Repurposing Ivermectin for COVID-19: Molecular Aspects and Therapeutic Possibilities
As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial ‘hints’ for IVM’s antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.
Introduction SARS-CoV-2 is a positive-sense RNA β-coronavirus, enclosing a capped polyadenylated 30 kb genome, which is the largest among RNA viruses (). SARS-CoV-2 binds to the ACE2 enzyme on the surface of the target host cell by way of its outer spike protein (S) (). The receptor-binding domain (RBD) on the S1 subunit interacts with the peptidase domain of ACE2. After partitioning into the host membrane, sequential enzymatic cleavages ultimately lead to the release of the viral genome into the cell (). The development of successful vaccines has been a priority in the pharmaceutical and scientific community (). However, the time between the initial SARS-CoV-2 outbreak in December 2019 until the pharmaceutical companies began vaccine distribution spanned over a year (5). During this period, two million people have died worldwide, according to the World Health Organization (WHO). Moreover, the increasing mutations detected in the S protein have raised concerns that virus evolution might outpace vaccine rollout and the time needed to reach herd immunity (). Additionally, while vaccines are the main stay for halting the pandemic, it remains critical to develop therapeutics to treat patients and reduce the disease burden. The drug ivermectin (IVM) has recently been shown to inhibit replication of SARS-CoV-2 in cell cultures (). IVM is a widely used drug, known best for its antiparasitic properties in both veterinary and human medicine. It was first discovered in the 1970s by microbiologist Satoshi Omura and parasitologist William Campbell (). Fifty years later, this same drug is suddenly at the forefront of the race against the current pandemic, namely via its unintentional inhibition of nuclear transport. It is important to understand and elucidate the ‘journey’ of how IVM emerged as a therapeutic agent against SARS-CoV-2, to follow this precedent and encourage repurposing available drugs for an increasing number of diseases. As such, we aim to highlight essential steps and components in the SARS-CoV-2 lifecycle, the significance of the nucleocapsid protein, the anecdotal evidence that hinted its potential as an anti-viral drug and its molecular mechanism of action. Finally, we summarize real-time results of current clinical trials. SARS-CoV-2 Lifecycle SARS-CoV-2 Nucleocapsid Protein Contains an Enhanced Nuclear Localization Signal As it happens, the SARS-CoV-2 N contains NLS motifs. Of great significance is the finding that NLS regions in the N gene of SARS-CoV viruses are highly variable compared to the NLS of other coronavirus clades ). Importantly, these changes occurred during the recent evolution of the highly pathogenic coronavirus clades- including SARS-CoV-2 ). Incidentally, the numerous nucleotide insertions and deletions within the NLS are associated with enhanced nuclear translocation. Three NLS motifs have been detected on the N of SARS-CoV-2, SARS-CoV, MERS-CoV and seasonal coronaviruses. Uniquely, as a result of the nucleotide variations found in SARS-CoV-2 and SARS-CoV-1, all three NLS motifs contain a distinctly higher overall positive charge among the peptides compared to the less virulent coronaviruses. The higher positive charge of NLS renders the entire N protein also more positively charged and subsequently enhances its efficacy (). It has been previously corroborated in animal studies that the enhanced translocation of viral Ns to the nucleus results in more severe pathogenicity (). Therefore, it is possible that these more positively charged Ns, which are characteristic of SARS-CoV-2, may be partially responsible for the associated detrimental effects. The Putative Role of the Nucleocapsid Protein Within the Nucleus It was previously shown that viral proteins that enter the nucleus might suppress host genes related to the anti-viral response, leading ultimately to increased pathogenicity (). This may also be the case with SARS-CoV-2, as in vitro studies indicated that the SARS-CoV-2 NP could interact with dsDNA, possibly due to its high positive charge and the negative charge of DNA (). Although the exact activity of the SARS-CoV-2 N within the nucleus has not been fully characterized, previous examination of several coronavirus Ns can offer insight (). The N of the coronavirus infectious bronchitis virus (IBV) was detected not only in the cytoplasm but also within the nucleolus. Nucleolus targeting was also shown with the SARS-CoV-1 N (). It is important to note that the presence of N in the nucleus was indispensable for the replication of IBV, highlighting that cytosolic activity was not sufficient. In another related coronavirus, mouse hepatitis virus (MHV), nuclear proteins were also implicated in its replication. MHV N was specifically detected in the nucleolus, which itself is formed during interphase of the cell cycle and allows formation of ribosomal RNA (rRNA) and ribosomal subunits. The reason for N targeting of the nucleolus is not entirely understood. However, it is possible that N associates with rRNAs, in order to ‘reserve’ their use for translation of sub-genomic RNA. It was also shown in vitro that N transfection into cells resulted in multi-nucleate cells, indicating the delay of cytokinesis (24). This would provide favorable and prolonged conditions for the virus intracellularly to continue to synthesize its genome and sub-genome, translate its proteins and enable sufficient virion packaging. Moreover, N is proposed to dampen the host cell’s antiviral transcriptional response within the nucleus (8). Nevertheless, confirming the presence SARS-CoV-2 N in the nucleolus and understanding its role would elucidate the pathogenicity of this virus. N is an essential component of newly formed virions as it ensures a proper ‘delivery’ of the replicated viral RNA genome within the developing envelope (28, 29). Moreover, it is essential for proper viral RNA dependent RNA polymerase activity, as demonstrated in Influenza A (29). As such, targeting the activity of N would offer a potent antiviral activity against SARS-CoV-2. In fact, N was shown to be an effective anti-viral target against Influenza A. One of the useful properties of N is its numerous binding sites, which have been shown to accommodate various drugs (29, 30). For example, compounds which can target the tail-loop binding pocket abrogate N oligomerization, while the compound F66 binds to the RNA-binding groove of the protein and is associated with improved survival in animal models infected with Influenza A (29). Figure 1 illustrates how the N of SARS-CoV-2 facilitates virus replication and mitigates the host cell response, thus further strengthening its position as a promising target of anti-viral drugs.
FIGURE 1 Figure 1 The importance of the SARS-CoV-2 nucleocapsid protein (N). The N exerts numerous functions that facilitate viral replication while mitigating the host cell response. Owing to its NLS motifs, the protein retains a relatively high positive charge, compared to the N of other coronavirus clades. This enhances its transport into the nucleus where it may silence host anti-viral genes while sequestering ribosomal subunits, possibly for viral mRNA translation, as demonstrated with the N of other related viruses. Moreover, the N is important for stabilizing the interaction between the viral mRNA and nsp3 protein, which facilitates genome replication. In addition, it tethers the newly emerged viral RNA to the viral envelope, ultimately allowing for its encapsulation and formation of new viral progeny. Given these features and its abundance in the infected cell, it would be a promising drug target against SARS-CoV-2. Ivermectin The Discovery of Ivermectin IVM was originally discovered from organisms that were isolated from soil samples collected from the woods nearby to Kitasato Institute in Kawana, Japan. Fermentation products released by a bacterium from the soil, which was later classified as Streptomyces acermitilis, appeared to exhibit antiparasitic activity (specifically against Nematospiroides dubius). Purification and isolation of the bioactive compounds showed naturally occurring macrocyclic lactones, and these were subsequently named avermectins. Avermectins are made up of four compounds, which exist as two variants: A1, A2, B1, and B2. Variants ‘A’ and ‘B’ indicate the presence of methoxy or hydroxyl groups, respectively, at the C5 position. Number ‘1’ describes the double bond between C22 and C23. On the other hand, number ‘2’ indicates the presence of hydrogen at C22 and a hydroxyl group at C23. B1 avermectins were proven to be most active on oral administration, and on this basis, IVM was chemically derived. IVM contains an 80:20 combination of 22,23-dihydro-acvermectin B1a and 22,23-dihydro-avermectin B1b. Its antiparasitic effects are primarily caused by high-affinity irreversible binding to glutamate-gated chloride (Cl-) channels located on nerve and muscle cells of nematode, which leads to hyperpolarization Ultimately, the increased permeability to Cl- results in paralysis and death of the nematode). Concluding Remarks and Perspectives
The available data from IVM clinical trials lack uniformity and have not established the optimal anti-viral dose. However, the evidence does support its safety and efficacy in improving survival rates, especially compared to the other aforementioned drugs. It is important to note that past research has demonstrated the importance of combined, rather than anti-viral monotherapy. Indeed, the use of a single drug does not efficiently suppress long-term replication of the virus (). As evident by the ongoing clinical trials for the treatment of COVID-19, the most efficient decrease in mortality (0%) was largely a result of multiple prescribed drugs including IVM, hydroxychloroquine and azithromycin or IVM and doxycyline Table 1. Given the wide use of numerous drugs to treat COVID-19 patients, it remains imperative to explore the optimal combination of various therapies.
Notably, the clinical outcomes upon prescribing IVM on its own did not result in significantly improved outcomes for COVID-19 patients and nor should it be particularly encouraged (). In fact, cross-resistance to other medications may be induced as a result of selective pressure resulting from a single medication (). This may be a likely event as RNA viruses are well noted for their pronounced capacity for mutations, a finding which has already been established also for SARS-CoV-2 (). Therefore, although IVM may contribute to the suppression of SARS-CoV-2 replication, it is important not to dismiss the risk of selecting for highly pathological and resistant viral strains when using a sole medication. That said, in a recent clinical trial that we have just concluded and is under review, we show that a single dose of IVM can significantly reduce the viral load in asymptomatic SARS-CoV-2 positive subjects. However, in these subjects, zinc and vitamin C were concomitantly used.
The available data thus far suggests a favorable outcome when using IVM in specific doses and in particular drug combinations. It remains imperative to establish the most effective doses, combination, and timing of drug administration as it may largely determine the therapeutic outcome. Although vaccines are currently being distributed, they do not guarantee complete protection against SARS-CoV-2. Therefore, it is important to establish therapeutic alternatives in the event that viral re-infection occurs. Given the promising emerging clinical data from IVM studies and the unprecedented public health threat that the pandemic poses, it is critical that further specific and well-designed studies are carried out to validate the therapeutic potential of IVM
Credited to Front. Immunol.