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.

Synergistic Antiviral Activity of Pamapimod and Pioglitazone against SARS-CoV-2 and Its Variants of Concern.

The SARS-CoV-2 pandemic remains a major public health threat, especially due to newly emerging SARS-CoV-2 Variants of Concern (VoCs), which are more efficiently transmitted, more virulent, and more able to escape naturally acquired and vaccine-induced immunity. Recently, the protease inhibitor Paxlovid® and the polymerase inhibitor molnupiravir, both targeting mutant-prone viral components, were approved for high-risk COVID-19 patients. Nevertheless, effective therapeutics to treat COVID-19 are urgently needed, especially small molecules acting independently of VoCs and targeting genetically stable cellular pathways which are crucial for viral replication. Pamapimod is a selective inhibitor of p38 Mitogen-Activated Protein Kinase alpha (p38 MAPKα) that has been extensively clinically evaluated for the treatment of rheumatoid arthritis. Signaling via p38 has recently been described as a key pathway for the replication of SARS-CoV-2. Here, we reveal that the combination of pamapimod with pioglitazone, an anti-inflammatory and approved drug for the treatment of type 2 diabetes, possesses potent and synergistic activity to inhibit SARS-CoV-2 replication in vitro. Both drugs showed similar antiviral potency across several cultured cell types and similar antiviral activity against SARS-CoV-2 Wuhan type, and the VoCs Alpha, Beta, Gamma, Delta, and Omicron. These data support the combination of pamapimod and pioglitazone as a potential therapy to reduce duration and severity of disease in COVID-19 patients, an assumption currently evaluated in an ongoing phase II clinical study.

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.

Lectin from Triticum vulgaris (WGA) Inhibits Infection with SARS-CoV-2 and Its Variants of Concern Alpha and Beta.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC50 of <10 ng/mL. WGA is effective upon preincubation with the virus or when added during infection. Pull-down assays demonstrate direct binding of WGA to SARS-CoV-2, further strengthening the hypothesis that inhibition of viral entry by neutralizing free virions might be the mode of action behind its antiviral effect. Furthermore, WGA exhibits antiviral activity against human coronavirus OC43, but not against other non-coronaviruses causing respiratory tract infections. Finally, WGA inhibits infection of the lung cell line Calu-3 with wild type and VoC viruses with comparable IC50 values. Altogether, our data indicate that topical administration of WGA might be effective for prophylaxis or treatment of SARS-CoV-2 infections.

The Saliva of Probands Sucking an Iota-Carrageenan Containing Lozenge Inhibits Viral Binding and Replication of the Most Predominant Common Cold Viruses and SARS-CoV-2.

PURPOSE: The aim of this study was to investigate whether sucking of an iota-carrageenan containing lozenge releases sufficient iota-carrageenan into the saliva of healthy subjects to neutralize representatives of the most common respiratory virus families causing common cold and SARS-CoV-2. PATIENTS AND METHODS: In this monocentric, open label, prospective clinical trial, 31 healthy subjects were included to suck a commercially available iota-carrageenan containing lozenge. Saliva samples from 27 subjects were used for ex vivo efficacy analysis. The study's primary objective was to assess if the mean iota-carrageenan concentration of the saliva samples exceeded 5 µg/mL, which is the concentration known to reduce replication of human rhinovirus (hRV) 1a and 8 by 90%. The iota-carrageenan concentration of the saliva samples was analyzed by UV-Vis spectroscopy. The antiviral effectiveness of the individual saliva samples was determined in vitro against a panel of respiratory viruses including hRV1a, hRV8, human coronavirus OC43, influenza virus A H1N1pdm09, coxsackievirus A10, parainfluenza virus 3 and SARS-CoV-2 using standard virological assays. RESULTS: The mean iota-carrageenan concentration detected in the saliva exceeds the concentration needed to inhibit 90% of hRV1a and hRV8 replication by 134-fold (95% CI 116.3-160.8-fold; p < 0.001). Thus, the study met the primary endpoint. Furthermore, the iota-carrageenan saliva concentration was 60 to 30,351-fold higher than needed to reduce viral replication/binding of all tested viruses by at least 90% (p < 0.001). The effect was most pronounced in hCoV OC43; in case of SARS-CoV-2, the IC90 was exceeded by 121-fold (p < 0.001). CONCLUSION: Sucking an iota-carrageenan containing lozenge releases sufficient iota-carrageenan to neutralize and inactivate the most abundant respiratory viruses as well as pandemic SARS-CoV-2. The lozenges are therefore an appropriate measure to reduce the viral load at the site of infection, hereby presumably limiting transmission within a population as well as translocation to the lower respiratory tract. TRIAL REGISTRATION: NCT04533906.

Quinine Inhibits Infection of Human Cell Lines with SARS-CoV-2.

While vaccination campaigns are ongoing worldwide, there is still a tremendous medical need for efficient antivirals against SARS-CoV-2 infection. Among several drug candidates, chloroquine (CQN) and hydroxychloroquine (H-CQN) were tested intensively, and any contentious therapeutic effect of both has been discussed controversially in the light of severe side effects and missing efficacy. Originally, H-CQN descended from the natural substance quinine, a medicinal product used since the Middle Ages, which actually is regulatory approved for various indications. We hypothesized that quinine also exerts anti-SARS-CoV-2 activity. In Vero cells, quinine inhibited SARS-CoV-2 infection more effectively than CQN, and H-CQN and was less toxic. In human Caco-2 colon epithelial cells as well as the lung cell line A549 stably expressing ACE2 and TMPRSS2, quinine also showed antiviral activity. In consistence with Vero cells, quinine was less toxic in A549 as compared to CQN and H-CQN. Finally, we confirmed our findings in Calu-3 lung cells, expressing ACE2 and TMPRSS2 endogenously. In Calu-3, infections with high titers of SARS-CoV-2 were completely blocked by quinine, CQN, and H-CQN in concentrations above 50 µM. The estimated IC50s were ~25 µM in Calu-3, while overall, the inhibitors exhibit IC50 values between ~3.7 to ~50 µM, dependent on the cell line and multiplicity of infection (MOI). Conclusively, our data indicate that quinine could have the potential of a treatment option for SARS-CoV-2, as the toxicological and pharmacological profile seems more favorable when compared to its progeny drugs H-CQN or CQN.

Iota-carrageenan neutralizes SARS-CoV-2 and inhibits viral replication in vitro.

In the absence of a vaccine and other effective prophylactic or therapeutic countermeasures the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) remains a significant public health threat. Attachment and entry of coronaviruses including SARS-CoV-2 is mainly mediated by the spike glycoprotein. Here, we show that iota-carrageenan can inhibit the cell entry of the SARS-CoV-2 spike pseudotyped lentivirus in a dose dependent manner. SARS-CoV-2 spike pseudotyped lentivirus particles were efficiently neutralized with an IC50 value of 2.6 µg/ml iota-carrageenan. Experiments with patient isolated wild type SARS-CoV-2 virus showed an inhibition of replication in a similar range. In vitro data on iota-carrageenan against various Rhino- and endemic Coronaviruses showed similar IC50 values and translated readily into clinical effectiveness when a nasal spray containing iota-carrageenan demonstrated a reduction of severity and duration of symptoms of common cold caused by various respiratory viruses. Accordingly, our in vitro data on SARS-CoV-2 spike pseudotyped lentivirus and replication competent SARS-CoV-2 suggest that administration of iota-carrageenan may be an effective and safe prophylaxis or treatment for SARS-CoV-2 infections.

Key Insights into Hand Biomechanics: Human Grip Stiffness Can Be Decoupled from Force by Cocontraction and Predicted from Electromyography.

We investigate the relation between grip force and grip stiffness for the human hand with and without voluntary cocontraction. Apart from gaining biomechanical insight, this issue is particularly relevant for variable-stiffness robotic systems, which can independently control the two parameters, but for which no clear methods exist to design or efficiently exploit them. Subjects were asked in one task to produce different levels of force, and stiffness was measured. As expected, this task reveals a linear coupling between force and stiffness. In a second task, subjects were then asked to additionally decouple stiffness from force at these force levels by using cocontraction. We measured the electromyogram from relevant groups of muscles and analyzed the possibility to predict stiffness and force. Optical tracking was used for avoiding wrist movements. We found that subjects were able to decouple grip stiffness from force when using cocontraction on average by about 20% of the maximum measured stiffness over all force levels, while this ability increased with the applied force. This result contradicts the force-stiffness behavior of most variable-stiffness actuators. Moreover, we found the thumb to be on average twice as stiff as the index finger and discovered that intrinsic hand muscles predominate our prediction of stiffness, but not of force. EMG activity and grip force allowed to explain 72 ± 12% of the measured variance in stiffness by simple linear regression, while only 33 ± 18% variance in force. Conclusively the high signal-to-noise ratio and the high correlation to stiffness of these muscles allow for a robust and reliable regression of stiffness, which can be used to continuously teleoperate compliance of modern robotic hands.