The redesign of the molecular scaffold of viral ion channel blockers

Figures

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  Fig. 1.. (A) The amino acid sequence alignment of the envelope protein (EP), the ion channel of SARS-CoV-2, shows no mutations between the different variants. Sequences were obtained from the National Center for Biotechnology Information [101], with repository codes shown in the figure. The transmembrane region is colored gray [32]. Sequence alignment was performed using Clustal Omega [102]. Positions with identical amino acids are marked with asterisks. (B) The compounds with an amphiphilic scaffold (AMS) investigated in the present study are arranged in increasing order of molecular weight. The compounds in the upper row are based on the AMA scaffold, while those in the lower row have a simplified (minimalist) scaffold. The labels of FDA-approved drugs (used as references) are highlighted in gray. The AMS, composed of a hydrophobic (sandy background) and hydrophilic (blue background) group, is highlighted in AMA.

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  Fig. 2.. The ion channel (EP) of SARS-CoV-2 binding AMS compounds. Cross-section of EP shown as a surface (left), with AMA (middle), and arterolane (ART) (right) represented with sticks. The blue and tan bands indicate hydrophilic and hydrophobic regions, respectively, based on the chemical properties of the amino acid residues lining the internal wall of EP. The hydrophobic adamantyl group of AMA fits into the hydrophobic bands of EP, while the hydrophilic amino group interacts with the hydrophilic bands via water molecules. These bridging water molecules are represented as red and white balls-and-sticks (not all are shown for clarity). Amino acids are labeled according to PDB entry 7k3g [32]. Hydrophobic bands (HBs) 1 and 2 are labeled, with their diameters shown, and the amino acids highlighted within each HB. The bottom of the figure points toward the extraviral space. Antiviral activity against the alpha variant in the present study is shown (measured by MTT assay), and literature activity values [46,82] are in brackets. Cytotoxicity values are also from the present manuscript.

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  Fig. 3.. The validation of the QMH-L binding affinity calculations based on complex structures of AMS compounds and ion channels M2 and EP generated by HydroDock in the previous [29] and the present study (Methods). (A) The correlation plot of experimental antiviral activity (pEC50) and calculated binding affinity (ΔGb, kcal/mol) is based on experimental EC50 data (Table S2) from the literature and QMH-L calculated values (Table S1) of the present study. (B) The correlation plot was also produced for the calculated EI (kcal/mol) values. The coefficient −1.364 comes from converting pEC50 to ΔGb using the equation ΔGb = RT lnEC50, where R is the gas constant and T = 298.15 K is the temperature. The points are labeled according to drug/target.

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  Fig. 4.. ART inhibits SARS-CoV-2 replication in vitro. (A) Wuhan, alpha, and delta strain-infected (labeled accordingly) cells were treated with ART. The concentration–response curves were generated from a representative biological experiment, in which each concentration was measured in 3 technical replicates. A sigmoid curve is fitted to the measured optical density data from MTT assays. The standard error of measurement of 3 replicates is shown with error bars. (B) Representative microscopy images show antiviral activity. The microscopy images illustrate the cytotoxic effects of viral infection and their reversal by arterolane treatment. Granule-like appearances indicate cell death.

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  Fig. 5.. The scaffold redesign strategy, including extension and simplification, resulting in the transformations from AMA to ETA to APC in the AMS series. Water molecules are omitted for clarity, but they are included in the detailed figures. The adamantyl group of AMA is depicted in a space-filling model, and the new groups added during the redesign are shown in ball-and-stick models.

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  Fig. 6.. Top results of the simplified scaffold redesign strategy from this study. (A) The binding modes of ETA, PAC, and APC to EP as produced by HydroDock. EP is shown as a surface, while ETA, PAC, and APC are represented as green sticks; water molecules are depicted as white and red balls and sticks. The blue and tan bands indicate hydrophilic and hydrophobic regions, respectively, based on the chemical properties of the amino acid residues lining the internal wall of EP. The bottom of the figure points toward the extraviral space. (B) The results from virology and cytology laboratory (MTT assay) tests of ETA, PAC, and APC. The orange curves indicate the inhibitory effects on SARS-CoV-2 replication, and the black curves show the cytotoxicity measurements. The concentration–response curves were generated from a representative biological experiment, in which each concentration was measured in 3 technical replicates. Error bars indicate the standard deviation of the technical replicates.

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  Fig. 7.. Synthesis of PAC (top) and ETA (bottom).