RESUMO
Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.
Assuntos
Fármacos Anti-HIV/farmacocinética , Barreira Hematoencefálica/metabolismo , Peptídeos/farmacocinética , Porfirinas/farmacocinética , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Transporte Biológico , Linhagem Celular , Descoberta de Drogas , Células HEK293 , HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Humanos , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Porfirinas/química , Porfirinas/farmacologiaRESUMO
Antiviral medicines to treat COVID-19 are still scarce. Porphyrins and porphyrin derivatives (PDs) usually present broad-spectrum antiviral activity with low risk of resistance development. In fact, some PDs are clinically approved to be used in anti-cancer photodynamic therapy and repurposing clinically approved PDs might be an alternative to treat COVID-19. Here, we characterize the ability of temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles. PDs light-dependent and -independent effect on SARS-CoV-2 infectivity were evaluated. PDs photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light dose. However, only temoporfin and verteporfin inactivated SARS-CoV-2 in the dark, being verteporfin the most effective. PDs treatment reduced viral load in infected Caco-2 cells, while not inducing cytotoxicity. Furthermore, light-independent treatment with temoporfin and verteporfin act on early stages of viral infection. Using lipid vehicles as membrane models, we characterized PDs interaction to the viral envelope. Verteporfin presented the lowest IC50 for viral inactivation and the highest partition coefficients (Kp) towards lipid bilayers. Curiously, although temoporfin and redaporfin presented similar Kps, redaporfin did not present light-independent antiviral activity, and only temoporfin and verteporfin caused lipid membrane disorder. In fact, redaporfin is located closer to the bilayer surface, while temoporfin and verteporfin are located closer to the centre. Our results suggest that viral envelope affinity, with penetration and destabilization of the lipid bilayer, seems critical to mediate PDs antiviral activity. Altogether, these findings open new avenues for the off-label application of temoporfin and verteporfin in the systemic treatment of COVID-19.
Assuntos
Antivirais , Reposicionamento de Medicamentos , Porfirinas , SARS-CoV-2 , Humanos , Porfirinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , Células CACO-2 , Tratamento Farmacológico da COVID-19 , Antineoplásicos/farmacologia , Envelope Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Células Vero , COVID-19/virologiaRESUMO
Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.
RESUMO
There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53â¯000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.
Assuntos
Vírus da Dengue , Infecções por HIV , HIV-1 , Proteínas do Capsídeo/genética , Humanos , Proteólise , Receptores CXCR4RESUMO
We have developed a nanocarrier consisting of large unilamellar vesicles (LUVs) for combined delivery of two human immunodeficiency virus type 1 (HIV-1) entry inhibitors, enfuvirtide (ENF) and protoporphyrin IX (PPIX). The intrinsic lipophilicity of ENF and PPIX, a fusion inhibitor and an attachment inhibitor, respectively, leads to their spontaneous incorporation into the lipid bilayer of the LUVs nanocarrier. Both entry inhibitors partition significantly toward LUVs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a 9:1 mixture of POPC:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), representative of conventional and immune-evasive drug delivery formulations, respectively. These colocalize in the core of lipid membranes. Dual-loaded nanocarriers are monodispersed and retain the size distribution, thermotropic behavior, and surface charge of the unloaded form. Combination of the two entry inhibitors in the nanocarrier resulted in improved synergy against HIV-1 entry compared to combination in free form, strongly when immune-evasive formulations are used. We propose that the improved action of the entry inhibitors when loaded into the nanocarriers results from their slow release at the site of viral entry. Overall, liposomes remain largely unexplored platforms for combination of viral entry inhibitors, with potential for improvement of current antiretroviral therapy drug safety and application. Our work calls for a reappraisal of the potential of entry inhibitor combinations and delivery for clinical use in antiretroviral therapy.
Assuntos
Enfuvirtida/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Protoporfirinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Humanos , Concentração Inibidora 50 , Lipossomos/química , Nanopartículas/química , PolietilenoglicóisRESUMO
The human APOBEC3 (A3) cytidine deaminases, such as APOBEC3G (A3G) and APOBEC3F (A3F), are potent inhibitors of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif (viral infectivity factor) binds A3 proteins and targets these proteins for ubiquitination and proteasomal degradation. As such, the therapeutic blockage of Vif-A3 interaction is predicted to stimulate natural antiviral activity by rescuing APOBEC expression and virion packaging. In this study, we describe a successful application of the Protein Fragment Complementation Assay (PCA) based on the enzyme TEM-1 ß-lactamase to study Vif-A3 interactions. PCA is based on the interaction between two protein binding partners (e.g., Vif and A3G), which are fused to the two halves of a dissected marker protein (ß-lactamase). Binding of the two partners reassembles ß-lactamase and hence reconstitutes its activity. To validate our assay, we studied the effect of well-described Vif (DRMR, YRHHY) and A3G (D128K) mutations on the interaction between the two proteins. Additionally, we studied the interaction of human Vif with other members of the A3 family: A3F and APOBEC3C (A3C). Our results demonstrate the applicability of PCA as a simple and reliable technique for the assessment of Vif-A3 interactions. Furthermore, when compared with co-immunoprecipitation assays, PCA appeared to be a more sensitive technique for the quantitative assessment of Vif-A3 interactions. Thus, with our results, we conclude that PCA could be used to quantitatively study specific domains that may be involved in the interaction between Vif and APOBEC proteins.
Assuntos
Citosina Desaminase/metabolismo , HIV-1/metabolismo , Análise Serial de Proteínas/normas , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminases APOBEC , Citidina Desaminase , Células HEK293 , Humanos , Análise Serial de Proteínas/métodos , Ligação Proteica/fisiologiaRESUMO
The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1ß secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1ß secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1ß secretion due to elevation of IL-1ß and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases.
Assuntos
Interleucina-1beta/metabolismo , Splicing de RNA/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Ativação Enzimática , Escherichia coli , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Humanos , Interleucina-1beta/genética , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Reprodutibilidade dos Testes , Fatores de Processamento de Serina-Arginina , Transcrição GênicaRESUMO
APOBEC proteins appeared in the cellular battle against HIV-1 as part of intrinsic cellular immunity. The antiretroviral activity of some of these proteins is overtaken by the action of HIV-1 Viral Infectivity Factor (Vif) protein. Since the discovery of APOBEC3G (A3G) as an antiviral factor, many advances have been made to understand its mechanism of action in the cell and how Vif acts in order to counteract its activity. The mainstream concept is that Vif overcomes the innate antiviral activity of A3G by direct protein binding and promoting its degradation via the cellular ubiquitin/proteasomal pathway. Vif may also inhibit A3G through mechanisms independent of proteasomal degradation. Binding of Vif to A3G is essential for its degradation since disruption of this interaction is predicted to stimulate intracellular antiviral immunity. In this paper we will discuss the different binding partners between both proteins as one of the major challenges for the development of new antiviral drugs.
RESUMO
The human APOBEC3G (A3G) is a potent inhibitor of HIV-1 replication and its activity is suppressed by HIV-1 virion infectivity factor (Vif). Vif neutralizes A3G mainly by inducing its degradation in the proteasome and blocking its incorporation into HIV-1 virions. Assessing the time needed for A3G incorporation into virions is, therefore, important to determine how quickly Vif must act to induce its degradation. We show that modelling the intracellular half-life of A3G can induce its Vif-independent targeting to the ubiquitin-proteasome system. By using various amino acids (X) in a cleavable ubiquitin-X-A3G fusion, we demonstrate that the half-life (t1/2) of X-A3G can be manipulated. We show that A3G molecules with a half-life of 13 min are incorporated into virions, whereas those with a half-life shorter than 5 min were not. The amount of X-A3G incorporated into virions increases from 13 min (Phe-A3G) to 85 min (Asn-A3G) and remains constant after this time period. Interestingly, despite the presence of similar levels of Arg-A3G (t1/2=28 min) and Asp-A3G (t1/2=65 min) into HIV-1 Deltavif virions, inhibition of viral infectivity was only evident in the presence of A3G proteins with a longer half-life (t1/2 > or = 65 min).