Oral Methioninase for Covid-19 Methionine-restriction Therapy

Abstract

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.

Coronavirus SARS-CoV-2 is responsible for the world-wide pandemic of Covid-19. At the present time there is no effective documented therapy. “Repurposed” drugs such as Kalera, a mixture of lopinavir and ritonvir, used as protease inhibitors for HIV and SARS, failed in a clinical trial for Covid-19 in China (1). Hydroxychloroquine, a malaria drug, also failed in a recent clinical trial in China (2). Ramdesivir an adenosine analog tested for Ebola on monkeys is being tested for Covid-19 (3). The in vitro activity of ramdesivir and hydroxychloroquine against SARS-CoV-2 is at micro-molar concentrations, which are quite high, indicating possible toxicity in vivo (4). An influenza drug called Arbidol (umifenovir) was not able to improve the clinical outcome of Covid-19 patients in China (5). Arbidol is an inhibitor that blocks the fusion of the influenza virus with the cell and is active against coronavirus in vitro (5). Tocilizumab (6), which targets the IL-6 receptor, is being tested for Covid-19-induced cytokine storm. A mesenchymal stem-cell therapy is also being tested for Covid-19 (7).

SARS-Cov-2 is a positive single-stranded RNA virus which uses its genomic RNA both for translation and replication (8-11). For proper RNA replication and translation, the cap of the viral RNA must be methylated (8-14). It appears that two methylation sites are present in the viral RNA of coronaviruses; one site is necessary for replication and translation, and the other site possibly for the viral RNA to evade the host intracellular immunity system which would degrade the RNA if not for the cap methylation (8-14). For example, Wang et al. showed that a designed peptide named TP29 targeted the 2’-O-methyltransferase of coronaviruses and inhibited both the transmethylation reactions and viral replication (8). Non-structural proteins (nsp) of the coronavirus, especially nsp10, nsp14 and nsp16 appear to be RNA methyltransferases or associate proteins of the methyltransferases (10-14). Methyltransferases use S-adenosylmethionine (SAM) which is the universal methyl donor, in order to transfer a methyl group to the viral genome (8-14). Synthesis of SAM depends on the availability of methionine. If methionine is in low concentration, the viral methyltransferases cannot complete their reaction due to the lack of SAM (15). Each time a methyl group is transferred to SAM, the resulting product is the methylated molecule, as well as S-adenosylhomocysteine (SAH) (8-15). The ratio of SAM to SAH is critical for whether methyltransferase reactions can proceed; if the SAM to SAH ratio is low, methyltransferase reactions will not occur, and the coronavirus RNA will not be methylated (8-15). As a result, the virus will not be able to replicate and the viral genomes present in the cell will be susceptible to degradation (8-15). The SAM to SAH ratio depends on the availability of methionine, which is converted to SAM by the enzyme methionine adenosyl transferase (MAT) (14,15). In addition, virally-infected cells have an increased requirement for methionine, and they cannot survive at low methionine levels (16). Therefore, restricting methionine availability may inhibit or eliminate the replication and translation of the virus and make it susceptible to be degraded in the host cells, as well as prevent proliferation of cells infected with SARS-CoV-2.

Another aspect of the pathology of SARS-CoV-2 is that it induces overactivation of T-cells and macrophages, resulting in a cytokine storm (17-19). A cytokine storm is one of the factors thought to be lethal in patients with a severe SARS-Cov-2 infection, especially in the lower part of the lungs (17-20). Since, upon activation, T-cells have an increased requirement for methionine (17-18), restriction of methionine may prevent or alleviate the cytokine storm.

The studies described above indicate that three important aspects of SARS-CoV-2 infection and pathogenicity require methionine. The question is how to sufficiently restrict methionine availability in the body. A low-methionine diet, such as a vegan diet can significantly lower the circulating levels of methionine (17,18,21). However low-methionine diets have to be extremely low in the amount of protein which can have adverse effects on patients. Previous studies in which patients were placed on a low-methionine diet, especially for cancer patients, have shown that it is difficult for them to maintain it (21). Therefore, alternative ways to lower methionine in the body are necessary.

A very promising and proven way to lower methionine in the body is by methioninase, which is an enzyme that degrades methionine (22). Our laboratory has developed methioninase over the past 30 years, which is coded by a gene originally present in the soil bacterium Pseudomonas putida. The gene has been cloned into E. coli enabling high levels of production of the enzyme. Methioninase is very robust, and highly stable even at 50˚C. In a previous study, we purified and administered this enzyme to cancer patients by intravenous infusion, and found that methionine levels in the blood were lowered by approximately 300-fold (23,24). On the other hand, long-term studies of intravenously administered methioninase in macaque monkeys have shown potential anaphylactic reactions, unless methioninase is modified by PEGylation; however, this is a difficult and expensive procedure (25).

A breakthrough occurred approximately 3 years ago when our laboratory found that methioninase could be successfully administered orally (26). Numerous studies have shown that oral methioninase is highly effective as an antitumor drug in mouse models of cancer (26,27). Oral administration of methioninase resulted in 80% depletion of methionine within 3 h (28). Oral methioninase restricts methionine in the gut and does not enter the blood stream, thereby eliminating immunological problems (28). In a previous study, we showed that oral administration of methioninase (5 mg twice a day), as a supplement, to both healthy people and cancer patients does not cause any side effects, even after months of administration (29).

It is our intention to make methioninase available now as a supplement and in the future as a drug after clinical trials, to patients with severe Covid-19 to alleviate their acute symptoms of viral pneumonia especially, in the lower respiratory system, which make it difficult or impossible to breathe without the assistance of a ventilator. Present data suggest that up to 80% of patients with Covid-19 who have to be ventilated do not survive (1) since there is no efficient therapy.

Recently there has been much publicity about the therapeutic efficacy of hydroxychloroquine against Covid-19. However, the results of the clinical trial that took place in China suggested that hydroxychloroquine had no benefit to the patients (2) while another two very recent trials also showed no benefit (30,31). The drug remdesivir (Gilead Sciences), which causes RNA chain termination and can be toxic, was provided on a compassionate use and may have some benefit for Covid-19 patients with assisted breathing (32). In Japan, the drug Avigan (favipiravir), which has been previously used for influenza, is now being promoted for Covid-19. Favipiravir is a purine analog that inhibits RNA polymerase, and is probably toxic (33). A randomized clinical trial of this drug for Covid-19 in China showed no improvement over Arbidol (34), which had failed in another clinical trial for Covid-19. Recently, a triple combination of interferon beta-1b, lopinavir-ritonavir and ribavirin showed moderate benefits to Covid-19 patients (35). A recent meta-study of 96,032 patients worldwide showed hydroxychloroquine and chloroquine are harmful when used for Covid-19 (36). Recently a double-blind, randomized, placebo-controlled trial of intravenous remdesivir for Covid-19 patients showed a moderate benefit of a decrease in recovery time from 15 to 11 days (37). A combination of oral methioninase and remdesivir may provide more benefit.

Conclusion

Based on the evidence reviewed here, a new therapeutic approach against Covid-19 that targets the biology of SARS-CoV-2 would be more beneficial than the use of repurposed drugs, developed for other diseases. Previous studies of oral administration of recombinant methioninase in healthy people and cancer patients have shown that it may be safely administered orally to patients without causing side effects (29). Therefore, targeting methionine with oral recombinant methioninase seems to be a promising strategy to treat Covid-19 patients. We are looking forward to promising results of methioninase treatment of Covid-19 patients.

Conflicts of Interest

None.

Authors’ Contributions

RMH and QH wrote the paper.

Acknowledgements

This paper is dedicated to the memory of AR Moossa, MD; Sun Lee, MD; Professor Li Jiaxi; and Masaki Kitajima, MD.