key: cord-0688289-qzeabqh4 authors: Cashman, Daniel P. title: Why the lower reported prevalence of asthma in patients diagnosed with COVID-19 validates repurposing EDTA solutions to prevent and manage treat COVID-19 disease date: 2020-06-26 journal: Med Hypotheses DOI: 10.1016/j.mehy.2020.110027 sha: 71d3304e436653791f6361654e14ff2941903b0a doc_id: 688289 cord_uid: qzeabqh4 There currently is no specific antiviral drug or a vaccine for SARS-CoV-2/COVID-19 infections; now exceeding 3,500,000 infections worldwide. In the absence of animal models to test drugs, we need to find molecular explanations for any unforeseen peculiarities in clinical data, especially the recent reports describing an unexpected asthma paradox. Asthma is considered a high medical risk factor for susceptibility to SARS-CoV-2/COVID-19 infection, yet asthma is not on the list of top 10 chronic health problems suffered by people who died from SARS-CoV-2/COVID-19. Resolving this paradox requires looking beyond the binary model of a viral receptor-binding domain (RBD) attaching to the ACE-2 receptor. A pBlast analysis revealed that the SARS-CoV-2 surface spike protein contains two calcium-dependent fusion domains that were recently discovered SARS-CoV-1. These viral calcium-dependent binding domains can facilitate membrane fusion only after cleavage by the host surface protease TMPRSS2. Importantly, TMPRSS2 also requires calcium for its SRCR (scavenger receptor cysteine-rich) domain and its LDLRA (LDL receptor class A) domain. Thus, the presence of EDTA excipients in nebulized β(2)-agonist medicines can disrupt SARS-CoV-2/COVID-19 infection and can explain the asthma paradox. This model validates repurposing EDTA in nebulizer solutions from a passive excipient to an active drug for treating COVID-19 infections. Repurposed EDTA delivery to respiratory tissues at an initial target dose of 2.4 mg per aerosol treatment is readily achievable with standard nebulizer and mechanical ventilator equipment. EDTA warrants further investigation as a potential treatment for SARS-CoV-2 /COVID-19 in consideration of the new calcium requirements for virus infection and the regular presence of EDTA excipients in common asthma medications such as Metaproterenol. Finally, the natural history of Coronavirus diseases and further analysis of the fusion loop homologies between the Betacorona SARS-CoV-2 virus and the less pathogenic Alphacorona HC0V-229E virus suggest how to engineer a hybrid virus suitable for an attenuated alpha-beta SARS-CoV-2/COVID-19 vaccine. Thus, replacing SARS-CoV-2 fusion loops (amino acids 816-855) with the less pathogenic HCoV-229E fusion loop (amino acids 923-982) may provide antigenicity of COVID-19, but limit the pathogenicity to the level of HCoV-229E. While the Coronavirus family of single-stranded enveloped RNA viruses is divided into four genera: α-CoVs, β-CoVs, γ-CoVs, and δ-CoVs, only alpha and beta can infect mammals. 1 After binding to their respective receptors, the Coronavirus viruses enter cells through endocytosis with the viral spike proteins driving the fusion of viral and endosomal membranes to enable insertion of the viral genome into the cytoplasm. 2 The less pathogenic Alphacorona virus 229E (HCoV-229E) was isolated from students suffering from the common cold in 1966. 3, 4 HCoV-229E is highly prevalent and most people experience acute infection during their childhood. 5 One study found 65 % of the children between the age of 2. 5 and 3 .5 years were seropositive for HCoV-229E. 6 The HCoV-229E virus binds to the aminopeptidase N receptor (CD13) 7 and enters the cell after cleavage by TMPRSS2 and fusion. 8 The more pathogenic SARS-CoV-1 and SARS-CoV-2 (COVID- 19) viruses belong to the β-genus. The Betacorona virus SARS-CoV2 is a positive-sense single-stranded ribonucleic acid (ssRNA) of approximately 29700 nucleotides in length, of about 80% identical to that of SARS-CoV-1 and approximately 96% identical to the bat coronavirus BatCoV RaTG13. 9 The Spike (S) protein is 1273 amino acid long and S viral envelope protein that has two main subunits (S1 and S2) which protrude outwards with a 'corona' like appearance and binds to the angiotensin-converting enzyme 2 (ACE2) receptors. 10, 11 The amino-terminal subunit is responsible for receptor binding and is labeled the S1 domain. The C-terminal part, labeled the S2 domain, contains the fusion machinery. More specifically, amino acids 318-510 of the S1 represent the receptor-binding domain (RBD) 4 that binds to ACE2. 12 CoV S proteins have two cleavage sites and protease cleavage is required for S2 fusion to the cell membrane. There is an S1/S2 site composed of the amino acids RSVR that is located at the border between the S1 and S2 subunits and an S2′ site, composed of the amino acids RSAR. In SARS-CoV-2, the S2' site is located at amino acid 815, just upstream of the putative fusion loop peptides present within the S2 subunit discussed below. In SARS-CoV-2/COVID-19 the type II transmembrane serine protease (TTSP) TMPRSS2 cleaves the S1-S2 subunits. 13 It is also noteworthy that TMPRSS2 has two calcium-binding domains; a SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) and a LDLRA (LDL receptor class A) domain (aa 113-148) that forms a binding site for calcium. 14 The SRCR is a conserved calcium-dependent domain in which binding was disrupted by EDTA. 15 Together, the LDLRA and SRCR-like domains that may serve as substrate recognition sites. Even now, the actual mechanism of virus membrane fusion is not completely understood. In the case of Coronaviruses (CoVs), it is not a simple two-step process of receptor binding (via the S1 domain) and membrane fusion (via the S2 domain containing the fusion peptide). Viral entry into host cells requires that there is a domain of the S protein that interacts with opposing hydrophobic cellular membranes called a fusion peptide or fusion loop. These fusion peptides (fusion loops, FL) are generally external amino acid domains that insert into the host membranes after major conformational changes of the virus S protein following proteolytic cleavage to initiate the process fusion with the host membrane. there are changes in the cleavage site position relative to the fusion peptide to modulate the fusion loop (FL). This process gives CoVs the unique flexibility to invade different cell types and host species. Additionally, the CoVs fusion process employs a calciumdependent fusion process that was only recently discovered for Rubella 16 and later described for SARS-CoV-1 infection. 17 While two fusion peptides (FLs) were found with SARS-CoV-1, influenza had no calcium-dependent membrane fusion process. The calcium dependent membrane-ordering results in more effective binding that can penetrate deeper into membranes. There are two FL domains in each SARS-CoV versus a single FL domain found in for HCoV-229E and Rubella shown in Table 2 below. Thus, this calcium-dependent requirement for the FL process may explain both the increased lethality of the beta CoVs and the apparent resistance of asthma patients to SARS-CoV-2 infection due to inhaling medications containing EDTA excipients. Since the drug development process from discovery of a new to approved drug generally takes over 10 years, it is unrealistic to expect development of a novel anticoronavirus drugs for SARS-CoV-2/COVID-19. The strategies for Coronavirus treatment regimens have mainly relied on combination therapies with drugs known to have acceptable safety profiles include IFNs, ribavirin, and corticosteroids. However, the data from past regimens indicates that the treatments were not effective in treating SARS. 18 Moreover, no perceived benefit, and possible deleterious effects were observed when corticosteroids (methylprednisolone) were given as treatment during the SARS and MERS epidemics. 19, 20 Additionally, recent clinical commentary indicates that corticosteroids should not be given routinely for the treatment of COVID-19. Accordingly, the asthma paradox is unlikely due to steroid treatments because recent admonitions against routine systematic corticosteroids for the treatment of COVID- 19 and prior reports indicate that systemic steroids for treating SARS-CoV-1 may have been harmful. 21 Ethylene Diamine Tetraacetic acid (EDTA) was first synthesized in1935 and EDTA has been employed as an excipient in bronchial dilator solutions for decades (e.g., Albuterol, Metaproterenol). EDTA has been added to nebulized bronchodilator solutions in the United States as both nonsterile and sterile-filled products. 22 Accordingly, Edetate disodium (Na2EDTA) is often present as preservative or stabilizing agents in nebulizer solutions used to treat asthma and chronic obstructive pulmonary disease. 23 Historically, common nebulizer therapies used by asthma and COPD patients have had concentrations of EDTA available in nebulizer solutions that vary from 0.1 to 0.5 mg/mL . 24 For example, Albuterol (manufactured by Dey Laboratories) contained 300 μg of EDTA, which is also far below the threshold dose for bronchoconstriction. Currently, Metaproterenol Inhalation Solution USP is expressly formulated with EDTA (edetate disodium) as a unit-dose bronchodilator to be administered by oral inhalation with the aid of an intermittent positive pressure breathing apparatus (IPPB). It contains 0.4% or 0.6% Metaproterenol sulfate in a sterile, acidic, aqueous solution containing edetate disodium, sodium chloride, hydrochloric acid, and/or sodium hydroxide for pH adjustment. 25 concentrations of EDTA (0.25 to 10.0 mg/mL) in a double-blind fashion. 26 [BMJ-1987] Here, EDTA produced concentration-dependent bronchoconstriction that did not resolve spontaneously within 1 hour. Mean EDTA PC 20 FEV 1 was 2.4 mg/mL (range 1.2 to 12.8 mg/mL). This study concluded that there was no significant difference in airway response to EDTA among volunteers receiving Beta2-agonist treatments. 27 To study off-target bronchoconstriction by EDTA, it was found that Albuterol (1 microgram/kg IV) significantly attenuated Na2EDTA-induced bronchoconstriction in canines. 28 Additionally, intravenous EDTA chelation therapy has been safely used for more than 50 years. 29 There were an estimated 500,000 visits for chelation therapy in the U.S. for 1993, 30 and 800,000 in 1997. 31 A Canadian survey found that 8% of patients who had undergone cardiac catheterization had used chelation therapy. 32 Recent medical articles indicate that there is markedly lower reported prevalence of asthma and COPD in patients diagnosed with COVID-19. 33, 34 To explain the unexpected observation that asthma patients and chronic The NCBI pBlast tool was used to test the hypothesis that SARS-CoV-2 contains a calcium-dependent fusion domain(s) similar to those that were recently discovered in both Rubella and SARS-CoV-1. An Amino Acids comparison of the two relevant amino acid regions in S protein of SARS-CoV-1 representing fusion loop 1 (FL1 = Amino Acids 798-819) and fusion loop 2 (FL2 = Amino Acids 835 -855) was conducted using the Protein Blast program from the National Center for Biotechnology Information. 35 Specifically, Table 1 and Table 2 and F2 fusion loops with HCoV-229E amino acids 923-982 can l maintain the AEC-2 tissue specificity and host range, yet it will effectively disturb fusion loop mechanism reduce the pathogenicity of SARS-CoV-2 to the level of HCoV-229. The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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