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

IF 5.3 2区医学 Q1 PHARMACOLOGY & PHARMACY

Biochemical pharmacology Pub Date : 2025-03-09 DOI:10.1016/j.bcp.2025.116866

Themistoklis Paraschiakos , Jing Li , Jonas Scholz , Soo-Ji Han , Marcus Deckers , Vivian Pogenberg , Jan Faix , Sabine Windhorst

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引用次数: 0

Abstract

Upregulation of the actin-severing protein Cofilin-1 is implicated in enhancing malignancy of various cancer types by promoting actin turnover and increasing cellular motility. Despite the importance of targeting Cofilin-1, currently there is a lack of inhibitors specifically targeting its actin-severing activity. To address this issue, we generated synthetic anti-Cofilin-1 nanobodies (Sybodies) that interfere with human Cofilin-1 binding to filamentous actin. We identified four high affinity Sybodies against human Cofilin-1 with dissociation constants (K_D) in the nanomolar range that inhibited G-actin sequestration, and actin-severing activity of Cofilin-1 in vitro. Notably, Sybody B12, with the lowest K_D of approximately 27 nM, competitively blocked actin binding to Cofilin-1, and also inhibited G-actin sequestration of murine Cofilin-1. The crystal structure of the Sybody-B12-Cofilin-1 complex, resolved at 1.8 Å, revealed that Sybody B12 binds to the G-actin binding site of Cofilin-1, showing that Sybody B12 engages the same binding site on Cofilin-1 as actin. Consistently, transient expression of mPlum-tagged Sybody B12 in human H1299 lung cancer cells inhibited the formation of enhanced green fluorescent protein (EGFP)-Cofilin-actin rods. Notably, stable expression of Sybody B12 did not affect viability of H1299 cells, and no compensatory up-regulation of Cofilin-2 or actin-depolymerization factor (ADF) mRNA were detectable in Sybody B12 expressing H1299 cells. Together, these findings suggest that Sybody B12 exhibits a strong potential as tool for inhibiting the interaction of Cofilin-1 with actin. In addition, it could serve as a promising lead structure for designing Cofilin-1 inhibitors in silico.

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来源期刊

Biochemical pharmacology 医学-药学

CiteScore

10.30

自引率

1.70%

发文量

420

审稿时长

17 days

期刊介绍： Biochemical Pharmacology publishes original research findings, Commentaries and review articles related to the elucidation of cellular and tissue function(s) at the biochemical and molecular levels, the modification of cellular phenotype(s) by genetic, transcriptional/translational or drug/compound-induced modifications, as well as the pharmacodynamics and pharmacokinetics of xenobiotics and drugs, the latter including both small molecules and biologics. The journal''s target audience includes scientists engaged in the identification and study of the mechanisms of action of xenobiotics, biologics and drugs and in the drug discovery and development process. All areas of cellular biology and cellular, tissue/organ and whole animal pharmacology fall within the scope of the journal. Drug classes covered include anti-infectives, anti-inflammatory agents, chemotherapeutics, cardiovascular, endocrinological, immunological, metabolic, neurological and psychiatric drugs, as well as research on drug metabolism and kinetics. While medicinal chemistry is a topic of complimentary interest, manuscripts in this area must contain sufficient biological data to characterize pharmacologically the compounds reported. Submissions describing work focused predominately on chemical synthesis and molecular modeling will not be considered for review. While particular emphasis is placed on reporting the results of molecular and biochemical studies, research involving the use of tissue and animal models of human pathophysiology and toxicology is of interest to the extent that it helps define drug mechanisms of action, safety and efficacy.