Mechanisms of inhibition of viral RNA replication by nucleotide analogs

Abstract

Nucleotide analogs are the cornerstone of direct acting antivirals used to control infection by RNA viruses. Here we review what is known about existing nucleotide/nucleoside analogs and the kinetics and mechanisms of RNA and DNA replication, with emphasis on the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in comparison to HIV reverse transcriptase and Hepatitis C RdRp. We demonstrate how accurate kinetic analysis reveals surprising results to explain the effectiveness of antiviral nucleoside analogs providing guidelines for the design of new inhibitors.

Keywords: Coronavirus; Enzyme specificity; Nucleoside analogs; Polymerase kinetics; Proofreading exonuclease; RNA-dependent RNA polymerase; Remdesivir; SARS CoV-2; Single-turnover kinetics.

Fig. 1

Structures of nucleoside/nucleotide analogs approved to treat HIV infections. Analogs are shown in their state before phosphorylation by cellular enzymes. Drug names are given in italics. Note that Tenofovir is a nucleotide analog with a phosphonate linkage to overcome the fact that cellular kinases are inefficient at adding the alpha phosphate to the structure lacking an intact ribose ring.

Fig. 2

Nucleoside analogs directed against RNA dependent RNA polymerases. Although each is administered as a prodrug, analogs are shown in their monophosphate form. Fialuridine is included here for comparison because it failed due to toxicity in a clinical trial for hepatitis B, which is a DNA virus.

Fig. 3

Pathway for nucleotide binding and incorporation. We show the pathway for nucleotide (N) binding to an enzyme-DNA complex with a primer n nucleotides in length (ED_n). After binding, there is a change in enzyme structure from an open to a closed (FD_n) state, leading to chemistry and release of pyrophosphate (PP).

Fig. 4

Free energy profiles. We compare dCTP (solid blue line) with 3TC (dashed green line). Note the change in specificity-determining step (highest barrier) in comparing dCTP with 3TC. This free energy profile was derived from the rate constants shown in Fig. 5.

Fig. 5

Rate constants comparing dCTP and 3TCTP. We show the rate constants governing incorporation of dCTP in comparison with 3TCTP along with the corresponding k_cat/K_m values. Note that even though 3TCTP is incorporated at a rate 650-fold slower than dCTP, the k_cat/K_m value is only tenfold lower .

Fig. 6

Kinetics of processive RNA polymerization. A solution of 2 μM NSP12/7/8 complex, 5 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mg^2 + was mixed with 250 μM each of ATP, CTP, and UTP to start the reaction. Reaction products were resolved and quantified by capillary electrophoresis . Smooth lines show the global fit of the data to define the rates of each sequential incorporation reaction .

Fig. 7

Kinetics of sequential UTP incorporation. A mixture containing 2 μM NSP12/7/8 complex, 6 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mg^2 + was mixed with varying concentrations of UTP (2.5–150 μM) to start the reaction. (A) Time dependence of conversion of 20 nt primer to 21 and then 22 nt by the incorporation of two UTP molecules at 20 μM UTP. (B) Concentration dependence of the first UTP incorporation. (C) UTP concentration dependence of the observed rate in (B) .

Fig. 8

Structure of the stalled SARS CoV-2 RdRp complex with four molecules of Remdesivir. We show the structure of the stalled complex formed after incorporation of four molecules of RMP (magenta), labeled from 1 to 4 to give the order of incorporation. The RNA is in the pre-translocated state with the fourth RMP remaining in the active polymerase site (Pol site). The red arrow shows the likely steric clash between the cyano group of RMP with the beta carbon of S861 upon translocation. Drawn using Pymol from PDBID: 7L1F.

Fig. 9

Role of the proofreading exonuclease. We illustrate three scenarios for the fate of a nucleotide analog after incorporation. (A) An obligate chain terminator is excised by the exonuclease, then replaced by a normal nucleotide. (B) A delayed chain terminator can be protected from the proofreading exonuclease by being buried by several normal nucleotides. (C) A lethal mutagen is stably incorporated and evades the proofreading function by fast extension, but in the next round of polymerization, copying the analog introduces mutations.