Proteases from dengue, West Nile and Zika viruses as drug targets

Proteases from flaviviruses have gained substantial interest as potential drug targets to combat infectious diseases caused by dengue, West Nile, Zika and related viruses. Despite nearly two decades of drug discovery campaigns, promising lead compounds for clinical trials have not yet been identified. The main challenges for successful lead compound development are associated with limited drug-likeness of inhibitors and structural ambiguity of the protease target. This brief review focuses on the available information on the structure of flavivirus proteases and their interactions with inhibitors and attempts to point the way forward for successful identification of future lead compounds.Christoph Nitsche thanks the Alexander von Humboldt Foundation for a Feodor Lynen Fellowship and the Free University of Berlin for a Rising Star Fellowship

Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags

C.N. thanks the Alexander von Humboldt Foundation for a Feo-dor Lynen fellowship. Financial support by the Australian ResearchCouncil is gratefully acknowledge

Characterisation of ten NS2B-NS3 proteases: Paving the way for pan-flavivirus drugs

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

The SARS-CoV-2 main protease as drug target

The unprecedented pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening global health. The virus emerged in late 2019 and can cause a severe disease associated with significant mortality. Several vaccine development and drug discovery campaigns are underway. The SARS-CoV-2 main protease is considered a promising drug target, as it is dissimilar to human proteases. Sequence and structure of the main protease are closely related to those from other betacoronaviruses, facilitating drug discovery attempts based on previous lead compounds. Covalently binding peptidomimetics and small molecules are investigated. Various compounds show antiviral activity in infected human cells.C.N. thanks the Australian Research Council for a Discovery Early Career Research Award (DE190100015). S.U. acknowledges a PROMOS scholarship from the German Academic Exchange Service

Targeting the protease of West Nile virus

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.C. N. thanks the Australian Research Council for a Discovery Early Career Research Award (DE190100015) and Discovery Project funding (DP200100348)

SARS-CoV-2 Papain-Like Protease: Structure, Function and Inhibition

Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.CN gratefully acknowledges funding by the Australian Research Council (DECRA: DE190100015; Discovery Project: DP200100348). The authors gratefully acknowledge Junming He (Australian National University) who designed and created the artwork for the frontispiec

Site-selective tagging of proteins by pnictogen-mediated self-assembly

Site-selective chemical protein modification is achieved by self-assembly of a specific di-cysteine motif, trivalent pnictogens (As, Sb or Bi) and an aromatic mercaptomethyl-based probe. The strategy is demonstrated with a quaternary complex involving Zika virus protease and a lanthanide ion, enabling paramagnetic nuclear magnetic resonance spectroscopy and luminescence measurements.Financial support by the Australian Research Council is gratefully acknowledge

Peptide–Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity

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 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.C.N. thanks the Australian Research Council for funding (DE190100015 and DP200100348). The work was supported by the DFG-funded Core Facility BioSupraMol. We thank Prof. Gottfried Otting for help with NMR spectroscopy, Dr. Josemon George for sample preparation, Dr. Christoph Arkona for plasmid transformation, Silke Bergemann for support with protein expression and purification, and Peter Demirel for providing a sample of Fmoc-Phe(4-Boc2-guanidino)-OH, development of the HPLC method, and MS support

Peptide-Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity

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 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.C.N. thanks the Australian Research Council for funding (DE190100015 and DP200100348). The work was supported by the DFG-funded Core Facility BioSupraMol. We thank Prof. Gottfried Otting for help with NMR spectroscopy, Dr. Josemon George for sample preparation, Dr. Christoph Arkona for plasmid transformation, Silke Bergemann for support with protein expression and purification, and Peter Demirel for providing a sample of Fmoc-Phe(4-Boc2- guanidino)-OH, development of the HPLC method, and MS support

Studying the Neurobiology of Social Interaction with Transcranial Direct Current Stimulation—The Example of Punishing Unfairness

Studying social behavior often requires the simultaneous interaction of many subjects. As yet, however, no painless, noninvasive brain stimulation tool existed that allowed the simultaneous affection of brain processes in many interacting subjects. Here we show that transcranial direct current stimulation (tDCS) can overcome these limits. We apply right prefrontal cathodal tDCS and show that subjects' propensity to punish unfair behavior is reduced significantl