RESUMEN
The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. IMPORTANCE: The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.
Asunto(s)
Microbiología del Aire , COVID-19 , Técnicas de Amplificación de Ácido Nucleico , ARN Viral , SARS-CoV-2 , SARS-CoV-2/genética , COVID-19/virología , COVID-19/transmisión , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Aerosoles , Técnicas de Diagnóstico MolecularRESUMEN
Seasonal or pandemic illness caused by influenza A viruses (IAVs) is a major public health concern due to the high morbidity and notable mortality. Although there are several approved drugs targeting different mechanisms, the emergence of drug resistance calls for new drug candidates that can be used alone or in combinations. Small-molecule IAV entry inhibitor, ING-1466, binds to hemagglutinin (HA) and blocks HA-mediated viral infection. Here, we show that this inhibitor demonstrates preventive and therapeutic effects in a mouse model of IAV with substantial improvement in the survival rate. When administered orally it elicits a therapeutic effect in mice, even after the well-established infection. Moreover, the combination of ING-1466 with oseltamivir phosphate or baloxavir marboxil enhances the therapeutic effect in a synergistic manner. Overall, ING-1466 has excellent oral bioavailability and in vitro absorption, distribution, metabolism, excretion, and toxicity profile, suggesting that it can be developed for monotherapy or combination therapy for the treatment of IAV infections.
Asunto(s)
Dibenzotiepinas , Virus de la Influenza A , Morfolinas , Piridonas , Tiepinas , Triazinas , Animales , Ratones , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Antivirales/uso terapéutico , Oxazinas/farmacología , Oxazinas/uso terapéutico , Piridinas , Tiepinas/farmacología , Tiepinas/uso terapéuticoRESUMEN
The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution, as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus like particles (VLP). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling study of BSL3 and BSL4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins including those of newly discovered pathogens and mutant variants; (v) the ability to introduce viral sequences to develop nucleic acid amplification assays.
RESUMEN
Ebola virus (EBOV) is one of the most deadliest agents already known, causing periodic epidemic of a severe hemorrhagic fever disease in Africa. Although two monoclonal antibody (mAb) drugs have recently received approval in the USA, additional therapeutics are still needed to combat potential outbreaks of resistance variants and other closely related ebola viruses. In this chapter, we describe the current understanding of the EBOV entry process and summarize the approaches, strategies, and advances in discovery and development of EBOV entry inhibitors, including therapeutic antibodies, peptides, small molecules, natural products, and other chemical structures.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Anticuerpos Monoclonales/uso terapéutico , Brotes de Enfermedades , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Humanos , Internalización del VirusRESUMEN
Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its fusion machinery. In this review, we summarize the advances in HA-targeted development of small molecule inhibitors. Moreover, we discuss the structural basis and mode of action of these inhibitors, and speculate upon future directions toward more potent inhibitors of membrane fusion and potential anti-influenza drugs.
RESUMEN
Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.
Asunto(s)
Antivirales/farmacología , Ebolavirus/efectos de los fármacos , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus/efectos de los fármacos , Células A549 , Animales , Chlorocebus aethiops , Ebolavirus/fisiología , Glicoproteínas/genética , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Interacciones Huésped-Patógeno , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/virología , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
Influenza hemagglutinin (HA) is a viral membrane bound protein that plays a critical role in the viral life cycle by mediating entry into target cells. HA exploits the lowering of the pH in the endosomal compartment to initiate a series of conformational changes that promote access of the viral genetic material to the cytoplasm, and hence viral replication. In this review we will first discuss what is known about the structural properties of HA as a function of pH. Next, we will discuss the dynamics and intermediate states of HA. We will then discuss the specific residues that are thought to be titrated by the change in pH and possible mechanisms for the pH triggered conformational changes. Finally, we will discuss small molecules that disrupt the pH trigger and thus serve as potential therapeutic strategies to prevent influenza infection.
RESUMEN
The SuperCam instrument suite provides the Mars 2020 rover, Perseverance, with a number of versatile remote-sensing techniques that can be used at long distance as well as within the robotic-arm workspace. These include laser-induced breakdown spectroscopy (LIBS), remote time-resolved Raman and luminescence spectroscopies, and visible and infrared (VISIR; separately referred to as VIS and IR) reflectance spectroscopy. A remote micro-imager (RMI) provides high-resolution color context imaging, and a microphone can be used as a stand-alone tool for environmental studies or to determine physical properties of rocks and soils from shock waves of laser-produced plasmas. SuperCam is built in three parts: The mast unit (MU), consisting of the laser, telescope, RMI, IR spectrometer, and associated electronics, is described in a companion paper. The on-board calibration targets are described in another companion paper. Here we describe SuperCam's body unit (BU) and testing of the integrated instrument. The BU, mounted inside the rover body, receives light from the MU via a 5.8 m optical fiber. The light is split into three wavelength bands by a demultiplexer, and is routed via fiber bundles to three optical spectrometers, two of which (UV and violet; 245-340 and 385-465 nm) are crossed Czerny-Turner reflection spectrometers, nearly identical to their counterparts on ChemCam. The third is a high-efficiency transmission spectrometer containing an optical intensifier capable of gating exposures to 100 ns or longer, with variable delay times relative to the laser pulse. This spectrometer covers 535-853 nm ( 105 - 7070 cm - 1 Raman shift relative to the 532 nm green laser beam) with 12 cm - 1 full-width at half-maximum peak resolution in the Raman fingerprint region. The BU electronics boards interface with the rover and control the instrument, returning data to the rover. Thermal systems maintain a warm temperature during cruise to Mars to avoid contamination on the optics, and cool the detectors during operations on Mars. Results obtained with the integrated instrument demonstrate its capabilities for LIBS, for which a library of 332 standards was developed. Examples of Raman and VISIR spectroscopy are shown, demonstrating clear mineral identification with both techniques. Luminescence spectra demonstrate the utility of having both spectral and temporal dimensions. Finally, RMI and microphone tests on the rover demonstrate the capabilities of these subsystems as well.
RESUMEN
HA plays a critical role in influenza infection and, thus HA is a potential target for antivirals. Recently, our laboratories have described a novel fusion inhibitor, termed CBS1117, with EC50 â¼3 µM against group 1 HA. In this work, we characterize the binding properties of CBS1117 to avian H5 HA by x-ray crystallography, NMR, and mutagenesis. The x-ray structure of the complex shows that the compound binds near the HA fusion peptide, a region that plays a critical role in HA-mediated fusion. NMR studies demonstrate binding of CBS1117 to H5 HA in solution and show extensive hydrophobic contacts between the compound and HA surface. Mutagenesis studies further support the location of the compound binding site proximal to the HA fusion peptide and identify additional amino acids that are important to compound binding. Together, this work gives new insights into the CBS1117 mechanism of action and can be exploited to further optimize this compound and better understand the group specific activity of small-molecule inhibitors of HA-mediated entry.
Asunto(s)
Antivirales/química , Hemaglutininas/ultraestructura , Animales , Antivirales/farmacología , Sitios de Unión/efectos de los fármacos , Aves/virología , Cristalografía por Rayos X/métodos , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Hemaglutininas/metabolismo , Humanos , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Aviar/metabolismo , Gripe Humana/metabolismo , Modelos Moleculares , Infecciones por Orthomyxoviridae , Internalización del Virus/efectos de los fármacosRESUMEN
Influenza A viruses (IAVs) cause seasonal flu and occasionally pandemics. The current therapeutics against IAVs target two viral proteins - neuraminidase (NA) and M2 ion-channel protein. However, M2 ion channel inhibitors (amantadine and rimantadine) are no longer recommended by CDC for use due to the emergence of high level of antiviral resistance among the circulating influenza viruses, and resistant strains to NA inhibitors (oseltamivir and zanamivir) have also been reported. Therefore, development of novel anti-influenza therapies is urgently needed. As one of the viral surface glycoproteins, hemagglutinin (HA) mediates critical virus entry steps including virus binding to host cells and virus-host membrane fusion, which makes it a potential target for anti-influenza drug development. In this study, we report the identification of compound CBS1116 with a 4-aminopiperidine scaffold from a chemical library screen as an entry inhibitor specifically targeting two group 1 influenza A viruses, A/Puerto Rico/8/34 (H1N1) and recombinant low pathogenic avian H5N1 virus (A/Vietnam/1203/04, VN04Low). Mechanism of action studies show that CBS1116 interferes with the HA-mediated fusion process. Further structure activity relationship study generated a more potent compound CBS1117 which has a 50% inhibitory concentration of 70 nM and a selectivity index of ~4000 against A/Puerto Rico/8/34 (H1N1) infection in human lung epithelial cell line (A549).
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Piperidinas/farmacología , Internalización del Virus/efectos de los fármacos , Células A549 , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Vaccination is the most prevalent prophylactic means for controlling seasonal influenza infections. However, an effective vaccine usually takes at least 6 months to develop for the circulating strains. Therefore, new therapeutic options are needed for the acute treatment of influenza infections to control this virus and prevent epidemics/pandemics from developing. We have discovered fast-acting, orally bioavailable acylated 4-aminopiperidines with an effective mechanism of action targeting viral hemagglutinin (HA). Our data show that these compounds are potent entry inhibitors of influenza A viruses. We present docking studies that suggest an HA binding site for these inhibitors on H5N1. Compound 16 displayed a significant decrease of viral titer when evaluated in the infectious assays with influenza virus H1N1 (A/Puerto Rico/8/1934) or H5N1 (A/Vietnam/1203/2004) strains and the oseltamivir-resistant strain with the most common H274Y mutation. In addition, compound 16 showed significant synergistic activity with oseltamivir in vitro.
Asunto(s)
Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Oseltamivir/farmacología , Piperidinas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Sitios de Unión , Perros , Sinergismo Farmacológico , Hemaglutininas Virales/química , Hemaglutininas Virales/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/química , Células de Riñón Canino Madin Darby , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/metabolismo , Unión Proteica , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Hemagglutnin (HA) mediates entry of influenza virus through a series of conformational changes triggered by the low pH of the endosome. The residue or combination of residues acting as pH sensors has not yet been fully elucidated. In this work, we assay pH effects on the structure of H5 HA by soaking HA crystallized at pH 6.5 in a series of buffers with lower pH, mimicking the conditions of the endosome. We find that HA1-H38, which is conserved in Group 1 HA, undergoes a striking change in side chain conformation, which we attribute to its protonation and cation-cation repulsion with conserved HA1-H18. This work suggests that x-ray crystallography can be applied for studying small-scale pH-induced conformational changes providing valuable information on the location of pH sensors in HA. Importantly, the observed change in HA1-H38 conformation is further evidence that the pH-induced conformational changes of HA are the result of a series of protonation events to conserved and non-conserved pH sensors.
Asunto(s)
Hemaglutininas/ultraestructura , Gripe Humana/genética , Orthomyxoviridae/ultraestructura , Internalización del Virus , Cristalografía por Rayos X , Endosomas/genética , Endosomas/ultraestructura , Hemaglutininas/química , Hemaglutininas/genética , Humanos , Concentración de Iones de Hidrógeno , Gripe Humana/patología , Gripe Humana/virología , Modelos Moleculares , Orthomyxoviridae/genética , Conformación ProteicaRESUMEN
15N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of 15N labeled amino acids in metabolite studies have not been pursued extensively, presumably due to line broadening present under standard experimental conditions. In this work, we show that lowering the temperature to -5 °C allows facile characterization of 15N-labeled amino acids. Further, we show that this technique can be exploited to measure 15NH3 produced in an enzyme catalyzed reaction and the transport and metabolism of individual amino acids in mammalian cell culture. With respect to 13C-labeled amino acids, 15N-labeled amino acids are less costly and enable direct characterization of nitrogen metabolism in complex biological systems by NMR. In summary, the present work significantly expands the metabolite pools and their reactions for study by NMR.
Asunto(s)
Aminoácidos/química , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Nitrógeno/química , Compuestos de Amonio/química , Línea Celular Tumoral , Humanos , TemperaturaRESUMEN
Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.
Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Proteínas Recombinantes/metabolismo , Animales , Asparaginasa/genética , Asparaginasa/metabolismo , Asparaginasa/farmacocinética , Línea Celular Tumoral , Femenino , Glutaminasa/metabolismo , Glutamina/sangre , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Pruebas de Toxicidad Aguda , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Two series of easily accessible anilines were identified as inhibitors of influenza A virus subtype H1N1, and extensive chemical synthesis and analysis of the structure-activity relationship were performed. The compounds were shown to interfere with low pH-induced membrane fusion mediated by the H1 and H5 (group 1) hemagglutinin (HA) subtypes. A combination of virus resistance, HA interaction, and molecular dynamics simulation studies elucidated the binding site of these aniline-based influenza fusion inhibitors, which significantly overlaps with the pocket occupied by some H3 HA-specific inhibitors, indicating the high relevance of this cavity for drug design.
Asunto(s)
Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Internalización del Virus/efectos de los fármacos , Compuestos de Anilina/metabolismo , Sitios de Unión , Células HeLa , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Conformación ProteicaRESUMEN
Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X/métodos , Hemaglutininas/química , Humanos , Concentración de Iones de Hidrógeno , Gripe Humana/metabolismo , Fusión de Membrana/fisiología , Modelos Moleculares , Mutagénesis , Péptidos/metabolismo , Conformación Proteica , Internalización del VirusRESUMEN
MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.
Asunto(s)
Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Sitios de Unión , Simulación por Computador , Ligandos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
Influenza A virus envelop protein hemagglutinin (HA) plays important roles in viral entry. We previously have reported that MBX2546, a novel influenza A virus inhibitor, binds to HA and inhibits HA-mediated membrane fusion. In this report, we show that (i) both binding and stabilization of HA by MBX2546 are required for the inhibition of viral infection and (ii) the binding of HA by MBX2546 represses the low-pH-induced conformational change in the HA, which is a prerequisite for membrane fusion. Mutations in MBX2546-resistant influenza A/PR/8/34 (H1N1) viruses are mapped in the HA stem region near the amino terminus of HA2. Finally, we have modeled the binding site of MBX2546 using molecular dynamics and find that the resulting structure is in good agreement with our results. Together, these studies underscore the importance of the HA stem loop region as a potential target for therapeutic intervention.
Asunto(s)
Acetanilidas/química , Antivirales/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/química , Fusión de Membrana/efectos de los fármacos , Sulfonamidas/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Bioensayo , Perros , Farmacorresistencia Viral/fisiología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Concentración de Iones de Hidrógeno , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Cinética , Células de Riñón Canino Madin Darby , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The viral envelope protein hemagglutinin (HA) plays a critical role in influenza entry and thus is an attractive target for novel therapeutics. The small molecule tert-butylhydroquinone (TBHQ) has previously been shown to bind to HA and inhibit HA-mediated entry with low micromolar potency. However, enthusiasm for the use of TBHQ has diminished due to the compound's antioxidant properties. In this work we show that the antioxidant properties of TBHQ are not responsible for the inhibition of HA-mediated entry. In addition, we have performed a structure-activity relationship (SAR) analysis of TBHQ derivatives. We find that the most promising compound, 3-tert-butyl-4-methoxyphenol, exhibits enhanced potency (IC50 = 0.6 µM), decreased toxicity (CC50 = 340 µM), and increased stability (t1/2 > 48 h). Finally, we have characterized the binding properties of 3-tert-butyl-4-methoxyphenol using NMR and molecular dynamics to guide future efforts for chemical optimization.
Asunto(s)
Antivirales/química , Hidroquinonas/química , Gripe Humana/virología , Antioxidantes/química , Antioxidantes/farmacología , Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Hidroquinonas/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.