In a recent study published in bioRxiv* preprint server, researchers evaluated the effect of a syntenin inhibitor on the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Background
Viruses depend on interacting with host factors to effectively induce infection and subsequently replicate. Therapeutic approaches aimed at such interactions allow the development of new and effective antiviral drugs. Syntenic is an important protein that regulates the architecture of major cell membranes due to its role in protein delivery. Due to this function, syntenin also has a beneficial effect on the human papillomavirus (HPV).
About the study
The present study is investigating the usefulness of the syntenin inhibitor KSL-128114 as a broad-spectrum inhibitor against viral infections, including coronavirus disease 2019 (COVID-19).
The team assembled synthetic fluorescein isothiocyanate (FITC) labeled peptides that corresponded to the C-terminus of the viral envelope protein (E), open reading frame (ORF)-3, and non-structural protein 11 (NSP11) from SARS-CoV-2. . These peptides helped evaluate the affinity for recombinantly expressed and purified PDZ1-PDZ2, SNX27 PDZ, and MPP5 PDZ, respectively.
In addition, the team investigated the consequences of viral infection and replication by inhibiting interaction with syntenins. This was achieved by penetrating cells with a KSL-128114 peptide-based inhibitor to assess the level of SARS-CoV-2 infection by the concentration of the inhibitor present in Vero E6 cells. In addition, the antiviral mechanism of the inhibitor was analyzed by experiments in which the time of addition of inhibition was evaluated. The effect of adding the inhibitor two hours before and one or three hours after infection was then calculated.
In addition, the effect of the inhibitor on the binding of the virus to the host cell or the entry of the virus into the host cell was investigated by treating the host cells with the inhibitor two hours prior to infection. The team then assessed viral ribonucleic acid (RNA) levels using quantitative polymerase chain reaction (qPCR) after one hour on ice or two hours after infection. In addition, infected Vero E6 and Calu-3 cells were treated with chloroquine and monitored for infection.
Results
The results of the study showed that PDZ1-2 syntenin binds with the highest affinity to the putative PDZ binding motif at the C-terminus of SARS-CoV-2 NSP11. Syntenin PDZ1-2 also showed low binding affinity for the peptide in the viral E protein. The observed affinity was low, but similar to that found in endogenous interactions with syntenins. The interaction noted with the oligomeric E protein may be improved by avidity effects in cellular conditions. Moreover, when MPP5 PDZ was added as a control, it preferentially bound to the viral E protein. On the other hand, SNX27 bound to all three peptides with comparatively lower affinity.
The inhibitor based on the KSL-128114 peptide effectively blocked the viral infection with virtually no effect on cell viability. Treatment of the cells with 30 μM of the inhibitor showed that the viral infection as well as the number of new viral particles released were significantly reduced. Although pre-infection inhibitor treatment had a marked effect on infection rates, the research team noted that post-infection treatment had no effect, indicating that the inhibitor inhibits the initial stages of viral infection that occur before infection. frequency of any interactions between viral proteins and syntenin.
Interestingly, the antiviral effect of a syntenin inhibitor cannot be due to inhibition of interactions between viral PDZ-binding motifs and intracellular PDZ proteins. This is because these interactions occur in the later stages of a viral infection. The results showed that the inhibitor blocks important endogenous interactions critical in the initial phases of the viral life cycle, such as the interaction between PDZ and angiotensin-converting enzyme 2 (ACE2) proteins during ACE2 endocytosis and recycling. Testing the affinity of the inhibitor for SNX27 showed that the inhibitor binds to SNX27 with an affinity 15 times lower than to syntenin.
The team hypothesized that PDZ1-2 syntenin probably binds to the ACE2 ending and plays a direct role in its transport. However, syntenin did not show any binding to ACE2, while treatment of ACE2 cells with a syntenin inhibitor had no effect on cell surface expression of ACE2. Instead, the team observed that treatment with the inhibitor reduced the expression of the syntenin cargo syndecan-1, suggesting that the inhibitor induces inhibition of syntenin-dependent endocytic transport.
The inhibitor did not cause any changes in SARS-CoV-2 binding in cells. In addition, the concentrations of both negative and positive single-stranded (ss) RNA were reduced, indicating suppression of viral entry. In addition, viral infection was significantly suppressed in chloroquine-treated infected cells, as chloroquine blocked endosome entry in Vero E6 cells lacking transmembrane serine protease 2 (TMPRSS2) expression.
Overall, the results of the study showed that the syntenin inhibitor KSL-128114 inhibits various viral infections, including COVID-19.
*Important note
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered definitive, guiding clinical practice/health-related behavior, or considered as established information.