Research Group of U. Schubert

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. 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 with low adverse effects have not been available yet. Moreover, though numerous vaccines have been approved worldwide, complete herd immunity might be difficult to achieve, as it appears that the vaccines do not confer sterile immunity. Thus, there is still an unmet urgent need to develop antivirals, which should be safe, widely available, and broadly acting against different VoCs of SARS-CoV-2 and other respiratory viruses.

We are currently working on the following main research topics:

The coevolution of viruses and humankind led to the mostly unexplored wisdom for the use of natural products in prevention and treatment of infectious diseases. Natural substances have been used for centuries to combat infections, even though in most cases the exact antiviral mechanism has not yet been clarified.

Natural antivirals can be used as prophylaxis and treatment to prevent progression of virus induced diseases for emerging viruses. As they commonly represent broadly acting antivirals they are important for pandemic preparedness. Moreover, in most cases, they are directed preferentially against host cell targets and thus chances for drug resistances are low. In addition, because of of their natural origin, they have fewer side effects than chemically designed drugs.

We tested the antiviral activity of several natural substances against SARS-CoV-2 which have previously been shown to be effective against other viruses in vitro and in vivo and which exhibit an excellent safety profile.

Quinine, an extract of the bark of the Chinchona tree (native to the Andes of South America), was used to treat feverish infections, particularly malaria, for hundreds of years almost worldwide. As a matter of fact, it can be considered as the oldest medication used as a pure molecular drug since the 17^th century. The first records for medical use of quinine date back to 1630, where in Peru the countess of Chinchon developed malaria and was successfully treated with an extract of the bark of the fever tree, which was later termed Chinchona bark. First structurally characterized in 1820 and chemically synthesized in 1944, it was the only antimalarial drug available. In the 19th century, British citizens and soldiers used tons of the Chinchona bark to protect themselves from malaria and thus permitted a stable British population in tropical colonies. Based on this natural product, Hydroxy-Chloroquine (H-CQN) was synthesized in 1946 and mainly used for the treatment of malaria. Nevertheless, and even until now, quinine is used for the treatment of severe and H-CQN-resistant cases of malaria tropica.

It is also approved for the treatment of calf cramps, and it is commonly used as an aromatic agent. For instance, it is added to bitter lemon and tonic water. Quinine has been commercialized since the 19th century in beverages known as Indian quinine tonic. The latter was the standard beverage for the pioneers in malaria-invested tropical areas. Particularly British soldiers mixed tonic water with lime and gin. Winston Churchill said: “The gin and tonic has saved more Englishmen’s lives, and minds, than all the doctors in the Empire”.

As an approved aromatic agent, quinine is added to bitter lemon and tonic water. According to the well-elaborated pharmacokinetics, 85 mg quinine, present in 1 L of, e.g., tonic water, could lead to a plasma concentration of ~0.5 µg/mL, corresponding to a molarity of ~1.5 µM, which would reach values close to the IC₅₀ values established in our in vitro systems.

We could show that in Vero cells quinine inhibited SARS-CoV-2 infection more effectively than Chloroquine (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. 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 became a treatment option for SARS-CoV-2, particularly as the toxicological and pharmacological profile seems more favorable when compared to its progeny drugs H-CQN or CQN.

Carrageenan is a high molecular weight sulfated polymer originated from red seaweed (Rhodophyta) that has been widely used in food, cosmetic and pharmaceutical industry and is commonly acclaimed as safe by the FDA. It represents a traditional Irish and Japanese medicine for the treatment of virus induced cold and pneumonia. Three main forms of carrageenans are commercially used: iota, kappa and lambda. They vary from each other in the degree of sulfation, solubility and gelling properties. In contrast to kappa- and lambda-carrageenan, preparations of iota-carrageenan are highly pure, have high molecular weight (MW ≥ 1100 kDa), cannot enter cells and thus are known to be biologically inert in terms of adverse and immunomodulatory effects. The EFSA and the FDA approved iota-carrageenan as food safe for a quantum satis of 75 mg/kg b.w. per day, i.e., 4500 mg for a 60 kg person, while with the nasal spray, only 2.3 mg/day is applied.

Safety and efficacy of iota-carrageenan has been investigated in at least six clinical trials. An independent meta-analysis confirms its high efficacy in the prevention of common cold.

The antiviral activity of iota-carrageenan is well established and has been demonstrated for a diversity of respiratory viruses. Thereby, the mechanism is based on its ability to form gels. Following application of iota-carrageenan to the nasopharyngeal mucosa, the polysaccharides interact with cations of the respiratory mucus and positively charged structures of the viral surface proteins, resulting in a change of conformation of their double helices by forming a gel. This gel builds a protective barrier between the infectious viruses and the epithelial cells of the respiratory tract and acts as a purely physical barrier. In addition, the gel envelops the viruses and prevents them from entering the cell. Finally, iota-carrageenan and trapped viruses are removed spontaneously from the nasopharynx by mucociliary clearance. Thus, iota-carrageenan, when applied contentiously in short intervals of approx. 30 minutes, can act as an inner mask.

Nasal sprays, throat sprays and lozenges containing iota-carrageenan have been approved as common cold preventions and treatment and have been sold in more than 30 countries worldwide.

We could show for the first time that iota- carrageenan exhibits antiviral activity not only against the SARS-CoV-2 Wuhan type but also the VOCs Alpha, Beta, Gamma, Delta and Omicron with comparable IC₅₀ values around 1 µg/mL. This effect was shown in human cell lines and in a SSPL (SARS-CoV-2 spike pseudotyped lentivirus particles) system. Thereby, the IC₅₀ values varied depending on the form of infection and cell lines used, ranging from 1.4–5 µg/mL iota-carrageenan in the SSPL particles system to 2.1–10.3 µg/mL in A549-ACE2/TMPRSS2 cells, and 0.04–0.15 µg/mL in Calu-3 human lung cells infected with the SARS-CoV-2 variants. Tox was controlled by water-soluble tetrazolium salt (WST)-1 assays in uninfected A549-ACE2/TMPRSS2 or Calu-3 cells under otherwise identical conditions as for infection experiments. Thereby we demonstrate that treatment with iota-carrageenan had no influence on the cell viability up to 100 µg/mL, which indicates a therapeutic window of at least 3-log stages.

A randomized, placebo-controlled, double-blinded, multicenter clinical study revealed that a nasal spray containing iota-carrageenan shows prophylactic efficacy in counteracting SARS-CoV-2 infection in healthcare workers caring for COVID-19 patients with a relative risk reduction of 79.8%.

The German and the Austrian Society of Hospital Hygiene recommend the use of iota-carrageenan for health care workers on COVID-19 stations.

In the context of a clinical study conducted in cooperation with the Marinomed AG, we investigate whether sucking of an iota-carrageenan containing lozenge releases sufficient iota-carrageenan into the saliva to neutralize SARS-CoV-2. Thereby, firstly the iota-Carrageenan amount in saliva after taking an iota-carrageenan containing lozenge was determined by NMR analysis. Then, the antiviral activity of iota-carrageenan containing saliva was determined by in vitro infection analysis. The results revealed that the salivary concentration of iota-carrageenan reached the concentration required to inhibit the virus replication of SARS-CoV-2 by 60-fold.

Lectins are proteins that bind specifically to carbohydrate structures. Due to their potential to interact with viral envelope glycoproteins, different plant- and bacteria-derived lectins have been reported to exhibit strong antiviral activity against a number of viruses, including SARS-CoV and MERS-CoV. In addition to the antiviral properties of lectins, numerous studies have highlighted their potential as antineoplastic agents active against different tumor cell lines, and several clinical trials have been conducted.

A common lectin belonging to the group of chitin-binding lectins composed of hevein domains is lectin from Triticum vulgaris, also known as Wheat Germ Agglutinin (WGA). WGA is one of the most extensively studied and characterized lectins, which is widespread in nutrition. Up to 0.5 g/kg lectin concentration are present in wheat germ resulting in 25g/center wheat flour. WGA binds specifically to N-Acetyl-D-glucosamine (GlcNAc) and was shown to interact with sialic acid residues. By now, only very rare  knowledge about its antiviral effects is available. For instance, WGA has been reported to inhibit the adsorption of human T-cell leukemia virus type 1 to the target cells.

We could show for the first time that WGA displays antiviral activity against SARS-CoV-2 Wuhan type with an IC₅₀ of approx.10 ng/mL. The IC₅₀ values for the VoC Alpha and Beta was approx. 100 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 RNA-viruses as human Rhinovirus serotype 1A and serotype 8, Coxsackievirus A10, Human Parainfluenza Virus Type 3 and Influenza virus A H1N1pdm09. This supports the idea that WGA interacts in a very specific way with certain Env glycoproteins from a selective number of viruses.

However, due to its unspecific interaction with glycosylated proteins, prophylactic or therapeutic usage would be limited to topical administration.

European black elderberries and extracts thereof have been used since ancient times in traditional medicine to treat upper respiratory infections. The antiviral effects of black elderberries have been evaluated in several in vivo and in vitro studies. Thereby, it was shown that liquid black elderberry extract inhibits in vitro the replication of the Influenza A virus (IAV), Feline Immunodeficiency Virus (FIV) and the human coronavirus HCoV-NL63. Moreover, clinical trials have shown that application of concentrated black elderberry extracts reduce symptom severity as well as the duration of viral infections, especially IAV and the Influenza B virus (IBV). Moreover, a juice concentrate of black elderberry suppressed viral replication in the bronchoalveolar lavage fluids of mice infected with human IAV. Due to the history of using European black elderberry, it can be considered as safe for ingestion or topical application.

The black elderberry extract used in our study is a standardized European black elderberry fruit extract (Sambucus nigra, variety ‘Haschberg’), termed ELDERcraft^®, with a total polyphenol content of 4.6% and a total anthocyanin content of 3.5%.

We investigated the antiviral activity of ELDERcraft^® against SARS-CoV-2 and its VoCs and explored the possible mode of action by performing time of addition experiments. The results revealed that the extract displayed a strong anti-SARS-CoV-2 activity against the Wuhan type as well as the VoCs Alpha, Beta, Gamma, Delta and Omicron with a comparable antiviral activity. Based on cytotoxicity data, a 2-log theoretical therapeutic window can be expected. The data accumulated so far suggest that Elderberries contain certain components that specifically interfere with SARS-CoV-2 replication at late stages of the replication cycle.

MP1032 is a phase-pure anhydrous polymorph of 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt that is very stable, water-soluble, and has good bioavailability via different routes of administration. It is currently under development at MetrioPharm (Zurich, Switzerland), reaching clinical Phase II for the oral treatment of psoriasis and showing a very good safety profile. The physiological action of MP1032 is based on a multi-target mechanism that depends on localized, self-limiting ROS (reactive oxygen species) scavenging activities and PARP-1-modulating properties. Efficacy has been demonstrated in preclinical models with excessively activated immune responses (such as lipopolysaccharide (LPS)-induced endotoxemia), suggesting MP1032 as a potential agent for the treatment/prevention of the SARS-CoV-2-induced cytokine storm.

We was able to demonstrate for the first time that MP1032 exhibits antiviral activity against the SARS-CoV-2 Wuhan type in Vero B4 cells. Thereby, MP1032 concentrations that effectively suppress SARS-CoV-2 replication did not affect cell viability. The cumulative results suggest that MP1032 exerts a dual mode of action: (i) immune-modulatory: it reduced cytokine storm and thus inflammation and organ failure and (ii) direct antiviral activity: reduced replication of SARS-CoV-2.

As part of an EU grant within the Horizon 2020 program, MP1032 entered a double-blinded, placebo-controlled, multicenter clinical trial to determine its efficacy against SARS-CoV-2 infection.

The role of p38 MAPK has  been revealed to be a key player in SARS-CoV-2 replication and inflammatory responses. SARS-CoV-2 binds and downregulates angiotensin-converting enzyme 2 (ACE2) as it enters the cell. ACE2 modulates the function of angiotensin II (Ang II). A number of reports indicate that the p38 MAPK pathway is important in Ang II signaling. Ang II increases blood pressure and inflammation, leading to tissue injury, particularly in alveoli. The interaction between the SARS-CoV-2 spike protein reduces the inhibition of Ang II, leading to tissue damage often observed in COVID-19 patients. The loss of ACE2 activity upon viral entry may allow Ang II mediated activation of p38 in the lungs and heart, resulting in excessive inflammation. Based on these data, p38 MAPK inhibitors previously developed for other indications may be repurposed as a potential therapy for COVID-19.

We investigated the efficacy of pamapimod (PAM), a potent and selective inhibitor of p38 MAPKα, previously developed for the treatment of rheumatoid arthritis, on SARS-CoV-2 replication in vitro. As previous data have suggested substantial crosstalk between the p38 MAPK and peroxisome proliferator-activated receptors γ (PPARγ) pathways, we have also investigated the combination of PAM with pioglitazone (Pio). Pio belongs to the family of thiazolidinedione (TZD) drugs that are used to lower glucose and HbA1C in type 2 diabetic patients. Pio also has broad anti-inflammatory activity, exemplified by its ability to significantly reduce interleukin-6 (IL-6) and tumor necrosis factor α (TNFα). Moreover, Pio has also been shown to inhibit monocyte gene and protein expression of IL-1b, IL-6, and IL-8 and lymphocyte IL-2, IL-6, and IL-8.

Our data show that PAM potently inhibited the replication of SARS-CoV-2 with IC₅₀ and IC₉₀ values of approximately 100 nM and 3 µM, respectively. PAM demonstrated similar antiviral activity against the SARS-CoV-2 Wuhan type and all VoCs across several cell lines. We also showed that treatment with Pio strongly reduced the release of SARS-CoV-2 progeny virions with an IC₅₀ of approximately 800 nM and IC₉₀ of ~10 µM. The combination of the two drugs demonstrated synergistic activity against the original SARS-CoV-2 strain, as well as against the most problematic VoCs, Delta and Omicron. Our data suggest that combined treatment with PAM and Pio should be considered as a potentially effective treatment to reduce severity and time to recovery in COVID-19 patients.

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. Moreover, it was shown that this viral protease can also act as a deubiquitinating enzyme.

We demonstrate for the first time 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 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.

Lipid levels in the host cells have been shown to promote SARS-CoV-2 replication, and since the start of COVID-19 pandemic, several studies have linked obesity and other components of the metabolic syndrome with severity of illness, as well as mortality in patients with COVID-19.

In cooperation with Prof. Claus Hellerbrand (Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg) we aimed to obtain insights into the pathophysiological mechanisms of these associations. First, we established an in vitro model simulating high fatty acid levels and showed that this situation induced the uptake of fatty acids and triglyceride accumulation in human Calu-3 lung cells. Importantly, we found that lipid accumulation significantly enhanced the replication of SARS-CoV-2 Wuhan type or the VoCs, Delta, in Calu-3 cells. In summary, these findings indicate that hyperlipidemia as found in patients with obesity promotes viral replication and herewith the disease course of COVID-19.

The major focus of this long-term project in cooperation with the lab of Dr. Ashok Balasubramanyam (Baylor College of Medicine, Houston, TX, USA) has been the functional characterization of the HIV-1 accessory protein Vpr. The 96 amino acid Vpr has multiple functions in HIV-1 pathogenesis, including virion incorporation, nuclear translocation of the HIV-1 preintegration complex, induction of cell cycle arrest at the G2/M phase, and the regulation of apoptosis.

HIV patients manifest adipose dysfunction characterized by accelerated lipolysis, hepatosteatosis, dyslipidemia, insulin resistance, and hyperglycemia. However, the in vivo mechanisms whereby HIV infection induces those defects in human adipose disorders have not been reported. Thus, in cooperation with the lab of Dr. Ashok Balasubramanyam the pathogenic role of Vpr in HIV-associated adipose dysfunction was investigated.

Persons living with HIV (PLWH) manifest chronic disorders of brown and white adipose tissues that lead to diabetes and metabolic syndrome. The mechanisms that link viral factors to defective adipose tissue function and abnormal energy balance in PLWH remain incompletely understood. We explored how Vpr contributes to adaptive thermogenesis in two mouse models and human adipose tissues. Uncoupling protein 1 (UCP1) gene expression was strongly increased in subcutaneous white adipose tissue (WAT) biopsy specimens from PLWH and in subcutaneous WAT of the Vpr mice, with nearly equivalent mRNA copy number. Histology and functional studies confirmed beige transformation in subcutaneous but not visceral WAT in the Vpr mice. Measurements of energy balance indicated Vpr mice displayed metabolic inflexibility and could not shift efficiently from carbohydrate to fat metabolism during day-night cycles. Furthermore, Vpr mice showed a marked inability to defend body temperature when exposed to 4°C. Importantly, Vpr couples higher tissue catecholamine levels with UCP1 expression independent of β-adrenergic receptors. Our data reveal surprising deficits of adaptive thermogenesis that drive metabolic inefficiency in HIV-1 Vpr mouse models, providing an expanded role for viral factors in the pathogenesis of metabolic disorders in PLWH.