RESUMO
On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.
Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Surtos de Doenças/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Assintomáticas/epidemiologia , Betacoronavirus/enzimologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Criança , Pré-Escolar , Proteínas do Envelope de Coronavírus , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , RNA-Polimerase RNA-Dependente de Coronavírus , Surtos de Doenças/estatística & dados numéricos , Feminino , Humanos , Lactente , Recém-Nascido , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Prevalência , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas do Envelope Viral/genética , Carga Viral , Proteínas não Estruturais Virais/genética , Adulto JovemRESUMO
Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.
Assuntos
Líquidos Iônicos , Isomerismo , Pirimidinas/química , EstereoisomerismoRESUMO
Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Oseltamivir/farmacologia , Oseltamivir/química , Neuraminidase , Simulação de Acoplamento Molecular , Virus da Influenza A Subtipo H5N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Glicosídeo HidrolasesRESUMO
To address drug resistance to influenza virus neuraminidase inhibitors (NAIs), a series of novel boron-containing N-substituted oseltamivir derivatives were designed and synthesized to target the 150-cavity of neuraminidase (NA). In NA inhibitory assays, it was found that most of the new compounds exhibited moderate inhibitory potency against the wild-type NAs. Among them, compound 2c bearing 4-(3-boronic acid benzyloxy)benzyl group displayed weaker or slightly improved activities against group-1 NAs (H1N1, H5N1, H5N8 and H5N1-H274Y) compared to that of oseltamivir carboxylate (OSC). Encouragingly, 2c showed 4.6 times greater activity than OSC toward H5N1-H274Y NA. Moreover, 2c exerted equivalent or more potent antiviral activities than OSC against H1N1, H5N1 and H5N8. Additionally, 2c demonstrated low cytotoxicity in vitro and no acute toxicity at the dose of 1000 mg/kg in mice. Molecular docking of 2c was employed to provide a possible explanation for the improved anti-H274Y NA activity, which may be due to the formation of key additional hydrogen bonds with surrounding amino acid residues, such as Arg152, Gln136 and Val149. Taken together, 2c appeared to be a promising lead compound for further optimization.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Aminoácidos/farmacologia , Animais , Antivirais/química , Boro/farmacologia , Ácidos Borônicos/farmacologia , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Neuraminidase , Oseltamivir/análogos & derivados , Oseltamivir/químicaRESUMO
Posaconazole (PCZ) is a clinically approved drug used predominantly for prophylaxis and salvage therapy of fungal infections. Here, we report its previously undescribed anti-human cytomegalovirus (HCMV) activity. By using antiviral assays, we demonstrated that PCZ, along with other azolic antifungals, has a broad anti-HCMV activity, being active against different strains, including low-passage-number clinical isolates and strains resistant to viral DNA polymerase inhibitors. Using a pharmacological approach, we identified the inhibition of human cytochrome P450 51 (hCYP51), or lanosterol 14α demethylase, a cellular target of posaconazole in infected cells, as a mechanism of anti-HCMV activity of the drug. Indeed, hCYP51 expression was stimulated upon HCMV infection, and the inhibition of its enzymatic activity by either the lanosterol analog VFV {(R)-N-(1-(3,4'-difluoro-[1,1'-biphenyl]-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide} or PCZ decreased HCMV yield and infectivity of released virus particles. Importantly, we observed that the activity of the first-line anti-HCMV drug ganciclovir was boosted tenfold by PCZ and that ganciclovir (GCV) and PCZ act synergistically in inhibiting HCMV replication. Taken together, these findings suggest that this clinically approved drug deserves further investigation in the development of host-directed antiviral strategies as a candidate anti-HCMV drug with a dual antimicrobial effect.
Assuntos
Infecções por Citomegalovirus , Preparações Farmacêuticas , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Citomegalovirus , Infecções por Citomegalovirus/tratamento farmacológico , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Humanos , Triazóis , Replicação ViralRESUMO
Isavuconazole is a broad-spectrum triazole antifungal drug recently approved for the therapy of both invasive aspergillosis and mucormycosis. To support a widespread therapeutic drug monitoring of isavuconazole, a simple, sensitive, and precise high-performance liquid chromatography method with UV detection was developed and fully validated for the quantification of this drug in human plasma. The method involved a combined protein precipitation-solid-phase extraction and a chromatographic separation on a Waters XTerra RP18 (150 × 4.6 mm, 3.5 µm) column using an isocratic mobile phase of ammonium acetate buffer (pH 8.0, 10 mm) and acetonitrile (45:55, v/v). The UV detection was performed at 285 nm. This method was linear (correlation coefficients ≥0.998), specific (no interference with plasma components or various potentially co-administrated drugs), sensitive (lower limit of quantification of 0.025 µg/mL), reproducible (coefficients of variation were ≤7.9%) and accurate (deviations ranged from -5.0 to 8.0%) over the range of 0.025-10 µg/mL. The method fulfilled all of the US Food and Drug Administration guidelines validation criteria and performed well in an international proficiency testing program. The assay was also successfully applied to routine therapeutic drug monitoring of patients and to drug stability investigations under various conditions.
Assuntos
Antifúngicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Nitrilas/sangue , Piridinas/sangue , Espectrofotometria Ultravioleta/métodos , Triazóis/sangue , Antifúngicos/química , Antifúngicos/farmacocinética , Estabilidade de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Nitrilas/química , Nitrilas/farmacocinética , Piridinas/química , Piridinas/farmacocinética , Reprodutibilidade dos Testes , Triazóis/química , Triazóis/farmacocinéticaRESUMO
The 4th European Seminars in Virology (EuSeV), which was focused on oncogenic and oncolytic viruses, was held in Bertinoro (Bologna), Italy, from June 10 to 12, 2016. This article summarizes the plenary lectures and aims to illustrate the main topics discussed at 4th EuSeV, which brought together knowledge and expertise in the field of oncogenic and oncolytic viruses from all over the world. The meeting was divided in two parts, "Mechanisms of Viral Oncogenesis" and "Viral Oncolysis and Immunotherapy," which were both focused on dissecting the complex and multi-factorial interplay between cancer and human viruses and on exploring new anti-cancer strategies. J. Cell. Physiol. 232: 2641-2648, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Pesquisa Biomédica , Transformação Celular Viral , Neoplasias/terapia , Vírus Oncogênicos/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Infecções Tumorais por Vírus/virologia , Virologia , Animais , Terapia Combinada , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/virologia , Vírus Oncogênicos/imunologia , Vírus Oncolíticos/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
UNLABELLED: Spontaneous bacterial peritonitis (SBP) is a common, life-threatening complication of liver cirrhosis. Third-generation cephalosporins have been considered the first-line treatment of SBP. In 2014, a panel of experts suggested a broader spectrum antibiotic regimen for nosocomial SBP, according to the high rate of bacteria resistant to third-generation cephalosporins found in these patients. However, a broader-spectrum antibiotic regimen has never been compared to third-generation cephalosporins in the treatment of nosocomial SBP. The aim of our study was to compare meropenem plus daptomycin versus ceftazidime in the treatment of nosocomial SBP. Patients with cirrhosis and nosocomial SBP were randomized to receive meropenem (1 g/8 hours) plus daptomycin (6 mg/kg/day) or ceftazidime (2 g/8 hours). A paracentesis was performed after 48 hours of treatment. A reduction in ascitic fluid neutrophil count <25% of pretreatment value was considered a treatment failure. The primary outcome was the efficacy of treatment defined by the resolution of SBP after 7 days of treatment. Thirty-two patients were randomized and 31 were analyzed. The combination of meropenem plus daptomycin was significantly more effective than ceftazidime in the treatment of nosocomial SBP (86.7 vs. 25%; P < 0.001). Ninety-day transplant-free survival (TFS) was not significantly different between the two groups. In the multivariate analysis, ineffective response to first-line treatment (hazard ratio [HR]: 20.6; P = 0.01), development of acute kidney injury during hospitalization (HR: 23.2; P = 0.01), and baseline mean arterial pressure (HR: 0.92; P = 0.01) were found to be independent predictors of 90-day TFS. CONCLUSION: The combination of meropenem plus daptomycin is more effective than ceftazidime as empirical antibiotic treatment of nosocomial SBP. Efficacy of the empirical antibiotic treatment is a strong predictor of 90-day survival in patients with nosocomial SBP.
Assuntos
Antibacterianos/administração & dosagem , Infecção Hospitalar/tratamento farmacológico , Mortalidade Hospitalar , Peritonite/diagnóstico , Peritonite/tratamento farmacológico , Adulto , Idoso , Ascite/complicações , Ascite/diagnóstico , Ceftazidima/administração & dosagem , Infecção Hospitalar/microbiologia , Daptomicina/administração & dosagem , Quimioterapia Combinada , Feminino , Hospitais Universitários , Humanos , Itália , Cirrose Hepática/complicações , Cirrose Hepática/diagnóstico , Masculino , Meropeném , Pessoa de Meia-Idade , Análise Multivariada , Peritonite/microbiologia , Peritonite/mortalidade , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Medição de Risco , Índice de Gravidade de Doença , Análise de Sobrevida , Tienamicinas/administração & dosagem , Resultado do Tratamento , Adulto JovemRESUMO
Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-triazole-3-carboxylate was used. [1,2,4]Triazolo[1,5-a]pyrimidine being a privileged scaffold, the procedures herein reported may be useful for the preparation of biologically active compounds. In this study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA polymerase PA-PB1 subunit heterodimerization.
Assuntos
Pirimidinas/síntese química , Triazóis/síntese química , Animais , Sobrevivência Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Células Madin Darby de Rim Canino/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Orthomyxoviridae/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Continuous veno-venous hemofiltration (CVVH) could affect the pharmacokinetic profile of linezolid (LZD). The aim of this study was to evaluate the LZD extracorporeal clearance using an in vitro CVVH model. METHODS: A sham miniaturized CVVH circuit (CARPEDIEM; Bellco, Mirandola, Italy) was set up with a polysulfone hemofilter (0.25 m; cutoff 50,000 Da) for 240 minutes using normal saline solution (0.9% wt/vol NaCl) and blood (n = 6) spiked with LZD. Drug solution samples were collected during CVVH at 10, 30, 60, 120, and 240 minutes. LZD levels were measured by high-performance liquid chromatography. RESULTS: Results were used to estimate pharmacokinetic parameters. The LZD baseline level decreased from 17.24 ± 0.54 to 9.73 ± 4.85 mg/L and from 11.75 ± 0.08 to 5.01 ± 0.67 mg/L in the first 10 minutes, and then increased to 13.2 ± 3.10 and 7.4 ± 0.71 mg/L in normal saline solution and blood, respectively. Mass balance analysis reported a rapid adsorption of LZD onto a polysulfone membrane followed by its release: a rebound phenomenon occurred. CONCLUSIONS: Although further studies are necessary to clarify this phenomenon, LZD level variations observed in our study should be considered to avoid antimicrobial underexposure. Several strategies are available for adjusting the dosage regimen of LZD, but therapeutic drug monitoring is highly recommended when it is used.
Assuntos
Hemofiltração , Linezolida/farmacocinética , Anti-Infecciosos/sangue , Anti-Infecciosos/farmacocinética , Humanos , Técnicas In Vitro , Linezolida/sangueRESUMO
Influenza viruses are major human pathogens responsible for respiratory diseases affecting millions of people worldwide and characterized by high morbidity and significant mortality. Influenza infections can be controlled by vaccination and antiviral drugs. However, vaccines need annual updating and give limited protection. Only two classes of drugs are currently approved for the treatment of influenza: M2 ion channel blockers and neuraminidase inhibitors. However, they are often associated with limited efficacy and adverse side effects. In addition, the currently available drugs suffer from rapid and extensive emergence of drug resistance. All this highlights the urgent need for developing new antiviral strategies with novel mechanisms of action and with reduced drug resistance potential. Several new classes of antiviral agents targeting viral replication mechanisms or cellular proteins/processes are under development. This review gives an overview of novel strategies targeting the virus and/or the host cell for counteracting influenza virus infection.
Assuntos
Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Hemaglutininas/imunologia , Hemaglutininas/metabolismo , Humanos , Influenza Humana/virologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/metabolismo , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Influenza viruses are the cause of yearly epidemics and occasional pandemics that represent a significant challenge to public health. Current control strategies are imperfect and there is an unmet need for new antiviral therapies. Here, we report the identification of small molecule compounds able to effectively and specifically inhibit growth of influenza A and B viruses in cultured cells through targeting an assembly interface of the viral RNA-dependent RNA polymerase. Using an existing crystal structure of the primary protein-protein interface between the PB1 and PA subunits of the influenza A virus polymerase, we conducted an in silico screen to identify potential small molecule inhibitors. Selected compounds were then screened for their ability to inhibit the interaction between PB1 and PA in vitro using an ELISA-based assay and in cells, to inhibit nuclear import of a binary PB1-PA complex as well as transcription by the full viral ribonucleoprotein complex. Two compounds emerged as effective inhibitors with IC(50) values in the low micromolar range and negligible cytotoxicity. Of these, one compound also acted as a potent replication inhibitor of a variety of influenza A virus strains in Madin-Darby canine kidney (MDCK) cells, including H3N2 and H1N1 seasonal and 2009 pandemic strains. Importantly, this included an oseltamivir-resistant isolate. Furthermore, potent inhibition of influenza B viruses but not other RNA or DNA viruses was seen. Overall, these compounds provide a foundation for the development of a new generation of therapeutic agents exhibiting high specificity to influenza A and B viruses.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , RNA Polimerase Dependente de RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Farmacorresistência Viral , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vírus da Influenza B/genética , Vírus da Influenza B/metabolismo , Concentração Inibidora 50 , Microscopia Confocal , Modelos Moleculares , Oseltamivir/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase Dependente de RNA/química , Células VeroRESUMO
The human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication, and here, we report the dissection of its mechanism of action against the viral IE2 protein. Using glutathione S-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (TBP), and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the major immediate-early promoter by preventing IE2 binding to the crs element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the early UL54 gene promoter, and it also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.
Assuntos
Aminoquinolinas/farmacologia , Antivirais/farmacologia , Citomegalovirus/crescimento & desenvolvimento , Proteínas Imediatamente Precoces/antagonistas & inibidores , Quinolonas/farmacologia , Replicação Viral/efeitos dos fármacos , Antígenos Virais , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Ciclina E/genética , Citomegalovirus/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Proteínas Virais/genéticaRESUMO
The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein-protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA-PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a-n) or several amino acid groups (compounds 3a-n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 µM in PA-PB1 ELISA, an EC50 value of 2.8 µM in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds.
RESUMO
In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against avian IV strains both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against avian IV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections.
RESUMO
Human cytomegalovirus (HCMV) is the viral leading cause of congenital defects in newborns worldwide. Many aspects of congenital CMV (cCMV) infection, which currently lacks a specific treatment, as well as the main determinants of neuropathogenesis in the developing brain during HCMV infection are unclear. In this study, we modeled HCMV infection at different stages of neural development. Moreover, we evaluated the effects of both approved and investigational anti-HCMV drugs on viral replication and gene expression in two different neural progenitor cell lines, i.e., human embryonic stem cells-derived neural stem cells (NSCs) and fetus-derived neuroepithelial stem (NES) cells. Ganciclovir, letermovir, nitazoxanide, and the ozonide OZ418 reduced viral DNA synthesis and the production of infectious virus in both lines of neural progenitors. HCMV infection dysregulated the expression of genes that either are markers of neural progenitors, such as SOX2, NESTIN, PAX-6, or play a role in neurogenesis, such as Doublecortin. Treatment with antiviral drugs had different effects on HCMV-induced dysregulation of the genes under investigation. This study contributes to the understanding of the molecular mechanisms of cCMV neuropathogenesis and paves the way for further consideration of anti-HCMV drugs as candidate therapeutic agents for the amelioration of cCMV-associated neurological manifestations.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Recém-Nascido , Humanos , Infecções por Citomegalovirus/tratamento farmacológico , Encéfalo , Drogas em Investigação , Células-Tronco , Antivirais/farmacologiaRESUMO
To date, Proteolysis Targeting Chimera (PROTAC) technology has been successfully applied to mediate proteasomal-induced degradation of several pharmaceutical targets mainly related to oncology, immune disorders, and neurodegenerative diseases. On the other hand, its exploitation in the field of antiviral drug discovery is still in its infancy. Recently, we described two indomethacin (INM)-based PROTACs displaying broad-spectrum antiviral activity against coronaviruses. Here, we report the design, synthesis, and characterization of a novel series of INM-based PROTACs that recruit either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. The panel of INM-based PROTACs was also enlarged by varying the linker moiety. The antiviral activity resulted very susceptible to this modification, particularly for PROTACs hijacking VHL as E3 ligase, with one piperazine-based compound (PROTAC 6) showing potent anti-SARS-CoV-2 activity in infected human lung cells. Interestingly, degradation assays in both uninfected and virus-infected cells with the most promising PROTACs emerged so far (PROTACs 5 and 6) demonstrated that INM-PROTACs do not degrade human PGES-2 protein, as initially hypothesized, but induce the concentration-dependent degradation of SARS-CoV-2 main protease (Mpro) both in Mpro-transfected and in SARS-CoV-2-infected cells. Importantly, thanks to the target degradation, INM-PROTACs exhibited a considerable enhancement in antiviral activity with respect to indomethacin, with EC50 values in the low-micromolar/nanomolar range. Finally, kinetic solubility as well as metabolic and chemical stability were measured for PROTACs 5 and 6. Altogether, the identification of INM-based PROTACs as the first class of SARS-CoV-2 Mpro degraders demonstrating activity also in SARS-CoV-2-infected cells represents a significant advance in the development of effective, broad-spectrum anti-coronavirus strategies.
Assuntos
COVID-19 , Proteases 3C de Coronavírus , Quimera de Direcionamento de Proteólise , Humanos , Proteólise , SARS-CoV-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antivirais/farmacologiaRESUMO
Influenza viruses (IV) are single-stranded RNA viruses with a negative-sense genome and have the potential to cause pandemics. While vaccines exist for influenza, their protection is only partial. Additionally, there is only a limited number of approved anti-IV drugs, which are associated to emergence of drug resistance. To address these issues, for years we have focused on the development of small-molecules that can interfere with the heterodimerization of PA and PB1 subunits of the IV RNA-dependent RNA polymerase (RdRP). In this study, starting from a cycloheptathiophene-3-carboxamide compound that we recently identified, we performed iterative cycles of medicinal chemistry optimization that led to the identification of compounds 43 and 45 with activity in the nanomolar range against circulating A and B strains of IV. Mechanistic studies demonstrated the ability of 43 and 45 to interfere with viral RdRP activity by disrupting PA-PB1 subunits heterodimerization and to bind to the PA C-terminal domain through biophysical assays. Most important, ADME studies of 45 also showed an improvement in the pharmacokinetic profile with respect to the starting hit.
RESUMO
Human papillomavirus (HPV)-induced cancers still represent a major health issue for worldwide population and lack specific therapeutic regimens. Despite substantial advancements in anti-HPV vaccination, the incidence of HPV-related cancers remains high, thus there is an urgent need for specific anti-HPV drugs. The HPV E7 oncoprotein is a major driver of carcinogenesis that acts by inducing the degradation of several host factors. A target is represented by the cellular phosphatase PTPN14 and its E7-mediated degradation was shown to be crucial in HPV oncogenesis. Here, by exploiting the crystal structure of E7 bound to PTPN14, we performed an in silico screening of small-molecule compounds targeting the C-terminal CR3 domain of E7 involved in the interaction with PTPN14. We discovered a compound able to inhibit the E7/PTPN14 interaction in vitro and to rescue PTPN14 levels in cells, leading to a reduction in viability, proliferation, migration, and cancer-stem cell potential of HPV-positive cervical cancer cells. Mechanistically, as a consequence of PTPN14 rescue, treatment of cancer cells with this compound altered the Yes-associated protein (YAP) nuclear-cytoplasmic shuttling and downstream signaling. Notably, this compound was active against cervical cancer cells transformed by different high-risk (HR)-HPV genotypes indicating a potential broad-spectrum activity. Overall, our study reports the first-in-class inhibitor of E7/PTPN14 interaction and provides the proof-of-principle that pharmacological inhibition of this interaction by small-molecule compounds could be a feasible therapeutic strategy for the development of novel antitumoral drugs specific for HPV-associated cancers.