RESUMO
Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Nucleotídeos de Uracila , Animais , Camundongos , Humanos , Vírus da Influenza A/genética , Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/genética , Furões , Infecções por Orthomyxoviridae/tratamento farmacológicoRESUMO
Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Vírus Sincicial Respiratório Humano , Humanos , Animais , Camundongos , Influenza Humana/tratamento farmacológico , Furões , SARS-CoV-2 , Infecções por Orthomyxoviridae/patologiaRESUMO
Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The â¼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L-P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5' end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.
Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Paramyxovirinae/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Genoma Viral , Metiltransferases/genética , Modelos Moleculares , Nucleoproteínas/química , Vírus da Parainfluenza 5/química , Paramyxovirinae/genética , Fosfoproteínas/química , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely, 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at nontoxic concentrations with 50% effective concentration (EC50) values ranging between 400 nM and 3.9 µM. Further studies indicated that these compounds inhibit a postentry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of nonstructural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical analyses, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.
Assuntos
Infecções por Alphavirus , Vírus Chikungunya , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Camundongos , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Proteínas não Estruturais Virais , Replicação ViralRESUMO
Influenza viruses are highly infectious and are the leading cause of human respiratory diseases and may trigger severe epidemics and occasional pandemics. Although antiviral drugs against influenza viruses have been developed, there is an urgent need to design new strategies to develop influenza virus inhibitors due to the increasing resistance of viruses toward currently available drugs. In this study, we examined the antiviral activity of natural compounds against the following influenza virus strains: A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and B/Lee/40. Papaverine (a nonnarcotic alkaloid that has been used for the treatment of heart disease, impotency, and psychosis) was found to be an effective inhibitor of multiple strains of influenza virus. Kinetic studies demonstrated that papaverine inhibited influenza virus infection at a late stage in the virus life cycle. An alteration in influenza virus morphology and viral ribonucleoprotein (vRNP) localization was observed as an effect of papaverine treatment. Papaverine is a well-known phosphodiesterase inhibitor and also modifies the mitogen-activated protein kinase (MAPK) pathway by downregulating the phosphorylation of MEK and extracellular signal-regulated kinase (ERK). Thus, the modulation of host cell signaling pathways by papaverine may be associated with the nuclear retention of vRNPs and the reduction of influenza virus titers. Interestingly, papaverine also inhibited paramyxoviruses parainfluenza virus 5 (PIV5), human parainfluenza virus 3 (HPIV3), and respiratory syncytial virus (RSV) infections. We propose that papaverine can be a potential candidate to be used as an antiviral agent against a broad range of influenza viruses and paramyxoviruses.IMPORTANCE Influenza viruses are important human pathogens that are the causative agents of epidemics and pandemics. Despite the availability of an annual vaccine, a large number of cases occur every year globally. Here, we report that papaverine, a vasodilator, shows inhibitory action against various strains of influenza virus as well as the paramyxoviruses PIV5, HPIV3, and RSV. A significant effect of papaverine on the influenza virus morphology was observed. Papaverine treatment of influenza-virus-infected cells resulted in the inhibition of virus at a later time in the virus life cycle through the suppression of nuclear export of vRNP and also interfered with the host cellular cAMP and MEK/ERK cascade pathways. This study explores the use of papaverine as an effective inhibitor of both influenza viruses as well as paramyxoviruses.
Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos , Infecções por Orthomyxoviridae , Orthomyxoviridae/metabolismo , Papaverina/farmacologia , Infecções por Paramyxoviridae , Paramyxovirinae/metabolismo , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Paramyxoviridae/metabolismo , Infecções por Paramyxoviridae/patologiaRESUMO
BACKGROUND AND AIMS: The present study is designed to evaluate addition of two different doses of dexmedetomidine (0.25 mcg/kg and 0.5 mcg/kg) as an adjuvant to bupivacaine in transversus abdominis plane block for post-operative analgesia in patients undergoing unilateral inguinal hernioplasty. MATERIAL AND METHODS: A total of 90 patients scheduled to undergo elective unilateral open inguinal hernioplasty were divided into three groups in a randomized triple blind way. In group B (n = 30), patients received TAP block using 22 ml of solution, consisting of 20 ml of 0.25% bupivacaine and 2 ml of normal saline; in group BD1 (n = 30), patients received TAP block using 22 ml of solution, consisting of 20 ml of 0.25% bupivacaine and dexmedetomidine 0.25 mcg/kg dissolved in 2 ml of normal saline; while in group BD2 (n = 30), patients received TAP block using 22 ml of solution, consisting of 20 ml of 0.25% bupivacaine and dexmedetomidine 0.5 mcg/kg dissolved in 2 ml of normal saline. RESULTS: Time to first analgesia was significantly prolonged in group BD2 (874.48 ± 118.28 minutes) as compared to BD1 (536.5 ± 60.35 minutes) and B (341.5 ± 46.22 minutes) (P < 0.0001). Total consumption of diclofenac was also reduced in BD2 (80.17 ± 19.34 mg) as compared with B (150 ± 0 mg) and BD1 (147.5 ± 13.69 mg) (P < 0.001). Patients in dexmedetomidine group were more sedated at 1-hour (P < 0.05). None of our patients required any intervention for hemodynamic changes which were significantly more in dexmedetomidine group. CONCLUSION: Dexmedetomidine in a dose of 0.5 mcg/kg is better than dose of 0.25 mcg/kg as an adjuvant to 0.25% bupivacaine in transversus abdominis plane block for post-operative pain relief in unilateral inguinal hernioplasty. However, it causes mores sedation and hemodynamic changes.
RESUMO
Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.
Assuntos
Nucleoproteínas/química , Vírus da Parainfluenza 5/química , Paramyxovirinae/química , Peptídeos/química , Fosfoproteínas/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Nucleoproteínas/metabolismo , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the K(m) value was found to be 2.63 ± 0.62 µM while the k(cat)/K(m) value was 4.97 × 10(4) M(-1) min(-1). Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCE The alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unappreciated trans-proteolytic activity of the enzyme and for the first time have developed a FRET-based protease assay for screening capsid protease inhibitors. Our structural studies unveil the structural features of the trans-active protease, which has been previously proposed to exist in the natively unfolded form (M. Morillas, H. Eberl, F. H. Allain, R. Glockshuber, and E. Kuennemann, J. Mol. Biol. 376:721-735, 2008, doi:http://dx.doi.org/10.1016/j.jmb.2007.11.055). The different enzymatic forms have been structurally compared to reveal conformational variations in the active and substrate binding sites. The flexible active-site residue Ser218, the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCPΔ2 (AVCP with a deletion of the last two residues at the C terminus) reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening, and structure-based development of antiviral drugs.
Assuntos
Alphavirus/enzimologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/isolamento & purificação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Moleculares , Conformação ProteicaRESUMO
Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD 50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU. Author Summary: Reduced sensitivity to FDA-approved influenza drugs is a major obstacle to effective antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate drug, 4'-FlU, against seasonal, pandemic, and highly pathogenic avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4'-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the drug. Testing of resistant variants against two chemically unrelated nucleoside analog inhibitors of influenza virus, conditionally approved favipiravir and the broad-spectrum SARS-CoV-2 drug molnupiravir, revealed cross-resistance of one cluster with favipiravir, whereas no viral escape from molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4'-FlU. These results demonstrate that partial viral escape from 4'-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.
RESUMO
The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Nucleotídeos de Uracila/farmacologia , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , COVID-19/virologia , Linhagem Celular , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Modelos Animais de Doenças , Feminino , Furões , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Mononegavirais/efeitos dos fármacos , Mononegavirais/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Transcrição Gênica , Nucleotídeos de Uracila/administração & dosagem , Nucleotídeos de Uracila/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The C-terminal protease domain of capsid protein from Aura virus expressed in a bacterial expression system has been purified to homogeneity and crystallized. Crystals suitable for X-ray diffraction analysis were obtained by the vapour-diffusion method using 0.1 M bis-tris and polyethylene glycol monomethyl ether 2000. Crystals of the C-terminal protease domain of capsid protein in complex with dioxane were also produced and crystal data were obtained. Both crystals belonged to space group C2, with unit-cell parameters a = 79.6, b = 35.2, c = 49.5 Å. High-resolution data sets were collected to a resolution of 1.81 Å for the native protein and 1.98 Å for the complex. Preliminary crystallographic studies suggested the presence of a single molecule in the crystallographic asymmetric unit, with a solvent content of 38.5%.
Assuntos
Alphavirus/enzimologia , Capsídeo/enzimologia , Dioxanos/química , Peptídeo Hidrolases/química , Cristalização , Cristalografia por Raios X , Dioxanos/metabolismoRESUMO
Near the end of 2019, a new betacoronavirus started to efficiently transmit between humans, resulting in the current COVID-19 pandemic. Unprecedented worldwide efforts were made to identify and repurpose antiviral therapeutics from collections of approved drugs and known bioactive compounds. Typical pitfalls of this approach (promiscuous/cytotoxic compounds leading to false positives), combined with bypassing antiviral drug development parameters due to urgency have resulted in often disappointing outcomes. A flood of publications, press-releases, and media posts, created confusion in the general public and sometime mobilized precious resources for clinical trials with minimal prospect of success. Breakthroughs have been made, not in the laboratory but in the clinic, resulting from the empiric identification of mitigators of clinical signs such as the discovery of improved disease management through immunomodulators. This opinion piece will aim to capture some of the lessons that we believe the COVID-19 pandemic has taught about drug repurposing screens.
Assuntos
Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos , Antivirais/farmacologia , COVID-19/epidemiologia , Gerenciamento Clínico , Avaliação Pré-Clínica de Medicamentos , Humanos , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.
RESUMO
The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i +3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. ONE-SENTENCE SUMMARY: 4'-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo .
RESUMO
Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre- and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies.
Assuntos
Antivirais/química , Antivirais/farmacologia , Descoberta de Drogas , Paramyxovirinae/fisiologia , Pneumovirus/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Sítios de Ligação , Descoberta de Drogas/métodos , Humanos , Modelos Moleculares , Infecções por Paramyxoviridae/tratamento farmacológico , Infecções por Paramyxoviridae/virologia , Paramyxovirinae/efeitos dos fármacos , Pneumovirus/efeitos dos fármacos , Infecções por Pneumovirus/tratamento farmacológico , Infecções por Pneumovirus/virologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Small heterocyclic molecules such as piperazine are potential pharmacotherapeutic agents and binding of these molecules to the hydrophobic pocket of capsid protein (CP) offers a new perspective for therapeutic intervention. Here, we report the crystal structure of CP from Aura virus (AVCP) in complex with piperazine at 2.2 Å resolution. Piperazine binds to the conserved hydrophobic pocket of CP where dioxane based antivirals bind. Comparative structural studies of the piperazine-bound AVCP structure with the apo, active and dioxane-bound AVCP structures provide insights into the conformational variations in the pocket. Additionally, the molecular docking studies showed that piperazine binds into the hydrophobic pocket of Chikungunya virus CP (CVCP) with more affinity than with AVCP. Furthermore, the antiviral activity of piperazine against Chikungunya virus (CHIKV) was investigated by plaque reduction and immunofluorescence assays. The AVCP-piperazine complex may serve as a lead scaffold for structure-based design of piperazine derivatives as alphaviral inhibitors. The antiviral properties of piperazine provide its usefulness for further investigations towards the development of piperazine based anti-alphaviral drugs.
Assuntos
Alphavirus/efeitos dos fármacos , Antivirais/farmacologia , Proteínas do Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Vírus Chikungunya/efeitos dos fármacos , Piperazinas/farmacologia , Alphavirus/química , Animais , Antivirais/metabolismo , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/química , Chlorocebus aethiops , Cristalização , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Piperazina , Piperazinas/metabolismo , Conformação Proteica , Células VeroRESUMO
INTRODUCTION: Clonidine has proved to be effective drug for postoperative analgesia but it's efficacy to alter neuroendocrine stress response and emergence agitation is unknown. This study was conducted to assess and compare the efficacy of caudal fentanyl vs. clonidine for analgesia, blunting of neuroendocrine stress responses (NESR) and emergence agitation (EA) following sevoflurane anaesthesia. MATERIALS AND METHODS: This prospective, randomized, double blind study enrolled 60 children undergoing infraumbilical surgery. Three groups of 20 each were assigned to receive caudal block with either bupivacaine 0.25% 1 ml/kg with normal saline (group I) or bupivacaine 0.25% 1 ml/kg and 1 microgram*kg-1fentanyl (group II), or bupivacaine 0.25% 1 ml/kg and 3 µg/kg clonidine [group III]. Postoperative analgesia, sedation, NESR, emergence agitation and side effects were observed. RESULTS: VAS score at two hours was significantly less in group III (0.60± 0.60) than in group I (1.80± 0.41) and group II (1.25± 0.44), the time to rescue analgesia was also significantly greater in group III (8.03+0.41hours) than groups I and II (4.15± 0.54 hours) and (6.18± 0.5hours) respectively. The EA scores were significantly better in Group III but patients were significantly more sedated postoperatively. Intraoperatively, NESR was blunted in all the groups and the markers of NESR were lowest in group III. CONCLUSION: Caudal clonidine in a dose of 3 µg/kg prolongs analgesia and decreases emergence agitation as compared to bupivacaine alone or with fentanyl 1µg/kg. Modulation of the neuroendocrine stress response was observed in all the investigated groups though the indicators were lowest in clonidine group.
RESUMO
Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 µM and 1.11 × 10(3) M(-1) sec(-1) respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus Chikungunya/fisiologia , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala/métodos , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Febre de Chikungunya/metabolismo , Febre de Chikungunya/virologia , Vírus Chikungunya/isolamento & purificação , Clonagem Molecular , Humanos , Cinética , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Inibidores de Proteases/isolamento & purificação , Conformação ProteicaRESUMO
Protein methyltransferase (CheR) catalyzes the methylation of the cytosolic domain of the membrane bound chemotaxis receptors, and plays a pivotal role in the chemotactic signal transduction pathway in bacteria. Crystal structure of CheR is available only from the gram-negative bacterium Salmonella typhimurium (StCheR), which contain a catalytic C-terminal domain, encompassing a ß-subdomain, connected via a linker to the N-terminal domain. The structural-functional similitude between CheR of the gram-negative and the gram-positive bacteria remains obscure. We investigated CheR, from a gram-positive bacterium, Bacillus subtilis (BsCheR), and have identified the functional roles of its N-terminal domain, by using the in silico molecular modeling and docking approach along with mass spectrophotometry and sequence analysis. The structural studies established that the N-terminal domain directly bound to S-Adenosyl-l-homocysteine (SAH). Structural and sequence analyses revealed that the α2 helix of the N-terminal domain was involved in the recognition of the methylation site of the chemotactic receptor. Additionally, immunoblot analysis showed that the purified BsCheR was phosphorylated. Further, mass spectrometry studies detected the phosphorylation at Thr3 position in the N-terminal domain of BsCheR. Phosphorylation of BsCheR suggested a regulatory role of the N-terminal domain, analogous to its antagonistic enzyme, the chemotaxis-specific methylesterase (CheB).
Assuntos
Bacillus subtilis/enzimologia , Proteínas Metiltransferases/metabolismo , Proteômica , Sequência de Aminoácidos , Bacillus subtilis/genética , Domínio Catalítico , Simulação por Computador , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Proteínas Metiltransferases/química , Proteínas Metiltransferases/genética , Estrutura Secundária de Proteína , S-Adenosil-Homocisteína/metabolismoRESUMO
The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphavirus life cycle that is essential to virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2.