RESUMEN
Flaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly4-Ser-Gly4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM.
Asunto(s)
Antivirales , Flavivirus , Serina Endopeptidasas , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Flavivirus/efectos de los fármacos , Flavivirus/enzimología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Antivirales/farmacología , Antivirales/química , Humanos , ARN Helicasas/metabolismo , ARN Helicasas/química , ARN Helicasas/genética , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
Antibiotic resistance is an escalating global health threat. Due to their diverse mechanisms of action and evasion of traditional resistance mechanisms, peptides hold promise as future antibiotics. Their ability to disrupt bacterial membranes presents a potential strategy to combat drug-resistant infections and address the increasing need for effective antimicrobial treatments. Amphipathic α-helical peptides possess a distinctive molecular structure with both charged/hydrophilic and hydrophobic regions that interact with the bacterial cell membrane, disrupting its structural integrity. The α-helical amphipathic peptide aurein 1.2, secreted by the Australian frog Litoria aurea, is one of the shortest known antimicrobial peptides, spanning only 13 amino acids. The primary objective of this study was to investigate stapled and photoswitchable modifications of short helical peptides employing biocompatible chemistry, utilising aurein 1.2 as a model system. We developed various stapled versions of aurein 1.2 using biocompatible conjugation chemistry between dicyanopyridine and 1,2-aminothiols. While the commonly employed stapling pattern for longer staples is i, i + 7, we observed superior helicity in peptides stapled at positions i, i + 8. Molecular dynamics simulations confirmed both stapling patterns to support an α-helical peptide conformation. Additionally, we utilised a cysteine-selective photosensitive staple, perfluoro azobenzene, to explore photoswitchable variants of aurein 1.2. A double-cysteine variant stapled at i, i + 7 indeed exhibited a change in overall helicity induced by light. We further demonstrated the applicability of this staple to attach to cysteine residues in i, i + 7 positions of a helix in a model protein. While some of the stapled variants displayed substantial increase in helicity, minimal inhibitory concentration assays revealed that none of the stapled aurein 1.2 variants exhibited increased antimicrobial activity compared to the wildtype.
Asunto(s)
Antiinfecciosos , Péptidos Antimicrobianos , Animales , Secuencia de Aminoácidos , Cisteína , Conformación Proteica , Australia , Péptidos/farmacología , Péptidos/química , Anuros , BacteriasRESUMEN
Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.
Asunto(s)
Péptidos de Penetración Celular , Humanos , Péptidos de Penetración Celular/química , Endocitosis/fisiología , Bismuto , Ciclismo , PinocitosisRESUMEN
Constrained peptides are promising next-generation therapeutics. We report here a fundamentally new strategy for the facile generation of bicyclic peptides using linear precursor peptides with three cysteine residues and a non-toxic trivalent bismuth(III) salt. Peptide-bismuth bicycles form instantaneously at physiological pH, are stable in aqueous solution for many weeks, and much more resistant to proteolysis than their linear precursors. The strategy allows the in situ generation of bicyclic ligands for biochemical screening assays. We demonstrate this for two screening campaigns targeting the proteases from Zika and West Nile viruses, revealing a new lead compound that displayed inhibition constants of 23 and 150â nM, respectively. Bicyclic peptides are up to 130â times more active and 19â times more proteolytically stable than their linear analogs without bismuth.
Asunto(s)
Bismuto/farmacología , Péptido Hidrolasas/metabolismo , Péptidos Cíclicos/farmacología , Inhibidores de Proteasas/farmacología , Bismuto/química , Relación Dosis-Respuesta a Droga , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Conformación Proteica , Virus del Nilo Occidental/enzimología , Virus Zika/enzimologíaRESUMEN
West Nile virus infections can cause severe neurological symptoms. During the last 25 years, cases have been reported in Asia, North America, Africa, Europe and Australia (Kunjin). No West Nile virus vaccines or specific antiviral therapies are available to date. Various viral proteins and host-cell factors have been evaluated as potential drug targets. The viral protease NS2B-NS3 is among the most promising viral targets. It releases viral proteins from a non-functional polyprotein precursor, making it a critical factor of viral replication. Despite strong efforts, no protease inhibitors have reached clinical trials yet. Substrate-derived peptidomimetics have facilitated structural elucidations of the active protease state, while alternative compounds with increased drug-likeness have recently expanded drug discovery efforts beyond the active site.
RESUMEN
In recent years, the Zika virus has emerged from a neglected flavivirus to a health-threatening pathogen that causes epidemic outbreaks associated with neurological disorders and congenital malformations. In addition to vaccine development, the discovery of specific antiviral agents has been pursued intensely. The Zika virus protease NS2B-NS3 catalyses the processing of the viral precursor polyprotein as an essential step during viral replication. Since the epidemic Zika virus outbreak in the Americas, several inhibitors of this protease have been reported. Substrate-derived peptides revealed important structural information about the active site, whilst more drug-like small molecules have been discovered as allosteric inhibitors.