A Non-Active-Site Inhibitor with Selectivity for Zika Virus NS2B-NS3 Protease.

Flavivirus infection usually results in fever accompanied by headache, arthralgia, and, in some cases, rash. Although the symptoms are mild, full recovery can take several months. Flaviviruses encode seven nonstructural proteins that represent potential drug targets for this viral family. Focusing on the Zika virus NS2B-NS3 protease, we uncovered a unique inhibitor, MH1, composed of aminothiazolopyridine and benzofuran moieties. MH1 inhibits ZVP with a biochemical IC50 of 440 nM and effectively blocks cleavage of ZVP substrates in cells. Surprisingly, MH1 inhibits the other flaviviral proteases at least 18-fold more weakly. This same phenomenon was observed in assays of the viral cytopathic effect, where only Zika virus showed sensitivity to MH1. This selectivity was unexpected since flaviviral proteases have high similarity in sequence and protein structure. MH1 binds at an allosteric site, as demonstrated by its ability to stabilize ZVP synergistically with an active site inhibitor. To understand its selectivity, we constructed a series of hybrid proteases composed of select segments of ZVP, which is sensitive to MH1, and dengue virus protease, which is essentially insensitive to MH1. Our results suggest that MH1 binds to the NS3 protease domain, disrupting its interaction with NS2B. These interactions are essential for substrate binding and cleavage. In particular, the unique dynamic properties of NS2B from Zika seem to be required for the function of MH1. Insights into the mechanism of MH1 function will aid us in developing non-active-site-directed, pan-flaviviral inhibitors, by highlighting the importance of evaluating and considering the dynamics of the NS2B regions.

Colorimetric Cotton Swab for Viral Protease Detection.

Reporting the activity of a specific viral protease remains an acute need for rapid point-of-care detection strategies that can distinguish active infection from a resolved infection. In this work, we present a simple colorimetric approach for reporting the activity of a specific viral protease through direct color conversion on a cotton swab, which has the potential to be extended to detect the corresponding virus. We use SARS-CoV-2 viral protease as a proof-of-concept model system. We use 4-aminomalachite green (4-AMG) as the base chromophore structure to design a CoV2-AMG reporter, which is selective toward the SARS-CoV-2 Mpro but does not produce any observable color change in the presence of other viral proteases. The color change is observable by the naked eye, as well as smartphone imaging, which affords a lower limit of detection. The simplicity and generalizability of the method could be instrumental in combating future viral outbreaks.

Comparative Study of Protein Aggregation Arrest by Zwitterionic Polysulfobetaines: Using Contrasting Raft Agents.

Protein aggregation has caused limitations in the study and development of protein-based biopharmaceuticals. We prepared different polysulfobetaine (poly-SPB) polymers via reversible addition fragmentation chain transfer (RAFT) polymerization. These polymers exhibited high efficiency in modulation of protein aggregation. We synthesized polysulfobetaines using two different RAFT agents, and analyzed the aggregation profile of lysozyme and insulin. In poly-SPBs, existence of a hydrophobic RAFT agent resulted in visible enhancement of the residual enzymatic activity of lysozyme, whereas it remained unaffected by the hydrophilic RAFT agent. In addition, these polymers resulted in significant suppression in the aggregation of insulin. Increase in the molecular weight of the polymer caused higher efficiency to perpetuate enzymatic activity of lysozyme upon thermal denaturation. The polymers arrested the formation of amyloid like fibrils of lysozyme and insulin, thus indicating their potential to inhibit aggregation. The results unambiguously demonstrate the importance of polysulfobetaine moiety and hydrophobicity in protein aggregation inhibition. This study gives insight into the protein aggregation inhibition by zwitterionic polymers, which have a potential to be developed as aggregation inhibitors in the future.