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Snakebite primarily impacts rural communities of Africa, Asia, and Latin America. The sharp-nosed viper (Deinagkistrodon acutus) is among the snakes of highest medical importance in Asia. Despite various studies on its venom using modern venomics techniques, a comprehensive understanding of composition and function of this species' venom remains lacking. We combined proteogenomics with extensive bioactivity profiling to present the first genome-level catalogue of D. acutus venom proteins and their exochemistry. Our analysis identified an unusually simple venom containing 45 components from 20 distinct protein families. Relative toxin abundances indicate that C-type lectin and C-type lectin-related protein (CTL), snake venom metalloproteinase (svMP), snake venom serine protease (svSP), and phospholipase A2 (PLA2) constitute 90 % of the venom. Bioassays targeting key aspects of viperid envenomation showed considerable concentration-dependent cytotoxicity, particularly in kidney and lung cells, and potent protease and PLA2 activity. Factor Xa and thrombin activities were minor, and no plasmin activity was observed. Effects on haemolysis, intracellular calcium (Ca2+) release, and nitric oxide (NO) synthesis were negligible. Our analysis provides the first holistic genome-based overview of the toxin arsenal of D. acutus, predicting the molecular and functional basis of its life-threatening effects, and opens novel avenues for treating envenomation by this highly dangerous snake.
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In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.
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BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.
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Aedes , Interferencia de ARN , ARN Bicatenario , Transfección , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Animales , Línea Celular , Aedes/genética , Silenciador del Gen , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/efectos de los fármacosRESUMEN
Spider-derived peptides with insecticidal, antimicrobial and/or cytolytic activities, also known as spider venom antimicrobial peptides (AMPs), can be found in the venoms of RTA-clade spiders. They show translational potential as therapeutic leads. A set of 52 AMPs has been described in the Chinese wolf spider (Lycosa shansia), and many have been shown to exhibit antibacterial effects. Here we explored the potential to enhance their antimicrobial activity using bioengineering. We generated a panel of artificial derivatives of an A-family peptide and screened their activity against selected microbial pathogens, vertebrate cells and insects. In several cases, we increased the antimicrobial activity of the derivatives while retaining the low cytotoxicity of the parental molecule. Furthermore, we injected the peptides into adult Drosophila suzukii and found no evidence of insecticidal effects, confirming the low levels of toxicity. Our data therefore suggest that spider venom linear peptides naturally defend the venom gland against microbial colonization and can be modified into more potent antimicrobial agents that could help to battle infectious diseases in the future.
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Venenos de Araña , Arañas , Animales , Venenos de Araña/química , Venenos de Araña/farmacología , Venenos de Araña/toxicidad , Drosophila/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Antiinfecciosos/farmacología , Insecticidas/farmacología , HumanosRESUMEN
Tuberculosis has remained one of the world's deadliest infectious diseases. The complexity and numerous adverse effects of current treatment options as well as the emergence of multi-drug resistant M. tuberculosis (Mtb) demand research and innovation efforts to yield new anti-mycobacterial agents. In this study, we synthesized a series of imidazo[1,5-a]quinolines, including 4 new analogs, and evaluated their activity against Mtb. Inspired by previous studies, we also designed 8 compounds featuring a coordinated metal ion, determined their absolute configuration by single-crystal X-ray diffraction and included them in the bioactivity study. Remarkably, the metal complexation of 5c with either Zn2+ or Fe2+ increased the Mtb inhibitory activity of the compound 12.5-fold and reduced its cytotoxicity. Ultimately, out of the 21 analyzed imidazo[1,5-a]quinoline analogs, two zinc complexes (C1 and C7) showed the strongest, specific activity against Mtb H37Rv in vitro (IC90 = 7.7 and 17.7 µM).
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The venoms of spiders from the RTA (retro-lateral tibia apophysis) clade contain diverse short linear peptides (SLPs) that offer a rich source of therapeutic candidates. Many of these peptides have insecticidal, antimicrobial and/or cytolytic activities, but their biological functions are unclear. Here, we explore the bioactivity of all known members of the A-family of SLPs previously identified in the venom of the Chinese wolf spider (Lycosa shansia). Our broad approach included an in silico analysis of physicochemical properties and bioactivity profiling for cytotoxic, antiviral, insecticidal and antibacterial activities. We found that most members of the A-family can form α-helices and resemble the antibacterial peptides found in frog poison. The peptides we tested showed no cytotoxic, antiviral or insecticidal activities but were able to reduce the growth of bacteria, including clinically relevant strains of Staphylococcus epidermidis and Listeria monocytogenes. The absence of insecticidal activity may suggest that these peptides have no role in prey capture, but their antibacterial activity may help to defend the venom gland against infection.
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Insecticidas , Venenos de Araña , Arañas , Animales , Ponzoñas , Péptidos/farmacología , Péptidos/química , Insecticidas/química , Antibacterianos/farmacología , Antibacterianos/química , Arañas/química , Venenos de Araña/farmacología , Venenos de Araña/químicaRESUMEN
Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.
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The Asian tiger mosquito Aedes albopictus is currently spreading across Europe, facilitated by climate change and global transportation. It is a vector of arboviruses causing human diseases such as chikungunya, dengue hemorrhagic fever and Zika fever. For the majority of these diseases, no vaccines or therapeutics are available. Options for the control of Ae. albopictus are limited by European regulations introduced to protect biodiversity by restricting or phasing out the use of pesticides, genetically modified organisms (GMOs) or products of genome editing. Alternative solutions are thus urgently needed to avoid a future scenario in which Europe faces a choice between prioritizing human health or biodiversity when it comes to Aedes-vectored pathogens. To ensure regulatory compliance and public acceptance, these solutions should preferably not be based on chemicals or GMOs and must be cost-efficient and specific. The present review aims to synthesize available evidence on RNAi-based mosquito vector control and its potential for application in the European Union. The recent literature has identified some potential target sites in Ae. albopictus and formulations for delivery. However, we found little information concerning non-target effects on the environment or human health, on social aspects, regulatory frameworks, or on management perspectives. We propose optimal designs for RNAi-based vector control tools against Ae. albopictus (target product profiles), discuss their efficacy and reflect on potential risks to environmental health and the importance of societal aspects. The roadmap from design to application will provide readers with a comprehensive perspective on the application of emerging RNAi-based vector control tools for the suppression of Ae. albopictus populations with special focus on Europe.
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Aedes , Dengue , Infección por el Virus Zika , Virus Zika , Animales , Humanos , Dengue/genética , Aedes/genética , Interferencia de ARN , Europa (Continente) , Mosquitos Vectores/genéticaRESUMEN
The venoms of ants (Formicidae) are a promising source of novel bioactive molecules with potential for clinical and agricultural applications. However, despite the rich diversity of ant species, only a fraction of this vast resource has been thoroughly examined in bioprospecting programs. Previous studies focusing on the venom of Central European ants (subfamily Myrmicinae) identified a number of short linear decapeptides and nonapeptides resembling antimicrobial peptides (AMPs). Here, we describe the in silico approach and bioactivity profiling of 10 novel AMP-like peptides from the fellow Central European myrmicine ants Myrmica rubra and Myrmica ruginodis. Using the sequences of known ant venom peptides as queries, we screened the venom gland transcriptomes of both species. We found transcripts of nine novel decapeptides and one novel nonapeptide. The corresponding peptides were synthesized for bioactivity profiling in a broad panel of assays consisting of tests for cytotoxicity as well as antiviral, insecticidal, and antimicrobial activity. U-MYRTX-Mrug5a showed moderately potent antimicrobial effects against several bacteria, including clinically relevant pathogens such as Listeria monocytogenes and Staphylococcus epidermidis, but high concentrations showed negligible cytotoxicity. U-MYRTX-Mrug5a is, therefore, a probable lead for the development of novel peptide-based antibiotics.
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Venenos de Hormiga , Antiinfecciosos , Hormigas , Animales , Hormigas/genética , Péptidos/química , Transcriptoma , Ponzoñas , Venenos de Hormiga/toxicidad , Venenos de Hormiga/químicaRESUMEN
Influenza is a severe contagious disease caused by influenza A and B viruses. The WHO estimates that annual outbreaks lead to 3-5 million severe infections of which approximately 10% lead to the death of the patient. While vaccination is the cornerstone of prevention, antiviral drugs represent the most important treatment option of acute infections. Only two classes of drugs are currently approved for the treatment of influenza in numerous countries: M2 channel blockers and neuraminidase inhibitors. In some countries, additional compounds such as the recently developed cap-dependent endonuclease inhibitor baloxavir marboxil or the polymerase inhibitor favipiravir are available. However, many of these compounds suffer from poor efficacy, if not applied early after infection. Furthermore, many influenza strains have developed resistances and lost susceptibility to these compounds. As a result, there is an urgent need to develop new anti-influenza drugs against a broad spectrum of subtypes. Natural products have made an important contribution to the development of new lead structures, particularly in the field of infectious diseases. Therefore, this article aims to review the research on the identification of novel lead structures isolated from natural resources suitable to treat influenza infections.
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Gripe Humana , Infecciones por Orthomyxoviridae , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Gripe Humana/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Recursos NaturalesRESUMEN
Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.
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Furina , Proproteína Convertasas , Secuencia de Aminoácidos , Carbamatos , Colorantes Fluorescentes , Oligopéptidos , Proteínas , Subtilisinas/metabolismoRESUMEN
Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin11-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.
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Antiinfecciosos , Proteínas de Artrópodos , Venenos de Artrópodos , Citotoxinas , Insecticidas , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antiinfecciosos/toxicidad , Áfidos/efectos de los fármacos , Arácnidos , Proteínas de Artrópodos/química , Proteínas de Artrópodos/farmacología , Proteínas de Artrópodos/toxicidad , Venenos de Artrópodos/química , Venenos de Artrópodos/farmacología , Venenos de Artrópodos/toxicidad , Citotoxinas/química , Citotoxinas/farmacología , Citotoxinas/toxicidad , Perros , Insecticidas/química , Insecticidas/farmacología , Insecticidas/toxicidad , Células de Riñón Canino Madin Darby , Alineación de SecuenciaRESUMEN
The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
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Células Epiteliales Alveolares/virología , Betacoronavirus/fisiología , Furina/genética , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Epiteliales Alveolares/citología , Animales , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Proteolisis , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Células Vero , Internalización del Virus , Replicación ViralRESUMEN
Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.
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Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Gripe Humana/virología , Serina Endopeptidasas/metabolismo , Animales , Bronquios/citología , Células Cultivadas , Células Epiteliales/virología , Técnicas de Silenciamiento del Gen , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Interacciones Huésped-Patógeno , Humanos , Gripe Humana/enzimología , Gripe Humana/metabolismo , Ratones , Infecciones por Orthomyxoviridae/enzimología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Alveolos Pulmonares/citología , Serina Endopeptidasas/genética , Regulación hacia Arriba , Replicación ViralRESUMEN
The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high-throughput screening format. The robust and cost-effective assay format of BPCS makes it suitable to identify novel specific small-molecule inhibitors against basic PCs for therapeutic application. Its cell-based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened 'target range' will enhance the likelihood to identify novel small-molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.
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Técnicas Biosensibles/métodos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Peptidomiméticos/farmacología , Proproteína Convertasas/metabolismo , Células A549 , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Técnicas Biosensibles/normas , Descubrimiento de Drogas/normas , Inhibidores Enzimáticos/química , Genes Reporteros , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Peptidomiméticos/química , Proproteína Convertasas/antagonistas & inhibidores , Sensibilidad y EspecificidadRESUMEN
Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the 15 possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogues using a targeted protecting group strategy to investigate the impact of the C19-C25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogues was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild-type tridegin and the analogues as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C19-C25 bond on conformational stability and binding mode. The strategy of selectively reducing disulfide bonds to facilitate large-scale synthesis, while retaining the functionality of disulfide-bonded peptides, has been demonstrated with our present study.
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Disulfuros/química , Factor XIIIa/antagonistas & inhibidores , Proteínas y Péptidos Salivales/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , Humanos , Isomerismo , Sanguijuelas , Simulación de Dinámica Molecular , Pliegue de Proteína , Estabilidad Proteica , Proteínas y Péptidos Salivales/químicaRESUMEN
The activation of viral glycoproteins by the host protease furin is an essential step in the replication of numerous pathogenic viruses. Thus, effective inhibitors of furin could serve as broad-spectrum antiviral drugs. A crystal structure of an inhibitory hexapeptide derivative in complex with furin served as template for the rational design of various types of new cyclic inhibitors. Most of the prepared derivatives are relatively potent furin inhibitors with inhibition constants in the low nanomolar or even sub-nanomolar range. For seven derivatives the crystal structures in complex with furin could be determined. In three complexes, electron density was found for the entire inhibitor. In the other cases the structures could be determined only for the P6/P5-P1 segments, which directly interact with furin. The cyclic derivatives together with two non-cyclic reference compounds were tested as inhibitors of the proteolytic activation and replication of respiratory syncytial virus in cells. Significant antiviral activity was found for both linear reference inhibitors, whereas a negligible efficacy was determined for the cyclic derivatives.
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Inhibidores Enzimáticos/farmacología , Furina/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Humanos , Compuestos Macrocíclicos/síntesis químicaRESUMEN
Bats harbor a plethora of viruses with an unknown zoonotic potential. In-depth functional characterization of such viruses is often hampered by a lack of virus isolates. The genome of a virus closely related to human mumps viruses (hMuV) was detected in African fruit bats, batMuV. Efforts to characterize batMuV were based on directed expression of the batMuV glycoproteins or use of recombinant chimeric hMuVs harboring batMuV glycoprotein. Although these studies provided initial insights into the functionality of batMuV glycoproteins, the host range, replication competence, immunomodulatory functions, virulence, and zoonotic potential of batMuV remained elusive. Here, we report the successful rescue of recombinant batMuV. BatMuV infects human cells, is largely resistant to the host interferon response, blocks interferon induction and TNF-α activation, and is neurotoxic in rats. Anti-hMuV antibodies efficiently neutralize batMuV. The striking similarities between hMuV and batMuV point at the putative zoonotic potential of batMuV.
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Quirópteros/virología , Evasión Inmune/inmunología , Virus de la Parotiditis/inmunología , Paperas/virología , Síndromes de Neurotoxicidad/etiología , Internalización del Virus , Replicación Viral , Animales , Femenino , Humanos , Virus de la Parotiditis/patogenicidad , Síndromes de Neurotoxicidad/patología , Ratas , Ratas Endogámicas LewRESUMEN
The proprotein convertase furin is a highly specific serine protease modifying and thereby activating proteins in the secretory pathway by proteolytic cleavage. Its substrates are involved in many diseases, including cancer and infections caused by bacteria and viruses. Understanding furin's substrate specificity is crucially important for the development of pharmacologically applicable inhibitors. Using protein X-ray crystallography, we investigated the extended substrate binding site of furin in complex with three peptide-derived inhibitors at up to 1.9 Å resolution. The structure of the protease bound with a hexapeptide inhibitor revealed molecular details of its S6 pocket, which remained completely unknown so far. The arginine residue at P6 induced an unexpected turnlike conformation of the inhibitor backbone, which is stabilized by intra- and intermolecular H-bonds. In addition, we confirmed the binding of arginine to the previously proposed S5 pocket (S51). An alternative S5 site (S52) could be utilized by shorter side chains as demonstrated for a 4-aminomethyl-phenylacetyl residue, which shows steric properties similar to those of a lysine side chain. Interestingly, we also observed binding of a peptide with citrulline at P4 substituting for the highly conserved arginine. The structural data might indicate an unusual protonation state of Asp264 maintaining the interaction with uncharged citrulline. The herein identified molecular interaction sites at P5 and P6 can be utilized to improve next-generation furin inhibitors. Our data will also help to predict furin substrates more precisely on the basis of the additional specificity determinants observed for P5 and P6.
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Furina/química , Sitios de Unión , Cristalografía por Rayos X , Furina/antagonistas & inhibidores , Furina/metabolismo , Células HEK293 , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Péptidos/química , Péptidos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Conformación Proteica , Especificidad por SustratoRESUMEN
The proprotein convertase furin is a potential target for drug design, especially for the inhibition of furin-dependent virus replication. All effective synthetic furin inhibitors identified thus far are multibasic compounds; the highest potency was found for our previously developed inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148). An initial study in mice revealed a narrow therapeutic range for this tetrabasic compound, while significantly reduced toxicity was observed for some tribasic analogues. This suggests that the toxicity depends at least to some extent on the overall multibasic character of this inhibitor. Therefore, in a first approach, the C-terminal benzamidine of MI-1148 was replaced by less basic P1 residues. Despite decreased potency, a few compounds still inhibit furin in the low nanomolar range, but display negligible efficacy in cells. In a second approach, the P2 arginine was replaced by lysine; compared to MI-1148, this furin inhibitor has slightly decreased potency, but exhibits similar antiviral activity against West Nile and Dengue virus in cell culture and decreased toxicity in mice. These results provide a promising starting point for the development of efficacious and well-tolerated furin inhibitors.