RESUMO
The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding.IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Produtos do Gene gag/metabolismo , HIV-1/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Precursores de Proteínas/metabolismo , Liberação de Vírus , Animais , Proteínas de Ligação ao Cálcio/genética , Gatos , Proteínas de Ciclo Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Produtos do Gene gag/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Domínios Proteicos , Precursores de Proteínas/genética , Dedos de ZincoRESUMO
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
Increasing evidence suggests that bone marrow derived mesenchymal stem cells (BM-MSCs) are recruited into the stroma of developing tumors where they contribute to progression by enhancing tumor growth and metastasis, or by inducing anticancer-drug resistance. Prostate cancer cells secrete ligands of epidermal growth factor receptor (EGFR) and EGFR signaling could play an important role in the cross-talk between mesenchymal stem cells and prostate cancer cells. In this study, we showed that treatment of human primary MSCs with conditioned medium (CM) derived from the bone metastatic PC3 carcinoma cells (PC3-CM) resulted in: a significant activation of EGFR; increased proliferation; increased osteoblastic but decreased adipocitic differentiation; inhibition of senescence induced by serum starvation; increased CCL5 secretion. These activities were significantly inhibited in the presence of the EGFR tyrosine kinase inhibitor gefitinib. PC3-CM directly inhibited osteoclastogenesis as well as the ability of osteoblasts to induce osteoclast differentiation. The increased MSCs migration by PC3-CM and PC3 cells was partially mediated by CCL5. MSC-CM increased the formation of colonies by PC3 cells and inhibited the anti-proliferative activity of Docetaxel. Activation of EGFR expressed on MSCs by PC3-CM enhanced their capability to increase PC3 cells proliferation and to inhibit Docetaxel activity. These findings, by showing that the tumor-promoting interactions between PC3 cells and MSCs are mediated, at least in part, by EGFR, suggest a novel application of the EGFR-tyrosine kinase inhibitors in the treatment of prostate cancer.
Assuntos
Comunicação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Neoplasias da Próstata/patologia , Quinazolinas/farmacologia , Taxoides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Quimiocina CCL5/metabolismo , Quimiotaxia/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Docetaxel , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
OBJECTIVE: Cisplatin-based chemotherapy has been shown to improve survival in cervical cancer; however, treatment is associated with tumor resistance and significant toxicity. Lipoplatin is a new liposomal formulation of cisplatin, developed to reduce cisplatin toxicity, to improve drug accumulation at tumor sites and to overcome drug resistance. The aim of this study is to analyze the antitumoral activity of lipoplatin against cisplatin-resistant cervical cancer cells and to investigate its mechanism of action. METHODS: The activity and mechanism of action of lipoplatin were studied in the ME-180 cervical cancer cell line and its cisplatin-resistant clone R-ME-180 and HeLa cells using cell proliferation assays, flow cytometry, ELISA assay, cell migration, spheroids and tumor xenograft. RESULTS: We demonstrated that lipoplatin exhibited a potent antitumoral activity on HeLa, ME-180 cells and its cisplatin-resistant clone R-ME-180. Lipoplatin inhibited cell proliferation in a dose-dependent manner and was more active than the reference drug cisplatin in R-ME-180 cells and induced apoptosis, as evaluated by Annexin-V staining and DNA fragmentation, caspases 9 and 3 activation, Bcl-2, and Bcl-xL down-regulation, but Bax up-regulation inhibited thioredoxin reductase (TrxR) enzymatic activity and increased reactive oxygen species (ROS) accumulation; reduced EGFR expression and inhibited both migration and invasion. R-ME-180, but not ME-180 cells, generated three-dimensional (3D)-multicellular spheroids expressing the cancer stem cell marker ALDH. The ability of R-ME-180 cells to form spheroids in vitro and tumors in nude mice was also remarkably decreased by lipoplatin. CONCLUSIONS: Overall, our results suggest that lipoplatin has potential for the treatment of cisplatin-resistant cervical cancer.
Assuntos
Cisplatino/administração & dosagem , Lipossomos/administração & dosagem , Neoplasias do Colo do Útero/tratamento farmacológico , Aldeído Desidrogenase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/biossíntese , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Papillomavirus Humano , Proteínas E7 de Papillomavirus/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Oncogênicas Virais/metabolismo , Proteínas Tirosina Fosfatases não ReceptorasRESUMO
Every year, dengue virus (DENV) causes one hundred million infections worldwide that can result in dengue disease and severe dengue. Two other mosquito-borne flaviviruses, i.e., Zika virus (ZIKV) and West Nile virus (WNV), are responsible of prolonged outbreaks and are associated with severe neurological diseases, congenital defects, and eventually death. These three viruses, despite their importance for global public health, still lack specific drug treatments. Here, we describe the structure-guided discovery of small molecules with pan-flavivirus antiviral potential by a virtual screening of ~1 million structures targeting the NS3-NS5 interaction surface of different flaviviruses. Two molecules inhibited the interaction between DENV NS3 and NS5 in vitro and the replication of all DENV serotypes as well as ZIKV and WNV and exhibited low propensity to select resistant viruses. Remarkably, one molecule demonstrated efficacy in a mouse model of dengue by reducing peak viremia, viral load in target organs, and associated tissue pathology. This study provides the proof of concept that targeting the flaviviral NS3-NS5 interaction is an effective therapeutic strategy able to reduce virus replication in vivo and discloses new chemical scaffolds that could be further developed, thus providing a significant milestone in the development of much awaited broad-spectrum antiflaviviral drugs. IMPORTANCE More than one-third of the human population is at risk of infection by different mosquito-borne flaviviruses. Despite this, no specific antiviral drug is currently available. In this work, using a computational approach based on molecular dynamics simulation and virtual screening of ~1 million small-molecule structures, we identified a compound that targets the interaction between the two sole flaviviral enzymes, i.e., NS3 and NS5. This compound demonstrated pan-serotype anti-DENV activity and pan-flavivirus potential in infected cells, low propensity to select viral resistant mutant viruses, and efficacy in a mouse model of dengue. Broad-spectrum antivirals are much awaited, and this work represents a significant advance toward the development of therapeutic molecules with extended antiflavivirus potential that act by an innovative mechanism and could be used alone or in combination with other antivirals.
Assuntos
Dengue , Flavivirus , Vírus do Nilo Ocidental , Infecção por Zika virus , Zika virus , Animais , Humanos , Camundongos , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Dengue/tratamento farmacológico , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/químicaAssuntos
Antineoplásicos/uso terapêutico , Auranofina/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Two years after its emergence, SARS-CoV-2 still represents a serious and global threat to human health. Antiviral drug development usually takes a long time and, to increase the chances of success, chemical variability of hit compounds represents a valuable source for the discovery of new antivirals. In this work, we applied a platform of variably oriented virtual screening campaigns to seek for novel chemical scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors. The study on the resulting 30 best hits led to the identification of a series of structurally unrelated Mpro inhibitors. Some of them exhibited antiviral activity in the low micromolar range against SARS-CoV-2 and other human coronaviruses (HCoVs) in different cell lines. Time-of-addition experiments demonstrated an antiviral effect during the viral replication cycle at a time frame consistent with the inhibition of SARS-CoV-2 Mpro activity. As a proof-of-concept, to validate the pharmaceutical potential of the selected hits against SARS-CoV-2, we rationally optimized one of the hit compounds and obtained two potent SARS-CoV-2 inhibitors with increased activity against Mpro both in vitro and in a cellular context, as well as against SARS-CoV-2 replication in infected cells. This study significantly contributes to the expansion of the chemical variability of SARS-CoV-2 Mpro inhibitors and provides new scaffolds to be exploited for pan-coronavirus antiviral drug development.
Assuntos
Antivirais , Proteases 3C de Coronavírus , Inibidores de Proteases , SARS-CoV-2 , Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologiaRESUMO
Interferon regulatory factor 4 (IRF4) expression is detected in many lymphoid and myeloid malignancies, and may be a promising therapeutic target. IRF4 is strongly expressed in classical Hodgkin lymphoma (cHL) and its expression is up-regulated by CD40L and down-regulated by both anti-proliferative and pro-apoptotic stimuli. This study analysed the effects of IRF4 silencing in a panel of HL-derived cell lines. We demonstrated that IRF4 down-modulation determined a remarkable decrease of both cell number and clonogenic growth in L-1236, L-428, KM-H2 and HDLM-2 cells, but not in IRF4-negative L-540 cells. IRF4 silencing induced apoptosis, as evaluated by caspase-3 activation and Annexin-V staining and up-regulation of the pro-apoptotic molecule Bax. CD40 engagement by both soluble and membrane bound-CD40L almost totally reduced IRF4 down-modulation and growth inhibition by IRF4 silencing in both L-1236 and L-428 cells. Finally, IRF4 silencing decreased CCL5 secretion in all HL cell lines tested and CCL17 in KM-H2 cells. Taken together, our results demonstrated that IRF4 down-modulation by IRF4 silencing was reversed by CD40 engagement, inhibited HL cells proliferation, induced apoptosis and decreased CCL5 secretion, thus suggesting that IRF4 may be involved in HL pathobiology.
Assuntos
Quimiocina CCL5/metabolismo , Doença de Hodgkin/metabolismo , Fatores Reguladores de Interferon/fisiologia , Proteínas de Neoplasias/fisiologia , Apoptose , Antígenos CD40/metabolismo , Proliferação de Células , Sobrevivência Celular , Citocinas/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Doença de Hodgkin/patologia , Humanos , Fatores Reguladores de Interferon/genética , Células Tumorais CultivadasRESUMO
High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas.
RESUMO
We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases.
Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Nitrilas/farmacologia , Piridinas/farmacologia , Esterol 14-Desmetilase/efeitos dos fármacos , Triazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Antifúngicos/farmacologia , Linhagem Celular , Infecções por Citomegalovirus/virologia , Reposicionamento de Medicamentos , Farmacorresistência Viral , Sinergismo Farmacológico , Quimioterapia Combinada , HumanosRESUMO
Human papillomavirus is the most common viral infectious agent responsible for cancer development in humans. High-risk strains are known to induce cancer through the expression of the viral oncogenes E6 and E7, yet we have only a partial understanding of the precise mechanisms of action of these viral proteins. Here we investigated the molecular mechanism through which the oncoprotein E6 alters the Hippo-YAP/TAZ pathway to trigger YAP/TAZ induction in cancer cells. By employing E6 overexpression systems combined with protein-protein interaction studies and loss-of-function approaches, we discovered that the E6-mediated targeting of hScrib, which supports YAP/TAZ upregulation, intimately requires E6 homodimerization. We show that the self-association of E6, previously reported only in vitro, takes place in the cytoplasm and, as a dimer, E6 targets the fraction of hScrib at the cell cortex for proteasomal degradation. Thus, E6 homodimerization emerges as an important event in the mechanism of E6-mediated hScrib targeting to sustain downstream YAP/TAZ upregulation, unraveling for the first time the key role of E6 homodimerization in the context of its transforming functions and thus paving the way for the possible development of E6 dimerization inhibitors.
RESUMO
Indomethacin (INM), a well-known non-steroidal anti-inflammatory drug, has recently gained attention for its antiviral activity demonstrated in drug repurposing studies against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Although the mechanism of action of INM is not yet fully understood, recent studies have indicated that it acts at an early stage of the coronaviruses (CoVs) replication cycle. In addition, a proteomic study reported that the anti-SARS-CoV-2 activity of INM could be also ascribed to its ability to inhibit human prostaglandin E synthase type 2 (PGES-2), a host protein which interacts with the SARS-CoV-2 NSP7 protein. Although INM does not potently inhibit SARS-CoV-2 replication in infected Vero E6 cells, here we have explored for the first time the application of the Proteolysis Targeting Chimeras (PROTACs) technology in order to develop more potent INM-derived PROTACs with anti-CoV activity. In this study, we report the design, synthesis, and biological evaluation of a series of INM-based PROTACs endowed with antiviral activity against a panel of human CoVs, including different SARS-CoV-2 strains. Two PROTACs showed a strong improvement in antiviral potency compared to INM. Molecular modelling studies support human PGES-2 as a potential target of INM-based antiviral PROTACs, thus paving the way toward the development of host-directed anti-CoVs strategies. To the best of our knowledge, these PROTACs represent the first-in-class INM-based PROTACs with antiviral activity and also the first example of the application of PROTACs to develop pan-coronavirus agents.
Assuntos
Antivirais/farmacologia , COVID-19/virologia , Indometacina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Chlorocebus aethiops , Reposicionamento de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Oncogênicas Virais/antagonistas & inibidores , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Cristalografia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Neoplasias/patologia , Neoplasias/virologia , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Esferoides Celulares , Relação Estrutura-AtividadeRESUMO
RNA-enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L-domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non-primate lentivirus, is strictly dependent for its budding on a "PSAP"-type L-domain, mapping in the carboxy-terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L-domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4-2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L-domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non-primate lentivirus shares with HIV-1 a novel mechanism of connection to the cellular budding machinery.
Assuntos
Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Gatos , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/genética , Genes gag , HIV-1/genética , HIV-1/fisiologia , Humanos , Vírus da Imunodeficiência Felina/genética , Mutagênese Sítio-Dirigida , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ubiquitinação , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
Currently, there are no therapeutic alternatives to DNA polymerase inhibitors to treat human cytomegalovirus (HCMV) infections, a major threat for immunocompromised patients and pregnant women. Here, we explored the potential to repurpose manidipine dihydrochloride (MND), a calcium antagonist clinically approved to treat hypertension, as a new anti-HCMV agent. MND emerged in a previous drug repurposing screen to find early inhibitors of HCMV replication, and now we confirm that it inhibits in the low micromolar range the replication of different HCMV strains, including clinical isolates and viruses resistant to approved DNA polymerase inhibitors. The antiviral activity of MND is specific for HCMV over different both DNA and RNA viruses. Further experiments in HCMV-infected cells testing the effects of MND on viral DNA synthesis and viral proteins expression revealed that it halts the progression of the virus cycle prior to viral DNA replication and E genes expression, but after IE proteins expression. According to these results, we observed that the overall antiviral activity of MND involves a specific interference with the transactivating functions of the viral Immediate-Early 2 (IE-2) protein, an essential viral transcription factor required for the progression of HCMV replication. Given that the inhibitory concentration against HCMV is in the range of clinically relevant concentrations of MND in humans, and the mechanism of action differs from that of the other available therapeutics, this already approved drug is an attractive candidate for repurposing in alternative anti-HCMV therapeutic protocols.
Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Animais , Linhagem Celular , Infecções por Citomegalovirus/tratamento farmacológico , Cães , Relação Dose-Resposta a Droga , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/metabolismo , Nitrobenzenos , Piperazinas , Regiões Promotoras Genéticas , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.
Assuntos
Antineoplásicos/farmacologia , Papillomavirus Humano 16/efeitos dos fármacos , Proteínas Oncogênicas Virais/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Papillomavirus Humano 16/metabolismo , Humanos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150mm×4.6mm, 3.5µm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10mM) and acetonitrile (56:44, v/v), and UV detection at 318nm. This assay proved to be sensitive (lower limit of quantification of 0.05µg/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.9%), and accurate (deviations ranged from -2.2 to 8.0% and from -6.5 to 9.2% for intra-day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.
Assuntos
Cromatografia de Fase Reversa/métodos , Cromatografia de Fase Reversa/normas , Hepacivirus , Imidazóis/sangue , Raios Ultravioleta , Carbamatos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Armazenamento de Medicamentos/métodos , Armazenamento de Medicamentos/normas , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Humanos , Imidazóis/farmacologia , Pirrolidinas , Reprodutibilidade dos Testes , Valina/análogos & derivadosRESUMO
We synthesized, characterized and tested in a panel of cancer cell lines, nine new bipyridine gold(III) dithiocarbamate-containing complexes. In vitro studies demonstrated that compounds 1, 2, 4, 5, 7 and 8 were the most cytotoxic in prostate, breast, ovarian cancer cell lines and in Hodgkin lymphoma cells with IC50 values lower than the reference drug cisplatin. The most active compound 1 was more active than cisplatin in ovarian (A2780cis and 2780CP-16) and breast cancer cisplatin-resistant cells. Compound 1 determined an alteration of the cellular redox homeostasis leading to increased ROS levels, a decrease in the mitochondrial membrane potential, cytochrome-c release from the mitochondria and activation of caspases 9 and 3. The ROS scavenger NAC suppressed ROS generation and rescued cells from damage. Compound 1 resulted more active in tumor cells than in normal human Mesenchymal stromal cells. Gold compounds were active independent of p53 status: exerted cytotoxic effects on a panel of non-small cell lung cancer cell lines with different p53 status and in the ovarian A2780 model where the p53 was knocked out. In conclusion, these promising results strongly indicate the need for further preclinical evaluation to test the clinical potential of these new gold(III) complexes.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Neoplasias/tratamento farmacológico , Compostos Organoáuricos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Acetilcisteína/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Sequestradores de Radicais Livres/farmacologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias/patologia , Compostos Organoáuricos/química , Compostos Organoáuricos/uso terapêutico , Piridinas/química , Espécies Reativas de Oxigênio/metabolismo , Tiocarbamatos/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Classical Hodgkin lymphoma (cHL) is characterized by few tumor cells surrounded by immune cells, fibroblasts, specialized mesenchymal stromal cells and endothelial cells, representing together with their products an active part of the disease. Hodgkin and Reed-Sternberg (HRS) cells can secrete cytokines/chemokines and angiogenic factors capable of recruiting and/or inducing the proliferation of the surrounding cells and can also interact with distant sites of the microenvironment by secreting exosomes. To escape from a useful anti-tumor response due to the recognition by T and NK cells, HRS cells down-regulate HLA molecules, produce immune suppressive cytokines that inhibit cytotoxic responses, and induce an immunosuppressive phenotype on T lymphocytes and Monocytes. HRS cells survive, proliferate and are protected from the cytotoxic effects of chemotherapy agents by soluble factors or by the direct contact with inflammatory and stromal cells of the tumor microenvironment (TME). A summary of the current knowledge about classical Hodgkin Lymphoma focusing on the cross-talk between tumor cells and the microenvironment leading to immune-escape, angiogenesis tumor growth/survival and drug resistance will be reviewed here.