RESUMO
Human adenoviruses can cause serious, disseminated infections in immunocompromised patients. For pediatric allogeneic stem cell transplant patients, the case fatality rate can reach 80%. Still, there is no available antiviral drug that is specifically approved by the Food and Drug Administration for the treatment of adenovirus infections. To fill this pressing medical need, we have developed NPP-669, a prodrug of cidofovir with broad activity against double-stranded DNA viruses, including adenoviruses. Here, we report on the in vivo anti-adenoviral efficacy of NPP-669. Using the immunosuppressed Syrian hamster as the model, we show that NPP-669 is highly efficacious when dosed orally at 1 mg/kg and 3 mg/kg. In a delayed administration experiment, NPP-669 was more effective than brincidofovir, a similar compound that reached Phase III clinical trials. Furthermore, parenteral administration of NPP-669 increased its efficacy approximately 10-fold compared to oral dosing without apparent toxicity, suggesting that this route may be preferable in a hospital setting. Based on these findings, we believe that NPP-669 is a promising new compound that needs to be further investigated.
Assuntos
Antivirais , Cidofovir , Citosina , Mesocricetus , Organofosfonatos , Pró-Fármacos , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Humanos , Cidofovir/farmacologia , Cidofovir/uso terapêutico , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Citosina/análogos & derivados , Citosina/farmacologia , Citosina/uso terapêutico , Adenovírus Humanos/efeitos dos fármacos , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/virologia , Modelos Animais de Doenças , Cricetinae , Administração OralRESUMO
DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize ß-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.
Assuntos
Infecções por Citomegalovirus , Organofosfonatos , Pró-Fármacos , Camundongos , Humanos , Animais , Antivirais/farmacocinética , Disponibilidade Biológica , Pró-Fármacos/farmacologia , Citosina , CidofovirRESUMO
The main protease (Mpro) represents one of the most effective and attractive targets for designing anti-SARS-CoV-2 drugs. In this study, we designed and synthesized a novel series of Ebselen derivatives by incorporating privileged fragments from different pockets of the Mpro active site. Among these compounds, 11 compounds showed submicromolar activity in the FRET-based SARS-CoV-2 Mpro inhibition assay, with IC50 values ranging from 233 nM to 550 nM. Notably, compound 3a displayed submicromolar Mpro activity (IC50 = 364 nM) and low micromolar antiviral activity (EC50 = 8.01 µM), comparable to that of Ebselen (IC50 = 339 nM, EC50 = 3.78 µM). Time-dependent inhibition assay confirmed that these compounds acted as covalent inhibitors. Taken together, our optimization campaigns thoroughly explored the structural diversity of Ebselen and verified the impact of specific modifications on potency against Mpro.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Azóis/farmacologia , Relação Estrutura-Atividade , Inibidores de Proteases/farmacologia , Antivirais/farmacologia , Simulação de Acoplamento MolecularRESUMO
Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC50s) ranging between 1.24 and 3.6 µM and a 50% cytotoxic concentration (CC50) of 100 to 150 µM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Infecções por Citomegalovirus , Infecções por Adenovirus Humanos/tratamento farmacológico , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Cricetinae , Infecções por Citomegalovirus/tratamento farmacológico , Humanos , Replicação ViralRESUMO
Syrian hamsters are permissive for the replication of species C human adenoviruses (HAdV-C). The virus replicates to high titers in the liver of these animals after intravenous infection, while respiratory infection results in virus replication in the lung. Here we show that two types belonging to species C, HAdV-C5 and HAdV-C6, replicate to significantly different extents and cause pathology with significantly different severities, with HAdV-C6 replicating better and inducing more severe and more widespread lesions. The virus burdens in the livers of HAdV-C6-infected hamsters are higher than the virus burdens in HAdV-C5-infected ones because more of the permissive hepatocytes get infected. Furthermore, when hamsters are infected intravenously with HAdV-C6, live, infectious virus can be isolated from the lung and the kidney, which is not seen with HAdV-C5. Similarly to mouse models, in hamsters, HAdV-C6 is sequestered by macrophages to a lesser degree than HAdV-C5. Depletion of Kupffer cells from the liver greatly increases the replication of HAdV-C5 in the liver, while it has only a modest effect on the replication of HAdV-C6. Elimination of Kupffer cells also dramatically increases the pathology induced by HAdV-C5. These findings indicate that in hamsters, pathology resulting from intravenous infection with adenoviruses is caused mostly by replication in hepatocytes and not by the abortive infection of Kupffer cells and the following cytokine storm.IMPORTANCE Immunocompromised human patients can develop severe, often lethal adenovirus infections. Respiratory adenovirus infection among military recruits is a serious problem, in some cases requiring hospitalization of the patient. Furthermore, adenovirus-based vectors are frequently used as experimental viral therapeutic agents. Thus, it is imperative that we investigate the pathogenesis of adenoviruses in a permissive animal model. Syrian hamsters are susceptible to infection with certain human adenoviruses, and the pathology accompanying these infections is similar to what is observed with adenovirus-infected human patients. We demonstrate that replication in permissive cells in a susceptible host animal is a major part of the mechanism by which systemic adenovirus infection induces pathology, as opposed to the chiefly immune-mediated pathology observed in nonsusceptible hosts. These findings support the use of compounds inhibiting adenovirus replication as a means to block adenovirus-induced pathology.
Assuntos
Infecções por Adenovirus Humanos/patologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/patogenicidade , Fígado/virologia , Carga Viral , Replicação Viral , Adenovírus Humanos/classificação , Adenovírus Humanos/fisiologia , Animais , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Humanos , Rim/virologia , Células de Kupffer/virologia , Fígado/patologia , Pulmão/virologia , Macrófagos/virologia , MesocricetusRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1005084.].
RESUMO
Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.
Assuntos
Infecções por Adenoviridae/imunologia , Adenovírus Humanos/patogenicidade , Modelos Animais de Doenças , Interferon Tipo I/imunologia , Fator de Transcrição STAT2/deficiência , Infecções por Adenoviridae/patologia , Adenovírus Humanos/imunologia , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Cricetinae , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Mesocricetus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT2/imunologiaRESUMO
Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.
Assuntos
Infecções por Adenoviridae/tratamento farmacológico , Adenovírus Humanos/efeitos dos fármacos , Antivirais/farmacologia , Ganciclovir/farmacologia , Hospedeiro Imunocomprometido , Proteínas Virais/antagonistas & inibidores , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/mortalidade , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/crescimento & desenvolvimento , Adenovírus Humanos/patogenicidade , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Citosina/análogos & derivados , Citosina/farmacologia , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Mesocricetus , Organofosfonatos/farmacologia , Análise de Sobrevida , Transaminases/sangue , Carga Viral/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 µM, with suppression at 50 µM reaching â¼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 µM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Nucleotidiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/antagonistas & inibidores , Aciclovir/farmacologia , Animais , Chlorocebus aethiops , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Citomegalovirus/crescimento & desenvolvimento , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/genética , DNA Viral/metabolismo , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 2/enzimologia , Herpesvirus Humano 2/crescimento & desenvolvimento , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Adenovirus infections of immunocompromised patients can cause life-threatening disseminated disease. While there are presently no drugs specifically approved to treat these infections, there are several compounds that showed efficacy against adenovirus in preclinical studies. For any such compound, low toxicity is an essential requirement. As cumulative drug effects can accentuate pathology, especially in patients with other morbidities, it is important to limit antiviral exposure to what is absolutely necessary. This is achievable by monitoring the virus burden of the patients and administering antivirals to suppress virus replication to a non-pathogenic level. We modeled such a system using Syrian hamsters infected with a replication-competent adenovirus vector, in which luciferase expression is coupled to virus replication. We found that virus replication could be followed in vivo in the same animal by repeated measurement of luciferase expression. To test the utility of an interrupted treatment regimen, we used NPP-669 and valganciclovir, two antiviral compounds with high and moderate anti-adenoviral efficacy, respectively. We found that short-term treatment of adenovirus-infected hamsters at times of peak virus replication can prevent virus-associated pathology. Thus, we believe that this animal model can be used to model different treatment regimens for anti-adenoviral compounds.
Assuntos
Infecções por Adenoviridae , Adenoviridae , Antivirais , Modelos Animais de Doenças , Mesocricetus , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Replicação Viral/efeitos dos fármacos , Infecções por Adenoviridae/tratamento farmacológico , Infecções por Adenoviridae/virologia , Cricetinae , Adenoviridae/efeitos dos fármacos , Adenoviridae/fisiologia , Carga Viral/efeitos dos fármacos , Humanos , Estudos LongitudinaisRESUMO
Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.
Assuntos
Adenina/análogos & derivados , Infecções por Adenoviridae , Infecções por Adenovirus Humanos , Organofosfonatos , Pró-Fármacos , Tirosina/análogos & derivados , Cricetinae , Animais , Humanos , Infecções por Adenovirus Humanos/tratamento farmacológico , Cidofovir/farmacologia , Cidofovir/uso terapêutico , Mesocricetus , Antivirais/uso terapêutico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Adenoviridae , Replicação Viral , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Infecções por Adenoviridae/tratamento farmacológico , Citosina/farmacologia , Citosina/uso terapêutico , Aminoácidos/farmacologia , Nucleotídeos/uso terapêuticoRESUMO
SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function. The N-substituted isatin derivative L-26 is a potential SARS-CoV-2 3CLpro inhibitor, but it has poor cell-based antiviral activity and high cytotoxicity. With L-26 as the lead compound, 58 isatin derivatives were prepared using click-chemistry-based miniaturized synthesis and their 3CLpro inhibitory activities were determined by a fluorescence resonance energy transfer-based enzymatic assay. Compounds D1N8 (IC50 = 0.44 ± 0.12 µM) and D1N52 (IC50 = 0.53 ± 0.21 µM) displayed excellent inhibitory potency against SARS-CoV-2 3CLpro, being equivalent to that of L-26 (IC50 = 0.30 ± 0.14 µM). In addition, the cytotoxicity of D1N8 (CC50 >20 µM) and D1N52 (CC50 >20 µM) decreased significantly compared with L-26 (CC50 <2.6 µM). Further molecular dynamics simulations revealed the potential binding interactions between D1N52 and SARS-CoV-2 3CLpro. These efforts lay a solid foundation for the research of novel anti-SARS-CoV-2 agents targeting 3CLpro.
RESUMO
The ongoing transmission of SARS-CoV-2 necessitates the development of additional potent antiviral agents capable of combating the current highly infectious variants and future coronaviruses. Here, we present the discovery of potent nonpeptide main protease (Mpro) inhibitors with prominent antiviral activity and improved pharmacokinetic properties. Three series of 1,2,4-trisubstituted piperazine derivatives were designed and synthesized, and the optimal GC-78-HCl demonstrated high enzyme-inhibitory potency (IC50 = 0.19 µM) and exhibited excellent antiviral activity (EC50 = 0.40 µM), reaching the same level as Nirmatrelvir (EC50 = 0.38 µM). Additionally, GC-78-HCl displayed potent antiviral activities against various SARS-CoV-2 variants as well as HCoV-OC43 and HCoV-229E, indicating its potential broad-spectrum anticoronaviral activity. Notably, the pharmacokinetic properties of GC-78-HCl were somewhat enhanced compared to those of the lead compound. Furthermore, the cocrystal and molecular docking elucidated the mechanism of action. In conclusion, we discovered a novel nonpeptidic Mpro inhibitor with promising antiviral activity and a favorable pharmacokinetic profile.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Antivirais/farmacologia , Antivirais/química , Piperazinas/farmacologiaRESUMO
Clusters of acute non HepA-E hepatitis cases in previously healthy children have been reported globally. At least, 1010 cases were identified in 35 countries, 5% of those cases required liver transplantation and 2% died. The exact cause is not yet known, but there is circumstantial evidence suggesting that human adenovirus F41 (HAdV-F41) might be playing a role. No antiviral drug has been approved for treating human adenovirus infections. Furthermore, HAdV-F41 is notoriously difficult to grow in cell culture, which hindered studying the efficacy of an antiviral compound against this virus. Here, we show that filociclovir (FCV), a nucleoside analog, is a potent inhibitor of HAdV-F41 in cell culture using 2 approaches, namely immunostaining of infected cells and virus yield reduction assay. The activity of FCV was compared to 3 other known antivirals: cidofovir (CDV), ganciclovir (GCV) and valganciclovir (VGCV). Among the 4 compounds examined in this study, FCV was the most potent, with an EC50 of 3.5 µM. These compounds can be ranked by potency as follows: FCV > CDV > GCV ≥ VGCV. In addition, FCV was 10-fold more potent than CDV in a virus yield reduction assay. This report provides timely and valuable methodologies to the research community for testing antivirals against HAdV-F41. Our findings also support the continued development of FCV for various therapeutic applications, including pediatric hepatitis, if a causal relationship is firmly established in the future.
Assuntos
Adenovírus Humanos , Humanos , Criança , Antivirais/farmacologia , Antivirais/uso terapêutico , Valganciclovir , Ganciclovir/uso terapêutico , Cidofovir/farmacologiaRESUMO
The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 µM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 µM), similar to that of remdesivir (EC50 = 2.27 µM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.
Assuntos
COVID-19 , Humanos , Antivirais/farmacologia , Antivirais/química , Simulação de Acoplamento Molecular , Piperazinas/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.
Assuntos
COVID-19 , Hepatite C Crônica , Antivirais/química , Antivirais/farmacologia , Caspase 3 , Catepsinas , Proteases 3C de Coronavírus , Cisteína Endopeptidases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ácido Orótico/análogos & derivados , Piperazinas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2RESUMO
We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp -158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.
Assuntos
Adenovírus Humanos/genética , Genoma Viral/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Virais/genética , Linhagem Celular , Humanos , Sítio de Iniciação de Transcrição , Proteínas Virais/análiseRESUMO
Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX - AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17-29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma.
Assuntos
Neoplasias Colorretais , Morte Celular Imunogênica , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , OxaliplatinaRESUMO
Model animals are indispensable for the study of human diseases, and in general, of complex biological processes. The Syrian hamster is an important model animal for infectious diseases, behavioral science and metabolic science, for which more experimental tools are becoming available. Here, we describe the generation and characterization of an interleukin-2 receptor subunit gamma (Il2rg) knockout (KO) Syrian hamster strain. In humans, mutations in IL2RG can result in a total failure of T and natural killer (NK) lymphocyte development and nonfunctional B lymphocytes (X-linked severe combined immunodeficiency; XSCID). Therefore, we sought to develop a non-murine model to study XSCID and the infectious diseases associated with IL2RG deficiency. We demonstrated that the Il2rg KO hamsters have a lymphoid compartment that is greatly reduced in size and diversity, and is impaired in function. As a result of the defective adaptive immune response, Il2rg KO hamsters developed a more severe human adenovirus infection and cleared virus less efficiently than immune competent wild-type hamsters. Because of this enhanced virus replication, Il2rg KO hamsters developed more severe adenovirus-induced liver pathology than wild-type hamsters. This novel hamster strain will provide researchers with a new tool to investigate human XSCID and its related infections.