ABSTRACT
Background: Antivirals are urgently needed to supplement SARS-CoV-2 vaccines and target SARS-CoV-2 variants of concern, particularly in resource-limited regions. Active derivatives from the medicinal plant Gunnera perpensa, already in use as a general antiviral in humans by traditional health practitioners in the Eastern Cape Province of South Africa, warrant further evaluation against SARS-CoV-2. Methods: Active constituents of Gunnera perpensa were identified using hyphenated analytical techniques and for ability to inhibit binding of recombinant SARS-CoV-2 spike with host ACE2 protein as assessed by AlphaScreen. Inhibition was tested against parental (USA-WA1/2020), beta (B.1.351), and delta (B.1.617.2) spike proteins using AlphaScreen and spike-expressing VSVΔG-GFP pseudoviruses. Infection of Vero cells was monitored by high-content imaging of GFP or nucleocapsid-positive Vero-E6 cells in pseudovirus and virus assays, respectively, at 2 days post-infection (dpi). Viral cytopathic effect (CPE) ± GC-376 or remdesivir was also monitored using resazurin viability dye at 4 dpi. All assays were described previously (PMID: 34543092). Synergism was assessed by the Bliss Independence model, and group differences were analyzed by two-sided, paired t-test. Results: Crude extracts from the leaves of Gunnera perpensa were confirmed to inhibit parental spike/ACE2 interactions with an IC50 of 37 ± 23 ng/mL. Bioassay-guided fractionation identified two ellagitannins, punicalin and punicalagin, which inhibited parental, beta, and delta spike/ACE2 binding with IC50s of 2.7 ± 0.6-5.8 ± 4.8 and 6.0 ± 4.5-19 ± 23 nM, respectively. Both compounds inhibited all spike variants in pseudovirus at low to mid micromolar concentrations (see Table). Notably, in CPE-based viral assays, a 1:1 molar mixture of punicalin and punicalagin significantly enhanced antiviral activity (EC50 = 2.9 μM vs. 11.6 and 46.8 μM for single compounds, p < 0.05), on par with activities of preclinical candidate GC-376 (1.3 μM) and remdesivir (2.8 μM;see Table). When combined in a 1:1 molar mixture, punicalin further significantly enhanced activity of GC-376 (EC50 = 0.6 μM, p < 0.05) and remdesivir (EC50 = 1.1 μM, p < 0.05). Conclusion: Punicalin and punicalagin inhibit entry and replication of SARS-CoV-2 variants in vitro and synergize when applied in combination and/or with GC-376 or remdesivir. Ellagitannins and medicinal plant extracts are promising new leads for SARS-CoV-2 antivirals in resource-limited regions.
ABSTRACT
Background: SARS-CoV-2 has caused a global pandemic, yet despite vaccine availability, it continues to inflict morbidity and mortality worldwide. The viral main protease (Mpro) is highly conserved across multiple coronaviruses and has a unique viral substrate specificity. Thus, highly selective Mpro inhibitors are expected to be safe, effective, and elude drug resistance for future coronaviruses. Methods: We used a conformationally restricted peptidomimetic to mimic the bioactive conformation of the Mpro-substrate complex to identify potent, selective Mpro inhibitors. We evaluated protease inhibition in biochemical assays, and cellular efficacy in Vero-E6 cells challenged with live virus representing parental (USA-WA1/2020), beta (B.1.351), and delta (B.1.617.2) variants by monitoring infection at day 2 post-infection measuring nucleocapsid-positive cells by high content imaging, and cytopathic effect (CPE) at day 4 post-infection using resazurin viability dye. Results were compared to reference compounds. Group differences were analyzed by two-sided, paired t-test. Results: AP-8-013 required a 2-hour incubation to achieve maximal dose-dependent Mpro inhibition with an IC50 = 230 ± 18 nM, reflecting its highly constrained conformation, compared to the more flexible Cpd 22 (AP-8-001;IC50 = 11 ± 0.7 nM) or GC-376 (IC50 = 18 ± 1.5 μM). Importantly, AP-8-013 showed exquisite selectivity for Mpro with no inhibition at key mammalian cysteine proteases, cathepsin B and L, or the serine protease thrombin, while Cpd 22 (Cat B IC50 = 24 ± 7.5 nM, Cat L IC50 = 1.8 ± 0.3 nM) or GC-376 (Cat B IC50 = 37 ± 1.5 nM, Cat L IC50 = < 1 nM) showed poor selectivity towards mammalian cysteine proteases. AP-8-013 was active in CPE cell-based assays with comparable potency to reference compounds, with EC50 = 4.7 μM compared to Cmp 22 (EC50 = 1.4 μM) or GC-376 (EC50 = 1.1 μM). Using intact SARS-CoV-2 infection-based assays, AP-8-013 significantly inhibited parental virus as well as beta and delta VOC (EC50s = 2.7, 2.5, and 6.0 μM, respectively). Finally, a 3:1 molar mixture of AP-8-013 and remdesivir significantly enhanced antiviral activity in CPE assays (EC50 = 1.3 μM;p < 0.05) when compared against either compound alone (EC50s = 4.7 and 3.3 μM, respectively). Conclusion: We have identified a novel drug-like Mpro inhibitor lead series which is highly selective over cysteine and serine proteases that can inhibit multiple SARS-CoV-2 VOC and increase the antiviral activity of remdesivir.