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1.
Signal Transduct Target Ther ; 9(1): 144, 2024 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-38853183

RESUMEN

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.


Asunto(s)
Piridinas , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Proteínas Virales de Fusión , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/genética , Piridinas/farmacología , Ratones , Animales , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Virus Sincitial Respiratorio Humano/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/antagonistas & inhibidores , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/genética , Antivirales/farmacología , Antivirales/química , Piperidinas/farmacología , Piperidinas/química , Ratones Endogámicos BALB C , Conformación Proteica , Dibenzocicloheptenos
2.
Molecules ; 27(2)2022 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-35056869

RESUMEN

Hendra virus (HeV) belongs to the paramyxoviridae family of viruses which is associated with the respiratory distress, neurological illness, and potential fatality of the affected individuals. So far, no competitive approved therapeutic substance is available for HeV. For that reason, the current research work was conducted to propose some novel compounds, by adopting a Computer Aided Drug Discovery approach, which could be used to combat HeV. The G attachment Glycoprotein (Ggp) of HeV was selected to achieve the primary objective of this study, as this protein makes the entry of HeV possible in the host cells. Briefly, a library of 6000 antiviral compounds was screened for potential drug-like properties, followed by the molecular docking of short-listed compounds with the Protein Data Bank (PDB) structure of Ggp. Docked complexes of top two hits, having maximum binding affinities with the active sites of Ggp, were further considered for molecular dynamic simulations of 200 ns to elucidate the results of molecular docking analysis. MD simulations and Molecular Mechanics Energies combined with the Generalized Born and Surface Area (MMGBSA) or Poisson-Boltzmann and Surface Area (MMPBSA) revealed that both docked complexes are stable in nature. Furthermore, the same methodology was used between lead compounds and HeV Ggp in complex with its functional receptor in human, Ephrin-B2. Surprisingly, no major differences were found in the results, which demonstrates that our identified compounds can also perform their action even when the Ggp is attached to the Ephrin-B2 ligand. Therefore, in light of all of these results, we strongly suggest that compounds (S)-5-(benzylcarbamoyl)-1-(2-(4-methyl-2-phenylpiperazin-1-yl)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide and 5-(cyclohexylcarbamoyl)-1-(2-((2-(3-fluorophenyl)-2-methylpropyl)amino)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide could be considered as potential therapeutic agents against HeV; however, further in vitro and in vivo experiments are required to validate this study.


Asunto(s)
Antivirales/química , Química Computacional/métodos , Proteínas Virales de Fusión/química , Antivirales/metabolismo , Efrina-B2/química , Efrina-B2/metabolismo , Virus Hendra/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Receptores Virales/química , Receptores Virales/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/metabolismo , Agua/química
4.
MAbs ; 13(1): 1912884, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33876699

RESUMEN

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antivirales/farmacología , Pulmón/virología , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Infecciones por Respirovirus/prevención & control , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos B/virología , Línea Celular , Modelos Animales de Enfermedad , Epítopos , Interacciones Huésped-Patógeno , Humanos , Huésped Inmunocomprometido , Pulmón/inmunología , Virus de la Parainfluenza 3 Humana/inmunología , Virus de la Parainfluenza 3 Humana/patogenicidad , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Sigmodontinae , Proteínas Virales de Fusión/inmunología
5.
J Gen Virol ; 102(1)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33054904

RESUMEN

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19/diagnóstico , Infecciones por Henipavirus/diagnóstico , Ensayos Analíticos de Alto Rendimiento , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , COVID-19/inmunología , COVID-19/virología , Fusión Celular , Convalecencia , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Sueros Inmunes/química , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Virus Nipah/inmunología , Virus Nipah/patogenicidad , Conformación Proteica , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/inmunología , Virus Sincitial Respiratorio Humano/patogenicidad , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Porcinos , Inhibidores de Proteínas Virales de Fusión/química , Inhibidores de Proteínas Virales de Fusión/metabolismo , Inhibidores de Proteínas Virales de Fusión/farmacología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología
6.
Bioorg Med Chem ; 28(24): 115818, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33190073

RESUMEN

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.


Asunto(s)
Antivirales/química , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Antivirales/metabolismo , Antivirales/farmacología , Sitios de Unión , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Isomerismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Ratones , Simulación de Dinámica Molecular , Mutación , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacología , Relación Estructura-Actividad , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Internalización del Virus/efectos de los fármacos
7.
Proc Natl Acad Sci U S A ; 117(44): 27637-27645, 2020 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-33087569

RESUMEN

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Aedes , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/uso terapéutico , Anticuerpos Antivirales/ultraestructura , Sitios de Unión/efectos de los fármacos , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/virología , Virus Chikungunya/inmunología , Chlorocebus aethiops , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Humanos , Inmunoglobulinas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Células Vero , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/inmunología , Acoplamiento Viral/efectos de los fármacos
8.
mBio ; 11(5)2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32934085

RESUMEN

Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.


Asunto(s)
Ebolavirus/química , Glicoproteínas/antagonistas & inhibidores , VIH-1/química , Hemaglutininas Virales/metabolismo , Subtipo H5N1 del Virus de la Influenza A/química , Ubiquitina-Proteína Ligasas/genética , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Ebolavirus/fisiología , Glicosilación , Células HEK293 , VIH-1/fisiología , Células HeLa , Células Hep G2 , Humanos , Subtipo H5N1 del Virus de la Influenza A/fisiología , Unión Proteica , Células THP-1 , Ubiquitina-Proteína Ligasas/metabolismo , Células Vero , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/metabolismo
9.
J Drug Target ; 28(10): 1046-1052, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32643453

RESUMEN

Attachment of a virus with a specific receptor on the cell surface is the first and foremost step in virus infection. In case of enveloped viruses, their interaction with the host cell receptor is mediated by viral encoded glycoproteins on its envelope, a host derived lipid bilayer. Since, virus entry is a multistep process, after receptor recognition, envelope proteins mediate internalisation of virus particles into the host cell. Envelope glycoproteins are the first proteins that the host immune system encounters upon infection. Thus, envelope proteins are important drug target with multiple strategies to inhibit entry of the virus into the host. Currently, there are very few drugs that function as envelope protein inhibitors which are approved for human use. Here, we reviewed different classes of envelope proteins of various viruses and emphasised the use of small molecules to inhibit fusion of envelope proteins. Based on the available information in the literature, envelope proteins can be important drug targets and small molecules inhibitors can serve as potential antiviral drugs to block viral infection at an initial stage.


Asunto(s)
Antivirales/farmacología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Membrana Celular/fisiología , Humanos , Concentración de Iones de Hidrógeno , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/metabolismo , Internalización del Virus/efectos de los fármacos
10.
N Engl J Med ; 383(5): 415-425, 2020 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-32726528

RESUMEN

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, and a need exists for prevention of RSV in healthy infants. Nirsevimab is a monoclonal antibody with an extended half-life that is being developed to protect infants for an entire RSV season with a single intramuscular dose. METHODS: In this trial conducted in both northern and southern hemispheres, we evaluated nirsevimab for the prevention of RSV-associated lower respiratory tract infection in healthy infants who had been born preterm (29 weeks 0 days to 34 weeks 6 days of gestation). We randomly assigned the infants in a 2:1 ratio to receive nirsevimab, at a dose of 50 mg in a single intramuscular injection, or placebo at the start of an RSV season. The primary end point was medically attended RSV-associated lower respiratory tract infection through 150 days after administration of the dose. The secondary efficacy end point was hospitalization for RSV-associated lower respiratory tract infection through 150 days after administration of the dose. RESULTS: From November 2016 through November 2017, a total of 1453 infants were randomly assigned to receive nirsevimab (969 infants) or placebo (484 infants) at the start of the RSV season. The incidence of medically attended RSV-associated lower respiratory tract infection was 70.1% lower (95% confidence interval [CI], 52.3 to 81.2) with nirsevimab prophylaxis than with placebo (2.6% [25 infants] vs. 9.5% [46 infants]; P<0.001) and the incidence of hospitalization for RSV-associated lower respiratory tract infection was 78.4% lower (95% CI, 51.9 to 90.3) with nirsevimab than with placebo (0.8% [8 infants] vs. 4.1% [20 infants]; P<0.001). These differences were consistent throughout the 150-day period after the dose was administered and across geographic locations and RSV subtypes. Adverse events were similar in the two trial groups, with no notable hypersensitivity reactions. CONCLUSIONS: A single injection of nirsevimab resulted in fewer medically attended RSV-associated lower respiratory tract infections and hospitalizations than placebo throughout the RSV season in healthy preterm infants. (Funded by AstraZeneca and Sanofi Pasteur; ClinicalTrials.gov number, NCT02878330.).


Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antivirales/administración & dosificación , Recien Nacido Prematuro , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Humano , Infecciones del Sistema Respiratorio/prevención & control , Proteínas Virales de Fusión/antagonistas & inhibidores , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Antivirales/efectos adversos , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Incidencia , Lactante , Recién Nacido , Inyecciones Intramusculares , Estimación de Kaplan-Meier , Masculino , Distribución de Poisson , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología
11.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165889, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32603829

RESUMEN

The novel Coronavirus disease of 2019 (nCOV-19) is a viral outbreak noted first in Wuhan, China. This disease is caused by Severe Acute Respiratory Syndrome (SARS) Coronavirus (CoV)-2. In the past, other members of the coronavirus family, such as SARS and Middle East Respiratory Syndrome (MERS), have made an impact in China and the Arabian peninsula respectively. Both SARS and COVID-19 share similar symptoms such as fever, cough, and difficulty in breathing that can become fatal in later stages. However, SARS and MERS infections were epidemic diseases constrained to limited regions. By March 2020 the SARS-CoV-2 had spread across the globe and on March 11th, 2020 the World Health Organization (WHO) declared COVID-19 as pandemic disease. In severe SARS-CoV-2 infection, many patients succumbed to pneumonia. Higher rates of deaths were seen in older patients who had co-morbidities such as diabetes mellitus, hypertension, cardiovascular disease (CVD), and dementia. In this review paper, we discuss the effect of SARS-CoV-2 on CNS diseases, such as Alzheimer's-like dementia, and diabetes mellitus. We also focus on the virus genome, pathophysiology, theranostics, and autophagy mechanisms. We will assess the multiorgan failure reported in advanced stages of SARS-CoV-2 infection. Our paper will provide mechanistic clues and therapeutic targets for physicians and investigators to combat COVID-19.


Asunto(s)
Enfermedades del Sistema Nervioso Central/patología , Infecciones por Coronavirus/patología , Neumonía Viral/patología , Animales , Antivirales/uso terapéutico , Betacoronavirus/aislamiento & purificación , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidad , COVID-19 , Enfermedades del Sistema Nervioso Central/complicaciones , Enfermedades del Sistema Nervioso Central/virología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Humanos , Pulmón/metabolismo , Pulmón/virología , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , SARS-CoV-2 , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo
12.
J Virol ; 94(18)2020 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-32611759

RESUMEN

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.


Asunto(s)
Ebolavirus/efectos de los fármacos , Proteína Niemann-Pick C1/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas Virales de Fusión/antagonistas & inhibidores , Virión/efectos de los fármacos , Animales , Sitios de Unión , Bioensayo , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacología , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Calor , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Proteína Niemann-Pick C1/química , Proteína Niemann-Pick C1/genética , Proteína Niemann-Pick C1/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacología , Toremifeno/química , Toremifeno/farmacología , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Virión/química , Virión/genética , Virión/metabolismo
13.
ACS Infect Dis ; 6(5): 922-929, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-32275393

RESUMEN

A series of five benzimidazole-based compounds were identified using a machine learning algorithm as potential inhibitors of the respiratory syncytial virus (RSV) fusion protein. These compounds were synthesized, and compound 2 in particular exhibited excellent in vitro potency with an EC50 value of 5 nM. This new scaffold was then further refined leading to the identification of compound 44, which exhibited a 10-fold improvement in activity with an EC50 value of 0.5 nM.


Asunto(s)
Antivirales , Bencimidazoles/farmacología , Virus Sincitial Respiratorio Humano , Proteínas Virales de Fusión/antagonistas & inhibidores , Antivirales/farmacología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Relación Estructura-Actividad
14.
Sci Rep ; 10(1): 4746, 2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32179788

RESUMEN

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-ß (Aß) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).


Asunto(s)
Antivirales/farmacología , Infecciones por Virus ADN/metabolismo , Virus ADN/efectos de los fármacos , Infecciones por Virus ARN/metabolismo , Virus ARN/efectos de los fármacos , Salicilatos/farmacología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Astrocitos/metabolismo , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , Infecciones por Virus ADN/virología , Virus ADN/genética , ADN Viral/genética , Células HEK293 , Humanos , Infecciones por Virus ARN/virología , Virus ARN/genética , Células Vero , Proteínas del Envoltorio Viral/biosíntesis , Proteínas Virales de Fusión/biosíntesis , Virión/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos
15.
mBio ; 11(1)2020 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-32047132

RESUMEN

The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.


Asunto(s)
Antivirales/farmacología , Proteína HN/metabolismo , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Proteínas Virales de Fusión/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Antivirales/aislamiento & purificación , Técnicas de Cultivo de Célula , Línea Celular , Descubrimiento de Drogas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Proteína HN/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Simulación del Acoplamiento Molecular , Virus de la Parainfluenza 3 Humana/fisiología , Infecciones por Paramyxoviridae/tratamiento farmacológico , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Virales de Fusión/metabolismo
16.
Artículo en Inglés | MEDLINE | ID: mdl-31712214

RESUMEN

Effective treatments for respiratory syncytial virus (RSV) infection are lacking. Here, we report a human proof-of-concept study for RV521, a small-molecule antiviral inhibitor of the RSV-F protein. In this randomized, double-blind, placebo-controlled trial, healthy adults were challenged with RSV-A Memphis-37b. After infection was confirmed (or 5 days after challenge virus inoculation), subjects received RV521 (350 mg or 200 mg) or placebo orally every 12 h for 5 days. The primary endpoint was area under the curve (AUC) for viral load, as assessed by reverse transcriptase quantitative PCR (RT-qPCR) of nasal wash samples. The primary efficacy analysis set included subjects successfully infected with RSV who received ≥1 dose of study drug. A total of 66 subjects were enrolled (n = 22 per group); 53 were included in the primary analysis set (RV521 350 mg: n = 16; 200 mg: n = 18; placebo: n = 19). The mean AUC of RT-qPCR-assessed RSV viral load (log10 PFU equivalents [PFUe]/ml · h) was significantly lower with RV521 350 mg (185.26; standard error [SE], 31.17; P = 0.002) and 200 mg (224.35; SE, 37.60; P = 0.007) versus placebo (501.39; SE, 86.57). Disease severity improved with RV521 350 mg and 200 mg versus placebo (P = 0.002 and P = 0.009, respectively, for AUC total symptom score [score × hours]). Daily nasal mucus weight was significantly reduced (P = 0.010 and P = 0.038 for RV521 350 mg and 200 mg, respectively, versus placebo). All treatment-emergent adverse events were grade 1 or 2. No subjects discontinued due to adverse events. There was no evidence of clinically significant viral resistance, and only three variants were detected. RV521 effectively reduced RSV viral load and disease severity in humans and was well tolerated. (This study has been registered at ClinicalTrials.gov under registration no. NCT03258502.).


Asunto(s)
Antivirales/farmacología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Proteínas Virales de Fusión/antagonistas & inhibidores , Adolescente , Adulto , Antivirales/farmacocinética , Área Bajo la Curva , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Placebos , Infecciones por Virus Sincitial Respiratorio/virología , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Carga Viral/efectos de los fármacos , Adulto Joven
17.
Nat Struct Mol Biol ; 26(10): 980-987, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31570878

RESUMEN

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antivirales/farmacología , Virus Hendra/efectos de los fármacos , Infecciones por Henipavirus/tratamiento farmacológico , Virus Nipah/efectos de los fármacos , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Células HEK293 , Virus Hendra/metabolismo , Infecciones por Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Humanos , Modelos Moleculares , Virus Nipah/metabolismo , Proteínas Virales de Fusión/metabolismo , Internalización del Virus/efectos de los fármacos
18.
J Med Chem ; 62(13): 6003-6014, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31194544

RESUMEN

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.


Asunto(s)
Antivirales/uso terapéutico , Benzazepinas/uso terapéutico , Quinazolinas/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Tiazepinas/uso terapéutico , Proteínas Virales de Fusión/antagonistas & inhibidores , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/síntesis química , Antivirales/farmacocinética , Benzazepinas/administración & dosificación , Benzazepinas/síntesis química , Benzazepinas/farmacocinética , Perros , Descubrimiento de Drogas , Femenino , Haplorrinos , Masculino , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Estructura Molecular , Quinazolinas/administración & dosificación , Quinazolinas/líquido cefalorraquídeo , Quinazolinas/síntesis química , Quinazolinas/farmacocinética , Ratas Wistar , Virus Sincitial Respiratorio Humano/química , Relación Estructura-Actividad , Sulfonas , Tiazepinas/administración & dosificación , Tiazepinas/líquido cefalorraquídeo , Tiazepinas/farmacocinética , Proteínas Virales de Fusión/química
19.
Virus Res ; 259: 28-37, 2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30296457

RESUMEN

Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that "top-stalk" CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all "bottom-stalk" F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on "top-stalk" F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.


Asunto(s)
Antivirales/farmacología , Virus del Moquillo Canino/efectos de los fármacos , Virus del Moquillo Canino/fisiología , Farmacorresistencia Viral , Proteínas Virales de Fusión/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Moquillo , Modelos Moleculares , Mutación , Conformación Proteica , Células Vero , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo
20.
ACS Nano ; 12(10): 9855-9865, 2018 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-30230818

RESUMEN

Measles remains one of the leading causes of child mortality worldwide and is re-emerging in some countries due to poor vaccine coverage, concomitant with importation of measles virus (MV) from endemic areas. The lack of specific chemotherapy contributes to negative outcomes, especially in infants or immunodeficient individuals. Fusion inhibitor peptides derived from the MV Fusion protein C-terminal Heptad Repeat (HRC) targeting MV envelope fusion glycoproteins block infection at the stage of entry into host cells, thus preventing viral multiplication. To improve efficacy of such entry inhibitors, we have modified a HRC peptide inhibitor by introducing properties of self-assembly into nanoparticles (NP) and higher affinity for both viral and cell membranes. Modification of the peptide consisted of covalent grafting with tocopherol to increase amphipathicity and lipophilicity (HRC5). One additional peptide inhibitor consisting of a peptide dimer grafted to tocopherol was also used (HRC6). Spectroscopic, imaging, and simulation techniques were used to characterize the NP and explore the molecular basis for their antiviral efficacy. HRC5 forms micellar stable NP while HRC6 aggregates into amorphous, loose, unstable NP. Interpeptide cluster bridging governs NP assembly into dynamic metastable states. The results are consistent with the conclusion that the improved efficacy of HRC6 relative to HRC5 can be attributed to NP instability, which leads to more extensive partition to target membranes and binding to viral target proteins.


Asunto(s)
Antivirales/farmacología , Virus del Sarampión/efectos de los fármacos , Nanopartículas/química , Péptidos/farmacología , Tocoferoles/farmacología , Antivirales/química , Pruebas de Sensibilidad Microbiana , Péptidos/química , Tocoferoles/química , Proteínas Virales de Fusión/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos
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