Inhibition of SARS CoV Envelope Protein by Flavonoids and Classical Viroporin Inhibitors

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus Betacoronavirus, family Coronaviridae. The SARS-CoV envelope protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na+ and high intracellular K+. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC50 values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC50 = 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.

Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium

“…We observed a similar response from two-electrode voltage clamp recordings in Xenopus laevis oocytes microinjected with E-protein cRNA (data not shown). Recent studies, on the other hand, have reported a pH-dependent current-voltage relationship where outward currents are elicited in acidic buffer conditions (Breitinger et al, 2021; Cabrera-Garcia et al, 2021). Given the lack of current response to mono- or divalent cations, we investigated the channel’s sensitivity and/or permeability to protons by decreasing the pH of our standard extracellular buffer and measuring the current in response to a voltage ramp.…”

Cell deaths: Involvement in the pathogenesis and intervention therapy of COVID-19

“…The loss of cellular ion homeostasis has been proven to promote NLRP3 inflammasome activation and cell pyroptosis during SARS-CoV-2 infection, thus ion-channel inhibitors (such as Amantadine, Memantine, 195 Rimantadine, 196 Tretinoin 197 ) could be utilized in treatment with COVID-19 patients. Amantadine displayed antiviral activity via directly blocking the ion channel encoded by SARS-CoV-2 E protein in vitro.…”

COVID-19 therapies: do we see substantial progress?

“…Another study demonstrated that protein E might also be a promising target for anti-COVID drugs. It was shown that some flavonoids, in particular epigallocatechin and quercetin, as well as rimantadine and 5-( N , N -hexamethylene)amiloride (HMA), may serve as inhibitors of the protein E channel [ 125 ] (Fig. 1 A).…”