A high affinity Sybody blocks Cofilin-1 binding to F-actin in vitro and in cancer cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Cofilin-1, an actin-severing protein, is upregulated in various cancers, promoting malignancy through increased actin turnover and cell motility.
• Targeting Cofilin-1 is crucial for cancer therapy, but specific inhibitors for its actin-severing activity are lacking.

Purpose of the Study:

• To generate and characterize novel synthetic nanobodies (Sybodies) that inhibit the actin-severing activity of Cofilin-1.
• To evaluate the potential of these Sybodies as therapeutic agents or lead structures for cancer treatment.

Main Methods:

• Generation of synthetic anti-Cofilin-1 nanobodies (Sybodies).
• Affinity determination using dissociation constants (K_D).
• In vitro inhibition assays for G-actin sequestration and actin-severing activity.
• Crystal structure determination of Sybody-Cofilin-1 complex.
• Inhibition of Cofilin-actin rod formation in cancer cells via transient and stable Sybody expression.

Main Results:

• Four high-affinity Sybodies against human Cofilin-1 were identified, with K_D in the nanomolar range.
• Sybody B12 demonstrated the lowest K_D (~27 nM) and competitively inhibited actin binding to Cofilin-1, also affecting murine Cofilin-1.
• Crystal structure revealed Sybody B12 binds to Cofilin-1's G-actin binding site, the same site targeted by actin.
• Transient Sybody B12 expression in H1299 lung cancer cells inhibited Cofilin-actin rod formation.
• Stable Sybody B12 expression did not impact cell viability or induce compensatory upregulation of related proteins (Cofilin-2, ADF).

Conclusions:

• Sybody B12 is a potent inhibitor of Cofilin-1's interaction with actin, offering a potential tool for research.
• Sybody B12 serves as a promising lead structure for the in silico design of novel Cofilin-1 inhibitors for cancer therapy.