Wiedmann F et al. (2024), SARS-CoV-2 ORF 3a-mediated currents are inhibit...

XB-ART-61015

Europace 2024 Oct 03;2610:. doi: 10.1093/europace/euae252.

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SARS-CoV-2 ORF 3a-mediated currents are inhibited by antiarrhythmic drugs.

Wiedmann F , Boondej E , Stanifer M , Paasche A , Kraft M , Prüser M , Seeger T , Uhrig U , Boulant S , Schmidt C .

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AIMS: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to cardiovascular complications, notably cardiac arrhythmias. The open reading frame (ORF) 3a of the coronavirus genome encodes for a transmembrane protein that can function as an ion channel. The aim of this study was to investigate the role of the SARS-CoV-2 ORF 3a protein in COVID-19-associated arrhythmias and its potential as a pharmacological target. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and cultured human fibroblasts were infected with SARS-CoV-2. Subsequent immunoblotting assays revealed the expression of ORF 3a protein in hiPSC-CM but not in fibroblasts. After intracytoplasmic injection of RNA encoding ORF 3a proteins into Xenopus laevis oocytes, macroscopic outward currents could be measured. While class I, II, and IV antiarrhythmic drugs showed minor effects on ORF 3a-mediated currents, a robust inhibition was detected after application of class III antiarrhythmics. The strongest effects were observed with dofetilide and amiodarone. Finally, molecular docking simulations and mutagenesis studies identified key amino acid residues involved in drug binding. CONCLUSION: Class III antiarrhythmic drugs are potential inhibitors of ORF 3a-mediated currents, offering new options for the treatment of COVID-19-related cardiac complications.

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	Graphical Abstract. Class III antiarrhythmic drugs are capable of inhibiting SARS-CoV-2 ORF 3a-mediated cation currents, thus offering novel therapeutic avenues for the management of cardiac arrhythmias associated with COVID-19. Created with the help of DALL-E 2, OpenAI.
	Figure 1. Time course of ORF 3a protein expression in SARS-CoV-2-infected human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts (hCFs). (A) Human-induced pluripotent stem cell-derived cardiomyocytes were subjected to SARS-CoV-2 infection [multiplicity of infection (MOI) of 1.0], and cells were harvested at various time points post-infection as specified. The upper section shows representative immunoblot signals for ORF 3a protein and β-actin as loading control. The lower section depicts the average ORF 3a protein signals, obtained from n = 3 independent experiments, normalized to β-actin and presented in arbitrary units (A.U.). (B) Both hiPSC-CMs and hCFs were exposed to SARS-CoV-2, and cells were harvested at designated time intervals. The top section illustrates the immunoblot signals for ORF 3a protein and β-actin from the respective cell lysates. The bottom section presents the average ORF 3a protein signals from n = 3 experiments, adjusted relative to β-actin expression and measured in A.U. Statistical significance is indicated by ***, representing a P < 0.001 from a two-tailed unpaired Student’s t-test. Data are expressed as mean ± standard error of the mean.
	Figure 2. The SARS-CoV-2 ORF 3a protein gives rise to outward potassium currents upon heterologous expression in X. laevis oocytes. (A) Protein sequence alignment of the ORF 3a protein sequences derived from SARS-CoV-2 and the classic SARS-CoV-1 variant (SARS2018). Differences in the amino acid sequence are highlighted in red *, conserved amino acid residue; :, conservative amino acid residue exchange; ., semiconservative amino acid residue exchange. (B) 3D visualization of the SARS-CoV-2 ORF 3a dimer based on the cryo-EM structure recently revealed by Kern et al.22 (PDB-ID: 6XDC); again the differences to the SARS-CoV-1 variant are highlighted in red. TM1–3, transmembrane domain 1–3. (C) Representative family of current trances recorded from X. laevis oocytes, heterologously expressing SARS-CoV-2 ORF 3a proteins by application of the pulse protocol depicted and respective uninjected control (ctrl) oocytes. Current–voltage relationship of SARS-CoV-2 ORF 3a protein-mediated current. Current amplitudes of ORF 3a-expressing X. laevis oocytes were quantified at the end of each 400 ms voltage pulse. Data are expressed as mean ± standard error of the mean from n = 5 cells. Dotted line represents zero current level, and scale bar is depicted as inset.
	Figure 3. Effects of clinically employed antiarrhythmic drugs on SARS-CoV-2 ORF 3a protein-mediated currents—class III antiarrhythmic drugs amiodarone and dofetilide block SARS-CoV-2 ORF 3a protein-mediated currents. (A) Representative macroscopic currents recorded under control conditions and after application of the respective antiarrhythmic drug as indicated (100 µM, 30 min) (B) Effects of the respective antiarrhythmics on SARS-CoV-2 ORF 3a protein-mediated currents, quantified at the end of the +80 mV test pulse (n = 3–6 cells). The drugs are arranged according to Vaughan Williams classes I–IV. Data are expressed as mean ± standard error of the mean from. (C and D) Time course of SARS-CoV-2 ORF 3a current inhibition by amiodarone (C) or dofetilide (D) and the partial reversibility of inhibition upon washout. In the right part of the panel, mean current levels are depicted: under control conditions, subsequent to a 30-min superfusion with the respective drug, and after a 20-min washout period. (E and F) Left: activation curves (total current amplitudes, quantified at the end of the 400 ms test pulse), investigated under isochronal recording conditions for control conditions and after administration of the respective drug. Center: normalized activation curves, investigated under isochronal recording conditions (current amplitudes normalized to the maximum current), investigated under isochronal recording conditions. Right: fraction of blocked step currents, plotted as function of the test pulse potential. Data are presented as mean ± standard error of the mean from n = 5 cells. Dotted lines represent zero current level, and scale bars are depicted as insets. Statistical significance is indicated by *, representing a P < 0.05 from two-tailed paired Student’s t-tests followed by Bonferroni correction.
	Figure 4. Concentration–response relationship for the effect of amiodarone on SARS-CoV-2 ORF 3a proteins heterologously expressed in X. laevis oocytes. (A–E) Time course of SARS-CoV-2 ORF 3a current inhibition by amiodarone, administered in a concentration of (A) 0.1 µM, (B) 1 µM, (C) 10 µM, and (D) 300 µM. Representative macroscopic current traces elicited before (Control) and after administration of amiodarone by application of the voltage pulse protocol depicted in (A) are shown at the top of the respective panels. (E) Concentration–response relationships for the effects of amiodarone on SARS-CoV-2 ORF 3a protein-mediated currents. The mean inhibitory concentration (IC50) is provided as inset. Data are expressed as mean ± standard error of the mean from n = 3–7 cells as indicated. Dotted lines represent zero current level, and scale bars are given as insets.
	Figure 5. Structural determinants of SARS-CoV-2 ORF 3a protein interaction with amiodarone and dofetilide. (A and B) Computational docking simulation revealing the interaction of amiodarone (A) and dofetilide (B) with the intracellular channel pore of the SARS-CoV-2 ORF 3a protein dimer. The simulation is based on the cryo-EM structure delineated by Kern et al.22 (PDB-ID: 7KJR). It highlights the critical roles of amino acids lysine 61 (K61) and aspartic acid 142 (D142) in forming the molecular drug-binding site for amiodarone (A) and the involvement of lysine 75 (K75) along with aspartic acid 142 (D142) in the interaction site for dofetilide (B). (C) To confirm the contribution of the said amino acids to the drug-binding site, mutant proteins SARS-CoV-2 ORF 3a K61A, D142A, and the double-mutant K61A/D142A were expressed in X. laevis oocytes and exposed to amiodarone (100 µM; 30 min as described above). (D) In a similar fashion, SARS-CoV-2 ORF 3a-K75A, D142A, and the double-mutant K75A/D142A were superfused with dofetilide (100 µM; 30 min as described above) upon heterologous expression in X. laevis oocytes. Data are presented as mean ± standard error of the mean. Chemical structures of amiodarone and dofetilide are depicted as insets. Statistical significance is indicated by *, representing a P < 0.05 from a two-tailed unpaired Student’s t-test vs. WT channels followed by Bonferroni correction.