Structural basis for small molecule binding to the SARS-CoV-2 nsp10-nsp14 ExoN complex

Area of Science:

• Virology
• Structural Biology
• Drug Discovery

Background:

• Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a global pandemic.
• The SARS-CoV-2 non-structural proteins 10 (nsp10) and 14 (nsp14) are crucial for viral replication and are potential drug targets.
• Nsp14's 3'-to-5' exoribonuclease (ExoN) activity, stimulated by nsp10, confers resistance to nucleoside analogue drugs by correcting errors in RNA synthesis.

Purpose of the Study:

• To elucidate the structural basis of nsp10-nsp14 complex function and identify novel inhibition strategies.
• To characterize conformational changes within the nsp10-nsp14 ExoN complex.
• To discover new fragment-based starting points for developing drugs targeting the SARS-CoV-2 nsp10-nsp14 interaction.

Main Methods:

• Crystallization of the nsp10-nsp14 ExoN complex to enable structural analysis.
• X-ray fragment screening to identify novel binding sites on the complex.
• Microscale thermophoresis to estimate binding affinities of identified fragments.
• Investigation of identified sites for potential inhibition of nsp10-nsp14 protein-protein interactions.

Main Results:

• The nsp10-nsp14 ExoN complex was crystallized, revealing distinct conformations and trapping key residues like His268 in different orientations.
• Five novel fragment binding sites were identified at the nsp10-nsp14 interface, hinge region, and on nsp10.
• One interface site showed a cluster of nine related fragments, enabling initial structure-activity relationship studies, and enantiomers selectively bound to different sites.

Conclusions:

• The identified fragments represent novel starting points for structure-based drug design against SARS-CoV-2.
• The discovered binding sites offer opportunities to develop inhibitors that disrupt the nsp10-nsp14 protein-protein interaction.
• Understanding the conformational dynamics and fragment binding provides a foundation for developing effective antiviral therapies.