Opportunities and Challenges in Targeting the Proofreading Activity of SARS-CoV-2 Polymerase Complex

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. While the development of vaccines and the emergence of antiviral therapeutics is promising, alternative strategies to combat COVID-19 (and potential future pandemics) remain an unmet need. Coronaviruses feature a unique mechanism that may present opportunities for therapeutic intervention: the RNA polymerase complex of coronaviruses is distinct in its ability to proofread and remove mismatched nucleotides during genome replication and transcription. The proofreading activity has been linked to the exonuclease (ExoN) activity of non-structural protein 14 (NSP14). Here, we review the role of NSP14, and other NSPs, in SARS-CoV-2 replication and describe the assays that have been developed to assess the ExoN function. We also review the nucleoside analogs and non-nucleoside inhibitors known to interfere with the proofreading activity of NSP14. Although not yet validated, the potential use of non-nucleoside proofreading inhibitors in combination with chain-terminating nucleosides may be a promising avenue for the development of anti-CoV agents.

Keywords: ExoN; NSP14; SARS-CoV-2; coronavirus; exonuclease; inhibitor; nucleoside; polymerase; proofreading.

Figure 1

High-throughput biochemical activity assays for NSP14 exonuclease activity. (A). Double-stranded RNA is incubated with the non-specific intercalator RiboGreen. Upon nuclease activity, the RiboGreen is released, corresponding to a loss of signal. (B). Traditional FRET assay using dsRNA substrates featuring a fluorescent emitter, such as Cy3, and a fluorescent quencher. Upon NSP14 nuclease activity, the two strands dissociate, releasing the fluorescent emitter generating a fluorescent signal. (C). A dsRNA substrate featuring a biotin on the 5′ end is digested by NSP14 exonuclease activity. The biotinylated substrates and products are immobilized onto NeutrAvidin, presenting self-assembled monolayers in a high-density biochip array format that are efficient substrates for MALDI-ToF-MS, a technique termed SAMDI.

Figure 2

Representative nucleoside analogs interfering with RNA proofreading.

Figure 3

Representative non-nucleoside NSP14 inhibitors. Compounds 102 (IC₅₀: 19.4 μM), 96 (IC₅₀: 17.4 μM), and 79 (IC₅₀: 22.0 μM) described in [72], and patulin (IC₅₀: 1.8 μM) and aurintricarboxylic acid (IC₅₀: 10.3 μM) described in [37].