Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity

Abstract

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4β1 and α4β7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α4β1 and α4β7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4β1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.

Myofibroblasts: Function, Formation, and Scope of Molecular Therapies for Skin Fibrosis

“…The accumulation of EDA-FN has been observed in multiple fibrotic disorders, including lung, liver, and skin [100,[105][106][107]. EDA-FN promotes myofibroblast differentiation by orchestrating LAP/LTBP release and activation of TGF-β1 [108], increasing matrix stress-strain tension [109,110], and by activating mechanotransducer-integrin signalling via FAK activation [111][112][113][114]. Research by Shinde et al [115] and Kohan et al [112] demonstrated that fibroblast-expressed integrins, α4β1 (VLA-4) and α4β7 (LPAM-1), mediate different roles in myofibroblast formation, respectively.…”

“…developed a small blocking polypeptide to bind and block EDA-FN interactions with the α4β1 binding cleft, with high specificity [114]. The polypeptide, AF38Pep, was designed to mimic the integrin α4β1 receptor site for the EDGIHEL motif of EDA-FN and was shown to interfere with TGF-β1-stimulated fibroblast-myofibroblast formation by the specific blockade of integrin α4β1 signalling, the inhibition of FAK activation, and the prevention of profibrotic gene transcription [114]. The aforementioned first generation of small blocking polypeptides have revealed the renewed promise of the specific interruption of integrin-mediated mechanotransduction and myofibroblast formation.…”

From the Matrix to the Nucleus and Back: Mechanobiology in the Light of Health, Pathologies, and Regeneration of Oral Periodontal Tissues

“…Finally, these proteins are linked to the actin cytoskeleton through an "actin binding layer", which consists of α-actinin, vasodilator-simulated phosphoprotein (VASP), and zyxin [62]. Beneath the listed proteins, many other cellular key players, such as sarcoma (Src)-family kinases or the extracellular signal-regulated kinases 1/2 (ERK1/2) have been shown to directly interact with FAs and regulate the activity and assembly status of its components (Figure 1A) [15,63,64].…”

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

“…It is found in a soluble form in liver and blood [ 9 ] and secreted by different cells such as endothelial cells and fibroblasts in insoluble form [ 10 ]. Several studies have underlined the considerable implication of FN in a variety of biological processes especially in cytoskeletal assembly, cell attachment, and migration [ 11 , 12 ], since its different domains are specific ligands for a variety of ECM proteins principally collagen [ 13 , 14 ] and heparin [ 15 ] as well as integrins cell surface receptors [ 16 , 17 , 18 ]. Therefore, controlling FN-integrins-specific interactions offers an innovative strategy to engineering biofunctionalized surfaces promoting cellular adhesion and stimulating cellular proliferation and migration [ 11 , 19 , 20 ].…”

“…Other observations indicate the loss of conformation while proceeding to adsorption of native FN on several supports leading to a reduced cell receptor response [ 27 ]. To circumvent these drawbacks, researchers tended to engineer small recombinant protein fragments deriving from functional ECM protein domains ensuring oriented bioactivity with reduced cost and improved affinity [ 18 , 28 ] as well as enabling the design of novel combined recombinants fragments for additional activities [ 22 , 29 , 30 , 31 ]. In this case, the fusion tag technology has been extensively used for recombinant fragments production to allow efficient purification with reduced time and cost when compared to native ECM protein purification [ 32 ].…”