Inhibitors of Deubiquitinating Enzymes Interfere with the SARS-CoV-2 Papain-like Protease and Block Virus Replication In Vitro

The ubiquitin proteasome system (UPS), particularly its deubiquitinating enzymes (DUBs), play a key role in the replication cycle of coronaviruses. The SARS-CoV-2 papain-like protease (Plpro) is known to process the viral polyproteins to form the replicase transcriptase complex and to counteract the host viral response. Recently, it was shown that this viral protease can also act as a deubiquitinating enzyme. In this study, we demonstrate that certain DUB-Inhibitors (DIs) interfere with SARS-CoV-2 replication. The DIs PR-619 and HBX41108 restrict SARS-CoV-2 in both Vero B4 and human Calu-3 lung cells where cells were infected with a Multiplicity of Infection (MOI) of 0.02. An in vitro protease assay using recombinant Plpro and Amido-4-methylcoumarin (AMC)-conjugated substrate revealed that PR-619 and HBX41108 are able to block the protease at concentrations where the interventions restricted virus replication. In contrast, DIs that do not inhibit Plpro had no influence on virus replication, which indicated that the protease might be at least one major target. Future vertical studies that would gain more insights into the mechanisms of how DUBs effect the replication of SARS-CoV-2 will further validate them as a potential therapeutic target

Saturation Mutagenesis by Efficient Free-Energy Calculation

Single-point mutations in proteins can greatly influence protein stability, binding affinity, protein function or its expression per se. Here, we present accurate and efficient predictions of the free energy of mutation of amino acids. We divided the complete mutational free energy into an uncharging step, which we approximate by a third-power fitting (TPF) approach, and an annihilation step, which we approximate using the one-step perturbation (OSP) method. As a diverse set of test systems, we computed the solvation free energy of all amino acid side chain analogues and obtained an excellent agreement with thermodynamic integration (TI) data. Moreover, we calculated mutational free energies in model tripeptides and established an efficient protocol involving a single reference state. Again, the approximate methods agreed excellently with the TI references, with a root-mean-square error of only 3.6 kJ/mol over 17 mutations. Our combined TPF+OSP approach does show not only a very good agreement but also a 2-fold higher efficiency than full blown TI calculations

Iota-Carrageenan Inhibits Replication of SARS-CoV-2 and the Respective Variants of Concern Alpha, Beta, Gamma and Delta

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC50 values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants

Iota-Carrageenan Inhibits Replication of the SARS-CoV-2 Variants of Concern Omicron BA.1, BA.2 and BA.5

Even with its endemic transition, the COVID-19 pandemic remains a public health threat, particularly in the light of emerging variants of concern (VoCs) and the need for pandemic preparedness in the future. In November 2021, the SARS-CoV-2 VoC Omicron emerged and its subvariants BA.1, BA.2 and BA.5 became predominant. Although the protease inhibitor Paxlovid® and the polymerase inhibitors Molnupiravir and Remdesivir were approved as specific antiviral treatment options for COVID-19 patients in the early stages after infection, effective prophylactically acting substances without adverse effects are not available yet. In a recent study, we demonstrated that iota-carrageenan, a sulfated polysaccharide extracted from red seaweed, efficiently inhibits the replication of the SARS-CoV-2 Wuhan Type and the VoCs Alpha, Beta, Gamma and Delta. Now, we extended this study by investigating the antiviral effects of iota-, lambda- and kappa-carrageenans on the VoC Omicron subvariants BA.1, BA.2 and BA.5. Using a VoC Omicron BA.1 spike pseudotyped murine leukemia virus (BA.1 MLVOMVLP) as well as patient-derived SARS-CoV-2 Omicron isolates BA.1, BA.2 and BA.5 (SARS-CoV-2OM BA.1, SARS-CoV-2OM BA.2 and SARS-CoV-2OM BA.5), we demonstrate that iota-carrageenan exhibits similar antiviral activity against all analyzed Omicron subvariants. As with other VoCs shown before, the biologically inert iota-carrageenan was more efficient than kappa- and lambda-carrageenan. Altogether, these results confirm that, independent of the current and potential future variants, the physical barrier provided by iota-carrageenan might be applicable for prophylaxis and early treatment of SARS-CoV-2 infections