Kill or corrupt: Mechanisms of action and drug-resistance of nucleotide analogues against SARS-CoV-2

Abstract

Nucleoside/tide analogues (NAs) have long been used in the fight against viral diseases, and now present a promising option for the treatment of COVID-19. Once activated to the 5'-triphosphate state, NAs act by targeting the viral RNA-dependent RNA-polymerase for incorporation into the viral RNA genome. Incorporated analogues can either 'kill' (terminate) synthesis, or 'corrupt' (genetically or chemically) the RNA. Against coronaviruses, the use of NAs has been further complicated by the presence of a virally encoded exonuclease domain (nsp14) with proofreading and repair capacities. Here, we describe the mechanism of action of four promising anti-COVID-19 NAs; remdesivir, molnupiravir, favipiravir and bemnifosbuvir. Their distinct mechanisms of action best exemplify the concept of 'killers' and 'corruptors'. We review available data regarding their ability to be incorporated and excised, and discuss the specific structural features that dictate their overall potency, toxicity, and mutagenic potential. This should guide the synthesis of novel analogues, lend insight into the potential for resistance mutations, and provide a rational basis for upcoming combinations therapies.

Fig. 1

Antiviral effects of nucleotide analogues following incorporation. Normal replication, showing extension of product RNA (top) against the template (bottom). Resulting, full-length product shown on right. Various effects of NA (red) incorporation, as either chain-terminators (killers), or RNA corruptors, shown below. Obligate chain-terminators lack the ribose 3′-OH, and therefore physically cannot be extended resulting in immediate arrest of synthesis. Non-obligate chain terminators carry a ribose 3′-OH, however still may result in early termination of synthesis, either in an immediate or delayed manner. RNA corruptors are inserted into RNA and extended to yield full-length products, without immediate effect. They can be further separated into chemical corruptors; whose chemical modifications may affect RNA secondary structure or regulatory elements, RNA-protein interactions, subsequent rounds of synthesis, translation etc.; and genetic corruptors; whose incorporation causes an accumulation of mutations throughout subsequent rounds of synthesis, resulting in error catastrophe/lethal mutagenesis.

Fig. 2

Remdesivir structure and mechanism of action. a) Structure of the prodrug Remdesivir (RDV/GS-5734, top left), its parent nucleoside (GS-441524, bottom left), and its active triphosphate form (RDV-TP/GS-443902, top right). ATP shown in the bottom right for comparison. b) SARS-CoV-2 nsp12 in post-incorporation, pre-translocated state, with incorporated RDV-monophosphate (RDV-MP) in −3 position (PDB 7B3C). Ser861 shown, predicted to sterically clash with the RDV 1′ cyano group preventing further translocation to the −4 position. c) Mechanisms of action of RDV-incorporation. RDV is incorporated in the place of ATP, and causes either delayed chain-termination (i+3), which may or may not be susceptible to nsp14-mediated excision (right), or chemical corruption, potentially impacting RNA secondary structure/regulatory elements, transcription/translation, and template-dependent inhibition during further rounds of synthesis (left).

Fig. 3

Favipiravir structure and mechanism of action. a) Structure of prodrug Favipiravir (T-705) and its active triphosphate form (FTP/T-705-TP) on left, and non-fluorinated versions T-1105 and T-1106 (ribonucleoside form) shown on right. b) Independent studies showing SARS-CoV-2 nsp12 with FTP occupying the +1 NTP binding position in the pre-incorporated state (PBD 7CTT top left, and PBD 7AAP top right). Key residues involved in stabilizing the NTP and metal ions indicated, showing their variability between the two structures. c) Mechanism of action of FTP incorporation. FTP is incorporated in the place GTP (and to a lesser extent ATP), and likely slows, but does not terminate RNA synthesis. Following incorporation in the place of GTP, FTP may template for either CTP or UTP, resulting in the gradual accumulation of G to A and C to U transitional mutations.

Fig. 4

Molnupiravir structure and mechanism of action. Structure of prodrug Molnupiravir (MK-448/EIDD-2801) and active triphosphate form (NHC-TP/EIDD-2061) in comparison with CTP. b) SARS-CoV-2 nsp12 in post-translocated state, showing NHC in template strand, opposite either AMP (PDB 7OZV) or GMP (PDB 7OZV) in −1 position. c) Mechanism of action of NHC-TP incorporation. NHC-TP is incorporated in the place of CTP, however once incorporated, can base-pair equally well with both GTP or ATP, resulting in the gradual accumulation of U to C and A to G transitional mutations.

Fig. 5

Bemnifosbuvir structure and mechanism of action. a) Structure of the phosphoramidate prodrugs Bemnifosbuvir (AT-527) and Sofosbuvir (SOF) and their active triphosphate forms AT-9010 and STP. NA prodrugs PSI-661, PSI-938 and BMS-986094 shown below for comparison. b) SARS-CoV-2 nsp12 in a chain-terminated synthesis state following incorporation of AT-9010 (−1 position) opposite a templating CTP (PDB 7ED5). A second AT-9010 in its triphosphate form occupies the NTP binding pocket in a pre-incorporation state, and is unable to be incorporated due to misalignment of the ribose and triphosphates. c) Mechanism of action of Bemnifosbuvir following incorporation by the viral RdRp. Incorporation causes immediate chain-termination, regardless of the identity or concentration of the incoming NTP. Loading of the next-templated nucleotide into the NTP binding site may increase resistance to nsp14-mediated excision.

Fig. 6

Structural features of the CoV RdRp active-site. Comparison of T-705-TP (PDB 7CTT, left panel) and AT-9010 (PDB 7ED5, middle panel) showing movement of key residues involved in stabilizing the ribose 2′-OH (Asn691, Asp623 and Ser682), and involved in the fidelity check of the base (Arg555). In the T-705 structure, motif B residue Ser682 is in a ‘closed’ conformation, and forms a hydrogen bond with the 2′-OH of the ribose. This is not possible with AT-9010 due to the 2′ ribose modification, and Ser682 is seen in the open conformation. Fingers domain residue Arg555 is more flexible in the SARS-CoV polymerase compared with other + RNA virus RNA polymerases.

Fig. 7

Resistance mutations against Remdesivir. SARS-CoV-2 RTC complex, with the nsp12 NiRAN domain shown in yellow, interface domain in orange, and polymerase domain in light blue, nsp7 in purple, and two nsp8 proteins in green (PDB 7C2K). Incorporated RDV monophosphate is in the −1 position, with incorporated GMP in the +1 position, pre-translocation. Resistance mutations are divided into two groups, domain I (black lined subdomain), located around the active-site, and domain II (red lined subdomain) located on or around motif D (dark blue). Close up of mutants shown in right panel.

Fig. 8

NTP binding pocket of the SARS CoV-2 nsp12 NiRAN domain. The NiRAN domain in complex with AT-9010 (PDB 7ED5, dark green) in the base-in conformation, occupying a small cavity, in comparison with ADP (PDB 7RE2, yellow) and GDP (PDB 7CYQ, purple) in the base-out conformation. Overlay shown on right.