Identifying Structural Features of Nucleotide Analogues to Overcome SARS-CoV-2 Exonuclease Activity

Area of Science:

• Virology
• Drug Discovery
• Molecular Biology

Background:

• The global spread of SARS-CoV-2 variants necessitates the development of novel, variant-resistant oral therapeutics.
• Combination therapies targeting viral RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) show potential for COVID-19 treatment.
• Nucleotide analogues must be efficiently incorporated by RdRp and resistant to ExoN excision to be effective SARS-CoV-2 inhibitors.

Purpose of the Study:

• To evaluate the resistance of various nucleotide analogues to ExoN-mediated removal from the 3' RNA terminus.
• To identify structural features of nucleotide analogues that confer resistance to ExoN excision.
• To provide insights for designing effective SARS-CoV-2 RdRp inhibitors with enhanced stability.

Main Methods:

• Selection and evaluation of nucleotide analogues with diverse structural modifications.
• In vitro assessment of ExoN excision resistance for analogues attached to the 3' RNA terminus.
• Comparative analysis of the impact of 2'-OH and 3'-OH groups on ExoN excision.

Main Results:

• Nucleotide analogues lacking both 2'- and 3'-OH groups exhibited the highest resistance to ExoN excision.
• Nucleotides with both 2'- and 3'-OH groups were efficiently removed by ExoN.
• The 3'-OH group was found to be more critical than the 2'-OH group for ExoN-mediated excision.

Conclusions:

• Structural modifications, particularly the absence of 2' and 3' hydroxyl groups, are crucial for developing ExoN-resistant nucleotide analogues.
• These findings offer valuable insights for designing novel RdRp inhibitors that are effectively incorporated and resistant to excision, potentially leading to new COVID-19 therapeutics.
• Further evaluation of these RdRp terminators, possibly in combination with ExoN inhibitors, is warranted in cell culture and animal models.