Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

Figures

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  Fig. 1.. SARS-CoV-2 replication cycle (56). The virus enters human cells via endocytosis by binding the ACE2 receptor and releasing its positive-sense RNA genome. The virus exploits the host machinery to facilitate efficient viral replication, which ultimately leads to progression of infection (57).

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  Fig. 2.. Secondary structure representation of the 5′-end (450 nt) of the SARS-CoV-2 RNA genome as determined by DMS chemical probing (18). Structure recreated in VARNA.

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  Fig. 3.. Conservation of 5′-end sequences encoding RNA structures in betacoronavirus genomes. Multiple sequence alignment showing percentage identity and sequence coverage within the 5′-UTR and adjacent region of representative species within genus Betacoronavirus. (A) SL1–3. (B) SL 4–6. Viral subgenus is indicated as shown in the key. Sequence numbering is standardized to the genome sequence start position of each taxon. Taxon labels show GenBank accession numbers and abbreviated virus names. OC43, human coronavirus OC43; RousettusBat, Rousettus bat coronavirus; MERS, Middle East respiratory syndrome–related coronavirus; Bat-Hibeco, Bat Hp-betacoronavirus.

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  Fig. 4.. Chemical structure of the three lead molecules derived from the focused library screen against OC43-infected Vero E6 cell screening.

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  Fig. 5.. Inhibition of SARS-CoV-2 infection by DMA leads without cellular toxicity. Vero E6 cells were infected with SARS-CoV-2 (MOI = 0.1) in the presence of DMSO or the indicated compounds for 3 days followed by normalized measurement of virus titer in culture supernatant (black bars). Results were generated with two independent experiments, each with two replicates. Cellular viability was assayed by measuring cellular ATP content in uninfected Vero E6 cells after 3 days of treatment with the indicated compounds (gray bars).

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  Fig. 6.. Vero E6 cells were cotransfected with luciferase constructs and cultured with various concentration of small molecule. (A) Vero E6 cells were cotransfected with CoV-2-5’UTR-FLuc-3’UTR. (B) Vero E6 cells were cotransfected with Cov-2–5′UTR–FLuc (left), and no change in inhibition was observed when compared to the presence of 3′-UTR (right, DMA-135). Luciferase activity was measured 2 days later. Mean values and SDs from three independent experiments are shown in the bar graphs. ***P < 0.001; **P < 0.01; N., not significant relative to the DMSO control, except for the 5′UTR-FLuc-3′UTR comparison in (B), which is relative to DMA-135 at 0.1 μM.

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  Fig. 7.. Single-point 13C-1H TROSY HSQC titrations reveal that DMA-132, DMA-135, and DMA-155 bind with moderate affinity and specificity to SARS-CoV-2 5′-region SLs. The spectra were recorded at 900 MHz in 100% D2O buffer of 25 mM K2HPO4 and 50 mM KCl at pH 6.2. Temperatures (298, 303, or 308 K) were optimized for each RNA construct to maximize the number of observed correlation peaks. The total RNA concentrations were set to 100 μM while titrating fivefold excess DMA.

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  Fig. 8.. Detailed interaction of DMA-155 with SL6. (A) Representation of SL6 secondary structure and labeled nucleotides (red). (B) 1H-1H NOESY spectra (900 MHz, tm = 250 ms) of free UG(2H), AC(2H3′-5″) selectively labeled SL6 (blue) and its DMA-155 complex (red), which were collected in 25 mM K2HPO4 and 50 mM KCl (pH 6.2) at 308 K in 100% D2O, show that DMA-155 has a degree of binding specificity. (C) 1H-1H NOESY spectra (900 MHz and tm = 250 ms) of free CUG(2H), A(2H3’-5″) selectively labeled SL6 (red), its DMA-155 complex (black), and the fully deuterated RNA complexed with DMA-155 (orange) were collected in 25 mM K2HPO4 and 50 mM KCl (pH 6.2) at 308 K in 100% D2O. On the secondary structure of SL6, the nucleotides highlighted in red represent the labeling scheme of the RNA.

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  Fig. 9.. Retrospective docking substantiates NMR findings. (A) Secondary structure representation of SL1 construct used in NMR studies. (B) SL1 3D model with binding pockets highlighted in different colors identified via ICM pocket finder. The model depicted is a representative conformer of the 15-confomer cluster generated for the internal loop motif. (C) conformation and binding pockets that yielded the best docking scores of the three hit molecules DMA-132 (red), DMA-135 (green), and DMA-155 (purple). (D) Secondary structure representation of SL6 construct used in NMR studies. (E) SL6 3D model with binding pockets (red and blue) identified via ICM pocket finder. Model depicted is a representative conformer of the 15-structure clusters built for internal loop motif. (F) Conformer that yielded the best docking scores for the three hit molecules, namely, DMA-132 (red), DMA-135 (green), and DMA-155 (purple) represented in space-filling model.