Amantadine inhibits known and novel ion channels encoded by SARS-CoV-2 in vitro

Abstract

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.

Reply to: How Many SARS-CoV-2 “Viroporins” Are Really Ion Channels?

“…1). This provides a novel and useful platform for future studies of ORF10 in a mammalian expression system and confirm ion channel activity of ORF10 described by us in oocytes 4 .…”

“…Future studies utilizing knock down or pharmacological inhibition of these could help to resolve this issue. However, considering that the viroporins have different pharmacological responses to the various inhibitors tested (see Table 1), we find it unlikely that a common endogenous ion channel (e.g., TMEM16A) is responsible for the observed inhibitory effects amongst the viroporins employed in our study 4 .…”

“…As integral membrane proteins, in some cases localized to the surface of a virus-infected cell, they are also considered as potential drug targets for future therapeutics against known and emerging viruses. In our recent analyses of transmembrane proteins in SARS-CoV-2 4 , we identified two novel proteins (ORF7b and ORF10) that like the two established viroporins (Protein E and Protein 3a) from this virus could mediate a current upon expression in X. laevis oocytes. We moreover identified inhibitors of the activities for all four proteins among known viroporin blockers.…”

“…REPLYING TO N. Harrison Communications Biology Matters arising [doi to be added later] 2022, we appreciate the constructive feedback on our paper 4 , the insightful suggestions and the additional experiments that confirm that amantadine also inhibits the engineered SARS-CoV-2 Protein E. In addition, the presented single channel recordings of SARS-CoV-2 Protein E reconstituted into artificial bilayers are important confirmatory data for ion channel activity of Protein E. We find that the method presented by Cabrera-Garcia et al 6 with engineered SARS-CoV-2 Protein E that enhances plasma membrane surface expression is an interesting approach, especially for generating a two-electrode voltage-clamp (TEVC) oocyte assay that can be used for screening drugs against putative viroporins. Hits would obviously need to be confirmed on wild type viroporins as such modifications of a given membrane protein is likely to alter its function and pharmacology.…”

Adverse effects of vaccinations and potential therapies against COVID-19

“…Amantadyna również należy do leków mających działania uboczne [134]. Chociaż istnieją pewne przesłanki, że może być ona skuteczna, to do tej pory nie udało się tego ani potwierdzić, ani wykluczyć [135]. Leki dobrze zbadane, które są skuteczne w I fazie choroby, nie dały wystarczających efektów.…”