Abstract
Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs) and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK), kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.
Highlights
Integrin inside-out activation is mainly regulated by the intracellular protein talin
KCH-1521 was selected for in vitro cellular evaluation after screening
Binding of synthesized KCH-1521 to talin was confirmed by surface plasmon resonance (SPR) analysis using purified glutathione-S-transferase-fused talin head domain at various concentrations of KCH-1521
Summary
Integrin inside-out activation is mainly regulated by the intracellular protein talin. Integrin is fully activated when outside-in signaling is activated in response to the extracellular matrix (ECM) such as fibronectin, laminin, and collagen [1] These stimulate numerous biological processes such as cell adhesion, survival, proliferation, and metastasis [2]. In response to integrin-talin interaction, FAK is activated via autophosphorylation on the tyrosine 397 residues [7] It leads to activation of its kinase function and downstream signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activating protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways and paxillin resulting in cytoskeletal and morphological changes [7,8]. Overexpression of talin in prostate cancer cells resulted in activation of AKT and MAPK under collagen and fibronectin attachment conditions [3]. We synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl) carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521, that is an N-acylurea derivative, and explored its effects and related mechanisms on HUVECs
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