Inhibition of Viral RNA-Dependent RNA Polymerases by Nucleoside Inhibitors: An Illustration of the Unity and Diversity of Mechanisms

Figures

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  Figure 1. The conserved motifs of the RdRP sequences in representative members of the Flaviviridae family. While HCV is prototypical of the Hepacivirus genus of this family, the others belong to the Flavivirus genus in which each virus is the namesake prototype of a cluster of viruses designated as “complex” such as the “Dengue virus complex” and the “yellow fever virus complex”. Note that the flavivirus NS5 sequences are very similar, whereas the lone Hepacivirus in this alignment is divergent, which affected the sequence alignment. The catalytic regions of the Flaviviridae NS5 sequences were aligned by Clustal Omega [35]; asterisk (*), colon (:), and period (.) respectively indicate identical, strongly similar, and weakly similar residues. In numbering the residues, the putative N-terminal residue of the processed NS5, liberated from the respective precursor polyprotein, was taken as 1. The motifs are color coded as well as named overhead. Motif G, which is upstream of Motif F, is not shown here, primarily because it is the most diverse in sequence and is characteristic of each virus. The loss of sequence similarity between the HCV and the other viruses can be seen here, going towards…

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  Figure 2. Metabolic activation of remdesivir [5,6,7]. This schematic depicts all the major steps of bioconversion of GS-5734, commonly known as remdesivir, into GS-443902, the final ATP analog that mimics ATP and is recognized by RdRP. In the final structure, the adenosine analog part is boxed in light green. The key cyano substitution (-CN) at the 1’ position of the ribose ring is shown in red; this CN is the primary reason behind the stalling of RdRP as it collides against Ser861 in the RNA channel of the RdRP, thus inhibiting viral transcription.

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  Figure 3. (A) Schematic drawing of stepwise transcription elongation in SARS-CoV-2. The RdRP (Nsp12) is shown as an ellipse; the 2’-CN substituents of R are indicated by asterisks (red). The thick arrowhead indicates the direction of RdRP translocation and growth of the product strand. As shown, the elongation complex stalls after four remdesivir monophosphates (R) are incorporated in the product strand (green color), generated by the copying of the template strand (black color). The translocating RdRP (light green oval) stalls when the 4th incorporated remdesivir (RMP) clashes with Ser861 in the RdRP catalytic tunnel [10,12,13,48,52]. Remdesivir—and in some cases, sofosbuvir (Section 2.2)—may also inhibit the RdRP of several other viruses [13] by a similar mechanism (see later). (B) Actual 3D structure (7l1f) of the stalled ternary complex, the relevant portion of which is presented in PyMol display. The Nsp12 portions are bluish gray in ribbon display, the RNA strand with H-bonds is depicted as an orange helix and green sticks, the 4 RMP moieties are pink, and the blocking Ser861 is labeled and is located within Motif B, which is mostly helical and in red.

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  Figure 4. Metabolic activation of sofosbuvir (GS-331077) through the intermediate nucleoside form. The steps are shown only briefly, as they are very similar to those for remdesivir, shown earlier (Figure 2). The final triphosphate form mimics UTP and is recognized by RdRP to be incorporated in the product RNA in place of U, which results in transcription termination [62,63]. The corresponding CTP analog (triphosphate from PSI-6130), discussed below (Section 2.2.1), is also generated essentially by the same pathway [62].

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  Figure 5. Structural location of sofosbuvir, bound inside viral RdRP catalytic pockets, generated from published structures using the EDock program [72] as described in Section 2.2.1. The structures are displayed by PyMOL. The RdRP is represented by the bluish gray ribbon display; the relevant Ser and Val are labeled and are located within Motif B, which is mostly helical and red in color; the single molecule of sofosbuvir (SOF) is shown as sticks. The “Measurement” function of PyMol was used to determine the theoretical atomic distance between the indicated amino acids in Motif B and the 2’C-methyl group of the drug (Figure 4), since this methyl group is the sole difference between the drug and the normal UTP. To clearly view the drug and the distance markers, each structure was rotated as needed; in addition, the “Hide” function of PyMol was used to remove portions of the catalytic channel, consisting mostly of loops.

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  Figure 6. (A) Metabolic activation of molnupiravir (EIDD-2801; or MK-4482) through the intermediate EIDD-1931. The final triphosphate form mimics CTP and is recognized by RdRP to be incorporated in the product RNA in place of C. The NHC (β-D-N4-hydroxycytidine) moiety is boxed in light green [79]. (B) The tautomeric forms (i.e., oxime and N-hydroxylamine) of NHC are shown hydrogen bonded with purine bases A and G, respectively.