Disulfide Bond Engineering of Soluble ACE2 for Thermal Stability Enhancement.

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals.
• Soluble ACE2 (sACE2) can inhibit SARS-CoV-2 by blocking spike protein interaction.
• Existing sACE2 therapeutics may lack sufficient stability against viral variants.

Purpose of the Study:

• To enhance the stability of soluble ACE2 fused to an Fc domain (sACE2-Fc) using structure-guided mutagenesis.
• To create a more robust variant-independent therapeutic candidate against SARS-CoV-2.

Main Methods:

• Structure-guided mutagenesis was used to introduce novel disulfide bonds into wild-type sACE2-Fc.
• Thermal shift assays were employed to screen for mutants with increased thermal stability.
• X-ray crystallography confirmed the structural integrity and disulfide bond formation in the selected mutant.

Main Results:

• A novel sACE2-Fc mutant with significantly increased melting temperature was identified.
• X-ray structure determination confirmed successful disulfide bond formation without major structural alterations.
• The thermostable sACE2-Fc mutant retained comparable spike protein binding affinity to the wild-type in molecular and cellular assays.

Conclusions:

• Engineered disulfide bonds can substantially improve the stability of sACE2-Fc.
• The thermostable sACE2-Fc mutant demonstrates preserved therapeutic potential against SARS-CoV-2.
• This enhanced sACE2-Fc represents a promising candidate for a variant-independent antiviral therapy.