Design of miniprotein inhibitors targeting complement C9 to block membrane attack complex assembly

Area of Science:

• Immunology
• Biochemistry
• Computational Biology

Background:

• Abnormal formation of the membrane attack complex (MAC) is linked to immune diseases.
• Complement C9 insertion is the final, rate-limiting step in MAC formation, but C9 is a challenging therapeutic target.
• Current research on blocking C9 is limited.

Purpose of the Study:

• To de novo design mini-protein inhibitors targeting soluble complement C9.
• To block the membrane insertion of C9, preventing MAC formation.
• To develop a potential therapeutic strategy for immune diseases.

Main Methods:

• Utilized deep learning for protein scaffold generation, sequence design, and structure prediction.
• Employed partial diffusion to optimize binding affinity to 700 pM.
• Verified design accuracy and specificity using X-ray crystallography and biochemical assays.

Main Results:

• Successfully designed mini-protein inhibitors that specifically block soluble complement C9 membrane insertion.
• Achieved high binding affinity (700 pM) for the optimized mini-protein inhibitor.
• Demonstrated in vivo efficacy in an acute hemolysis inhibition assay, outperforming eculizumab.

Conclusions:

• De novo designed C9 mini-protein inhibitors offer a novel therapeutic approach.
• These inhibitors are effective against hemolysis even after complement activation.
• Potential application in preventing and treating immune diseases linked to abnormal complement activation.