , Fosfolípidos/biosíntesis , Biosíntesis de Proteínas , ARN Viral/biosíntesis , Receptores Virales/metabolismo , Compartimentos de Replicación Viral/fisiología , Compartimentos de Replicación Viral/ultraestructura , Internalización del Virus , Replicación Viral
4.
Nat Microbiol ; 4(12): 2523-2537, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31527793

RESUMEN

Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.


Asunto(s)
Enterovirus/metabolismo , Enterovirus/patogenicidad , Histona Metiltransferasas/metabolismo , Metiltransferasas/metabolismo , Animales , Sistemas CRISPR-Cas , Enfermedades Virales del Sistema Nervioso Central/virología , Modelos Animales de Enfermedad , Encefalitis Viral , Enterovirus/genética , Infecciones por Enterovirus/virología , Histona Metiltransferasas/genética , Ratones , Mielitis/virología , Enfermedades Neuromusculares/virología , Proteolisis , Proteínas Virales , Replicación Viral
5.
ACS Chem Biol ; 13(12): 3251-3258, 2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30133247

RESUMEN

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.


Asunto(s)
Proteínas Bacterianas/metabolismo , Replicación del ADN/fisiología , Nucleósido-Fosfato Quinasa/metabolismo , Ribonucleótido Reductasas/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/citología , Bacillus subtilis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Proteínas Portadoras/metabolismo , Análisis Discriminante , Endopeptidasa Clp/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica/métodos , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Nucleósido-Fosfato Quinasa/genética , Proteínas Recombinantes de Fusión , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Ácidos Teicoicos/antagonistas & inhibidores , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo
6.
J Med Chem ; 60(6): 2425-2438, 2017 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-28248504

RESUMEN

The emergence of antibiotic-resistant bacterial species, such as vancomycin-resistant enterococci (VRE), necessitates the development of new antimicrobials. Here, we investigate the spectrum of antibacterial activity of three phenylthiazole-substituted aminoguanidines. These compounds possess potent activity against VRE, inhibiting growth of clinical isolates at concentrations as low as 0.5 µg/mL. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis. Transposon mutagenesis suggested three possible targets: YubA, YubB (undecaprenyl diphosphate phosphatase (UPPP)), and YubD. Both UPPP as well as undecaprenyl diphosphate synthase were inhibited by compound 1. YubA and YubD are annotated as transporters and may also be targets because 1 collapsed the proton motive force in membrane vesicles. Using Caenorhabditis elegans, we demonstrate that two compounds (1, 3, at 20 µg/mL) retain potent activity in vivo, significantly reducing the burden of VRE in infected worms. Taken altogether, the results indicate that compounds 1 and 3 warrant further investigation as novel antibacterial agents against drug-resistant enterococci.


Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Tiazoles/química , Tiazoles/farmacología , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Animales , Caenorhabditis elegans , Pared Celular/efectos de los fármacos , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Resistencia a la Vancomicina
7.
PLoS One ; 12(8): e0182821, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28797064

RESUMEN

Bacterial resistance to antibiotics remains an imposing global public health challenge. Of the most serious pathogens, methicillin-resistant Staphylococcus aureus (MRSA) is problematic given strains have emerged that exhibit resistance to several antibiotic classes including ß-lactams and agents of last resort such as vancomycin. New antibacterial agents composed of unique chemical scaffolds are needed to counter this public health challenge. The present study examines two synthetic diphenylurea compounds 1 and 2 that inhibit growth of clinically-relevant isolates of MRSA at concentrations as low as 4 µg/mL and are non-toxic to human colorectal cells at concentrations up to 128 µg/mL. Both compounds exhibit rapid bactericidal activity, completely eliminating a high inoculum of MRSA within four hours. MRSA mutants exhibiting resistance to 1 and 2 could not be isolated, indicating a low likelihood of rapid resistance emerging to these compounds. Bacterial cytological profiling revealed the diphenylureas exert their antibacterial activity by targeting bacterial cell wall synthesis. Both compounds demonstrate the ability to resensitize vancomycin-resistant Staphylococcus aureus to the effect of vancomycin. The present study lays the foundation for further investigation and development of diphenylurea compounds as a new class of antibacterial agents.


Asunto(s)
Antibacterianos/farmacología , Carbanilidas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Antibacterianos/uso terapéutico , Carbanilidas/uso terapéutico , Humanos , Meticilina/farmacología , Meticilina/uso terapéutico , Resistencia a la Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Vancomicina/farmacología , Vancomicina/uso terapéutico
8.
Eur J Med Chem ; 139: 665-673, 2017 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-28846967

RESUMEN

The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages.


Asunto(s)
Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Tiazoles/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Bacillus subtilis/citología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Línea Celular , Pared Celular/metabolismo , Relación Dosis-Respuesta a Droga , Macrófagos/microbiología , Staphylococcus aureus Resistente a Meticilina/citología , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ratones , Estructura Molecular , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química
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