The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models

Stegmann, Kim M.; Dickmanns, Antje; Gerber, Sabrina; Nikolova, Vella; Klemke, Luisa; Manzini, Valentina; Schlösser, Denise; Bierwirth, Cathrin; Freund, Julia; Sitte, Maren; Lugert, Raimond; Salinas, Gabriela; Meister, Toni Luise; Pfaender, Stephanie; Görlich, Dirk; Wollnik, Bernd; Groß, Uwe; Dobbelstein, Matthias

doi:10.1016/j.virusres.2021.198469

Cited by 29 publications

(32 citation statements)

References 63 publications

(71 reference statements)

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“…This cell line is very susceptible to viral infection, because it expresses high levels of the SARS‐CoV‐2 receptor ACE2 (Li et al , 2003 ) and is deficient in its interferon response (Emeny & Morgan, 1979 ). For inoculation, we employed a patient‐derived SARS‐CoV‐2 isolate that carries the infection‐enhancing D614G mutation of the Spike (Stegmann et al , 2021 ; preprint: Zhang et al , 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…Nanobodies were serial‐diluted on a separate 96‐well plate, using supplemented DMEM GlutaMax™ medium (2% FBS). A patient‐derived SARS‐CoV‐2 strain (D614G), which was previously sequenced and described (Stegmann et al , 2021 ) was used for virus infection. Virus stocks corresponding to 2.5 × 10 6 RNA copies of SARS‐CoV‐2 were pre‐incubated with VHH antibodies 1 h prior to infection of cells.…”

Section: Methodsmentioning

confidence: 99%

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Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

et al. 2021

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Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

et al. 2021

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“…The lung oxidative balance, which is disrupted by MTX administration, may be further disrupted by COVID-19-based oxidative stress. Although there are no studies in the literature linking lung tissue damage caused by COVID-19 and MTX, the new preclinical evaluation of MTX for the treatment of COVID-19 has not been successful (Stegmann et al, 2021). SARS CoV-2 uses the human angiotensin I converting enzyme 2 (ACE2) for entry into the cells with its spike protein.…”

Section: Discussionmentioning

confidence: 99%

“…Stegman et al revealed that SARS CoV-2 replication was inhibited by MTX in therapeutic concentrations of 1 μM, which led to a more than a 1000-fold decrease in virus progeny in Vero C1008 cells and Calu3 cells. They also stated that the inhibitory effect of MTX was primarily based on reduced purine synthesis(Stegmann et al, 2021). When whey protein was used as an inhibitor of furin activity and lung of group C + W was used as a source of furin, inhibition of furin was higher in the lung of group C + W compared with the C group.…”

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confidence: 99%

Inhibitory effect of whey protein concentrate on SARS‐CoV‐2‐targeted furin activity and spike protein‐ACE2 binding in methotrexate‐induced lung damage

Tufan

Sivas

Gurel-Gokmen

et al. 2022

Journal of Food Biochemistry

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This study aims to investigate the effects of whey proteins on SARS CoV‐2 in methotrexate‐induced lung tissue damage in rats. To determine the possible effects, rats were divided into four groups as control, control + whey, methotrexate (20 mg/kg, i.p.) and methotrexate + whey. Whey protein concentrate (2 g/kg, oral gavage) was administered for 10 days. Cytokine levels were measured and protein electrophoresis was carried out in serum samples. Lipid peroxidation, nitric oxide and glutathione level, and superoxide dismutase and glutathione S transferase activities were determined in lung samples. Inhibition of SARS CoV‐2‐targeted lung furin activity and SARS CoV‐2 spike protein‐angiotensin converting enzyme binding with whey protein concentrate were also measured in each group. In conclusion, whey protein concentrate improved methotrexate‐induced lung damage and inhibited lung furin activity targeting SARS‐CoV‐2 S1/S2 site cleavage and SARS CoV‐2 spike protein‐angiotensin converting enzyme binding. Whey proteins are potential protective candidates that inhibit SARS CoV‐2‐related interactions, even in methotrexate‐induced lung injury. Practical applications Whey proteins have anticarcinogenic, antihypertensive, antioxidant, antibacterial, antiviral, and immunomodulating properties due to the protein, bioactive peptide, and essential amino acid content. Methotrexate is a folate antagonist and inhibits cell proliferation and purine synthesis. The combined use of whey protein concentrate and methotrexate may be an alternative in the development of new strategies to the treatment approaches against COVID‐19. In addition, according to the results of this study, it is thought that the protective effect of whey proteins in healthy conditions before encountering the SARS CoV‐2 may be higher than those who have never used it.

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“…Remdesivir in combination with methotrexate (MTX) led to synergistic impairment of the replication of SARS-CoV-2 as MTX improves remdesivir efficacy in treated Vero cells by reducing the RNA virus in the cell supernatant by more than 98% while each drug alone reduces the RNA virus by 50%–75% ( 81 ).…”

Section: Remdesivir Combined With Repurposed Drugsmentioning

confidence: 99%

Remdesivir and Its Combination With Repurposed Drugs as COVID-19 Therapeutics

Chatterjee

Thakur

2022

Front. Immunol.

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The SARS-CoV-2 virus needs multiple copies for its multiplication using an enzyme RNA-dependent RNA polymerase (RdRp). Remdesivir inhibits viral RdRp, controls the multiplication of the virus, and protects patients. However, treatment of COVID-19 with remdesivir involves adverse effects. Many ongoing clinical trials are exploring the potential of the combination of remdesivir with repurposed drugs by targeting multiple targets of virus and host human simultaneously. Better results were obtained with the remdesivir–baricitinib combination treatment for COVID-19 compared to the treatment with remdesivir alone. Notably, recovery from COVID-19 was found to be 8 days less via the remdesivir–baricitinib combination treatment as compared to remdesivir treatment alone. Furthermore, the mortality rate via the remdesivir–baricitinib combination treatment was lower compared to the remdesivir-only treatment. Remdesivir targets the SARS-CoV-2 enzyme while baricitinib targets the host human enzyme. Simultaneously, remdesivir and baricitinib as a combination inhibit their target viral RdRp and human Janus kinase, respectively. Ongoing trials for the combination of drugs will suggest in the future whether they may reduce the recovery time, reduce the mortality rate, and improve patient clinical status for noninvasive ventilation. In the future, simultaneously targeting virus replication enzymes and host human kinases may be the strategy for SARS-CoV-2 therapeutics.

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The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models

Cited by 29 publications

References 63 publications

Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

Inhibitory effect of whey protein concentrate on SARS‐CoV‐2‐targeted furin activity and spike protein‐ACE2 binding in methotrexate‐induced lung damage

Remdesivir and Its Combination With Repurposed Drugs as COVID-19 Therapeutics

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