Molecular determinants of the SARS-CoV-2 fusion peptide activity

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2), emerged in late 2019 and quickly spread worldwide. SARS-CoV-2 is an enveloped virus and its entry into host cells is mediated by the spike glycoprotein (S-protein) [1]. The S-protein is composed of two subunits (S1 and S2) that contain essential domains for the viral entry mechanism, such as the fusion peptide (FP) which inserts into and disturbs the host cell membrane promoting the fusion between viral and host membranes. Despite its relevance for viral entry, there is still no consensus among scientists for its location on the S-protein and amino acid sequence, although two major candidate regions have been proposed [2, 3]. To shed light on this matter, we combined computational and experimental methods to characterize and compare the effect of the two putative SARS-CoV-2 FPs. We performed a systematic analysis of the SARS-CoV-2 putative FPs, using Molecular Dynamics simulations, to dissect how these peptides interact with the membrane. In parallel, we evaluated the putative FPs behavior in membrane model systems applying biophysical techniques. Since both FPs revealed modest fusogenic activity, we hypothesized that a longer FP or a cooperation among the individual FPs might be required to achieve fusion between viral and host membranes. Given the pivotal role of the FP to viral entry, our work provides relevant insights on the SARS-CoV-2 entry mechanism.

Targeting brain-resident viruses across the blood-brain barrier in vitro using peptideporphyrin conjugates

The recent SARS-CoV-2 pandemic brought awareness to the permanent dangers of viral infections and outbreaks. Beyond its inherent infections, several viruses such as Dengue (DENV), Zika (ZIKV), HIV and even SARS-CoV-2 have the potential to infect the brain, causing more aggressive and irreversible injuries. These brain infections are particularly hard to treat not only because the number of efficient antiviral drugs against these viruses is scarce, but also due to the restrictive permeability of the blood- brain barrier (BBB) that hinders brain drug-intake. To overcome these issues, we designed peptide-drug conjugates formed by covalent attachment of a BBB peptide shuttle and a broad-spectrum antiviral porphyrin drug. We synthesized eighteen novel peptide-porphyrins conjugates (PPCs) and tested their activity in vitro, both in BBB-translocation and antiviral capacity against DENV, ZIKV, HIV and SARS-CoV-2. Cytotoxicity towards pharmacologic relevant cell lines was also studied. After careful fine-tuning of the on-resin synthetic chemistry, DIC/Oxyma coupling has emerged as preferred method, bearing a 99% conjugation yield. Ten PPCs inactivate at least two different viruses in vitro, a selection criterion for further evaluation, with IC50s ranging between 0.5 to 33 lM. Although all ten PPCs efficiently translocate the cellular BBB model in vitro, a set of seven stand out as the most druggable since they are not cytotoxic towards all cell lines tested. Overall, peptide-porphyrin conjugation shows to be an innovative and promising strategy to treat viral brain infections.

Repurposing drugs to fight COVID-19: SARSCoV- 2 envelope targeting by clinically approved porphyrins and analogues

Despite mitigation measures and vaccination programs, there are still very few medicines to treat COVID-19. Porphyrins and analogues (P&A) usually present broad-spectrum antiviral activity. Some are clinically approved for photodynamic therapy in cancer. Therefore, repurposing clinically approved P&A might be an alternative to treat COVID-19. In this work, we evaluate the ability of the clinically approved temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles, characterizing their mechanism of action. Loss of infectivity of P&A treated SARS-CoV-2 was assessed by plaque assay. P&A photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light doses. However, only temoporfin and verteporfin were able to inactivate SARS-CoV-2 in the dark, verteporfin being the most effective. Next, P&A dark antiviral mechanism was characterized starting from P&A interaction with membrane models. P&A partition, membrane-insertion depth, lipid-membrane disruption and changes in membrane ordering were investigated using fluorescent spectroscopy. Among all tested P&A, verteporfin presented the highest partition coefficient, Kp. Curiously, temoporfin and redaporfin presented similar Kp values, although redaporfin did not present dark antiviral activity. Noteworthy, redaporfin was located closer to the surface of the lipid bilayer and both temoporfin and verteporfin were located closer to the centre. Finally, only temoporfin and verteporfin induced reduction of GP (laurdan-generalized polarization), with transition from an ordered phase to a liquidcrystalline phase. Our results suggest that dark antiviral activity is dependent on P&A interaction with viral envelope. Membrane affinity, penetration, and destabilization are critical for P&A dark antiviral activity. Furthermore, dark anti-SARS-CoV-2 activity opens the possibility for off-label P&A application in the systemic treatment of COVID-19.