high-throughput-antiviral-assays-to-screen-for-inhibitors-zika-virus

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

University of Sao Paulo

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)₂, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller-Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)₂ were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys/Lys-(p-BthTX-I)₂), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)₂, and des-Lys/Lys-(p-BthTX-I)₂ were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys/Lys-(p-BthTX-I)₂ and (p-BthTX-I)₂ degraded  biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic  in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)₂ are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)₂ and Its Serum Degradation Product against Multidrug-Resistant Bacteria.

An important aspect of host-pathogen interactions is the interference of secreted proteins with the fibrinolytic system. Herein, we describe a modified ELISA method used to evaluate the interaction of a recombinant Schistosoma mansoni protein with plasminogen (PLG). Using this protocol, we demonstrated that a secreted protein, recombinant venom allergen-like protein 18 (rSmVAL18) acts as a plasminogen receptor increasing its activation into plasmin in the presence of the urokinase-type plasminogen activator (uPA). PLG binding was determined by immobilizing human PLG in the plate and incubating with the recombinant protein; competitive binding with a lysine analog demonstrated the interaction of the protein lysine residues with PLG Kringle domains. To assess the activation of S. mansoni recombinant protein-bound PLG, the amidolytic activity of generated plasmin was measured using the D-Val-Leu-Lys 4-nitroanilide dihydrochloride substrate.

A Modified ELISA Method to Evaluate the Interaction of Schistosoma mansoni Proteins with Plasminogen.

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.

Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses.

The 2015/16 Zika virus (ZIKV) epidemic led to almost 1 million confirmed cases in 84 countries and was associated to the development of congenital microcephaly and Guillain-Barré syndrome. More recently, a ZIKV African lineage was identified in Brazil raising concerns about a future outbreak. The long-term consequences of viral infection emphasizes the need for the development of effective anti-ZIKV drugs. In this study, we developed and characterized a ZIKV replicon cell line for the screening of viral replication inhibitors. The replicon system was developed by engineering the IRES-Neo cassette into the 3' UTR terminus of the ZIKV Rluc DNA construct. After in vitro transcription, replicon RNA was used to transfect BHK-21 cells, that were selected with G418, thus generating the BHK-21-RepZIKV_IRES-Neo cell line. Through this replicon-based cell system, we identified two molecules with potent anti-ZIKV activities, an imidazonaphthyridine and a riminophenazine, both from the MMV/DNDi Pandemic Response Box library of 400 drug-like compounds. The imidazonaphthyridine, known as RO8191, showed remarkable selectivity against ZIKV, while the riminophenazine, the antibiotic Clofazimine, could act as a non-nucleoside analog inhibitor of viral RNA-dependent RNA polymerase (RdRp), as evidenced both in vitro and in silico. The data showed herein supports the use of replicon-based assays in high-throughput screening format as a biosafe and reliable tool for antiviral drug discovery.

Discovery of an imidazonaphthyridine and a riminophenazine as potent anti-Zika virus agents through a replicon-based high-throughput screening.

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys, Lys,Lys-(pBthTX-I)K () and derivatives against SARS-CoV-2 are reported. The lead peptide  and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC = 28-65 µM) and mostly low cytotoxic effect (CC > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (M) and Papain-Like protease (PL) inhibitory activities of the peptides were assessed. The synthetic peptides showed PL inhibition potencies (ICs = 1.0-3.5 µM) and binding affinities ( = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against M (IC > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PL substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.

Article	Year
Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)₂ and Its Serum Degradation Product against Multidrug-Resistant Bacteria. Molecules (Basel, Switzerland)\| PubMed ID: 29113051	2017
A Modified ELISA Method to Evaluate the Interaction of Schistosoma mansoni Proteins with Plasminogen. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32452005	2020
Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses. Viruses\| PubMed ID: 32486283	2020
Discovery of an imidazonaphthyridine and a riminophenazine as potent anti-Zika virus agents through a replicon-based high-throughput screening. Virus research\| PubMed ID: 33887282	2021
Non-Toxic Dimeric Peptides Derived from the Bothropstoxin-I Are Potent SARS-CoV-2 and Papain-like Protease Inhibitors. Molecules (Basel, Switzerland)\| PubMed ID: 34443484	2021

Rafaela S. Fernandes

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