G4s potential targets for COVID-19
Interestingly, a multi-domain polypeptide in SARS-CoV, including some structurally organized domains, within the Nsp3, represents a SARS unique macrodomain that could bind G4s and plays a key role in viral replication and transcription machinery [16, 17]. These findings suggest that targeting G4s in the viral genomes may lead to the exciting possibility of affecting replication of coronaviruses, including the recently emerged SARS-CoV-2. Based on the evidence above, we speculate that the use of G4-mediated antiviral drugs may represent a significant turning point in the therapeutic management of COVID-19. Several algorithms for detecting expected matching patterns for G4s formation have been already described [12].
Here, we used the QGRS Mapper, a web-based server database [14], that generates detailed information on composition and distribution of PG4FS in COVID-19.
We analyzed the occurrence of PG4FS in the genome of SARS-CoV-2 by Wuhan and then the QGRS data were ranked based on the G-score. In accordance with the findings by Lavezzo et al. in SARS-CoV [13], we identified 25 PG4FS in the SARS-CoV-2 genome (Table 1). This finding suggests that G4s ligands could represent a potential therapeutic strategy for COVID-19.
Although this data supports the observation that G4s ligands may exert a reasonably antiviral activity, the use of these compounds in clinical trials is far from being adopted because of their low selectivity profile and poor drug-like properties.
Alternative approaches to target G4s in COVID-19
Interestingly, the therapeutic potential of G4s in coronavirus could be also reversed on molecules that block the activity of RNA helicases of the virus. This class of proteins can unwind G4s with a mechanism often required before virus or cellular machineries can act on the nucleic acid substrate, thus suggesting their important role in the virus replication [18].
The nsp13 is a helicase that could unwind the 3′ end of the nascent RNA primer and facilitate transfer to the complementary region of the 5′-leader genomic sequence in SARS-CoV, thus regulating virus replication [19]. The functional relevance of helicases activities highlights that inhibitors or modulators for these enzymes are potentially important as therapeutic agents [20]. Tanner et al. [21] found that Bananin and three of their derivatives, exhibit activity as non-competitive inhibitors on nsp13 helicase, probably by an allosteric mechanism. Cho et al. [22], identified a novel small compound (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione also designed as EMMDPD) able to inhibit ATP hydrolysis, as well as dsDNA unwinding activities of the SARS-CoV helicase, without any cell cytotoxicity.
Unfortunately, for many of these compounds, no further evidence exists on action mechanisms or potential translational application in clinical practice.
Interestingly, DNA aptamers, composed by two distinct classes of G4s and non-G4s forming sequences against the SARS-CoV helicase, were isolated using a SELEX (systematic evolution of ligands by exponential enrichment) approach [23]. These aptamers are oligonucleotides emerging as a class of molecules that may interact with viral components and interfere with viral replication [24].
While the identification of therapeutic aptamers requires further studies, we have to explore alternative plausible therapeutic strategies to face the emergency caused by COVID-19.
One possible alternative strategy is to inhibit viral helicase activity by using Food and Drug Administration (FDA) approved drugs with scientific evidence of this molecular effect, and, with no toxic effects.
Interestingly, after a search in the DrugBank database (https://www.drugbank.ca/, version 5.1.5, released 2020-01-03), we retrieved 18 entries for FDA approved compounds that have the term “helicase” as a target (Table 2). It is plausible that most of these molecules have already been tested during this emergency period caused by COVID-19, with unknown large-scale effect.
In addition to these molecules, we found other two FDA-approved small compounds with experimental evidence of activity on viral helicases, guanidine hydrochloride (GuHCl) and Ebselen.
GuHCl, a well-known small compound FDA-approved to treat the symptoms of muscle weakness and fatigability associated with Eaton-Lambert syndrome, was found able to inhibit activities of RNA helicase-like proteins, such as NS3 in Norovirus and VP35 in Ebola virus [25, 26]. However, this drug may present several common side effects such as nausea, diarrhea, stomach cramps, abnormal liver function tests and loss of appetite.
Ebselen is an additional molecule that may inhibit the RNA helicase NS3 of Hepatitis C Virus (HCV) [27]. In this study, after a screening of 727 compounds in the NIH clinical collection sets and their testing for inhibitory activity on NS3 helicase binding nucleic acids, the authors found that Ebselen was able to disrupt NS3/nucleic acid interactions and impair virus replication. Interestingly, Ebselen is a selenium-containing compound that was tested in different Phase 1 and 2 clinical trials on patients with Meniere’s Disease or in subjects affected by type1/2 diabetes (https://www.drugbank.ca/drugs/DB12610). A double-blind, placebo-controlled, Phase 2 trial conducted in 83 healthy adults aged 18-31, demonstrated that Ebselen treatment was well tolerated [28]. Interestingly, very recently, preliminary data demonstrated that Ebselen strongly inhibit SARS-CoV2 growth in a cell-based assay (https://www.biorxiv.org/content/10.1101/2020.02.26.964882v1.full) suggesting this as a molecule of particular interest for further investigation in clinical trials.