Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic.

PURPOSE: To develop PEGylated variants of pUR4/FUD (FUD), a fibronectin assembly inhibitor, using 10 kDa, 20 kDa, and 40 kDa PEGs to evaluate their binding affinity and inhibitory potency. METHODS: The FUD peptide was recombinantly expressed, purified, and PEGylated at the N-terminus using 10 kDa, 20 kDa, and 40 kDa methoxy-PEG aldehyde. The PEGylates were purified and fractionated using ion-exchange chromatography. The molecular weight and degree of PEGylation of each conjugate was verified using MALDI-TOF. The binding affinity of each PEG-FUD conjugate was studied using isothermal titration colorimetry (ITC) and their inhibitory potency was characterized by a cell-based matrix assembly in vitro assay. RESULTS: The 10 kDa, 20 kDa, and 40 kDa PEG-FUD conjugates were synthesized and isolated in good purity as determined by HPLC analysis. Their molecular weight was consistent with attachment of a single PEG molecule to one FUD peptide. The binding affinity (Kd) and the fibronectin fibrillogenesis inhibitory potency (IC50) of all PEG-FUD conjugates remained nanomolar and unaffected by the addition of PEG. CONCLUSIONS: Retention of FUD fibronectin binding activity following PEGylation with three different PEG sizes suggest that PEG-FUD holds promise as an effective anti-fibrotic with therapeutic potential and a candidate for further pharmacokinetic and biodistribution studies.

Fibronectin-targeted FUD and PEGylated FUD peptides for fibrotic diseases.

Tissue fibrosis is characterized by excessive deposition of extracellular matrix (ECM) molecules. Fibronectin (FN) is a glycoprotein found in the blood and tissues, a key player in the assembly of ECM through interaction with cellular and extracellular components. Functional Upstream Domain (FUD), a peptide derived from an adhesin protein of bacteria, has a high binding affinity for the N-terminal 70-kDa domain of FN that plays a crucial role in FN polymerization. In this regard, FUD peptide has been characterized as a potent inhibitor of FN matrix assembly, reducing excessive ECM accumulation. Furthermore, PEGylated FUD was developed to prevent rapid elimination of FUD and enhance its systemic exposure in vivo. Herein, we summarize the development of FUD peptide as a potential anti-fibrotic agent and its application in experimental fibrotic diseases. In addition, we discuss how modification of the FUD peptide via PEGylation impacts pharmacokinetic profiles of the FUD peptide and can potentially contribute to anti-fibrosis therapy.

PEGylated pUR4/FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine Unilateral Ureteral Obstruction model of kidney disease.

Fibronectin is a blood and extracellular matrix glycoprotein that plays important roles in wound healing and fibrosis since it controls the deposition of collagen and other extracellular matrix molecules and is a substrate for infiltrating lymphocytes. Using a high-affinity fibronectin-binding peptide (FUD/pUR4) that inhibits fibronectin deposition into extracellular matrix (ECM), we tested the ability of a PEGylated FUD/pUR4 (PEG-FUD) to inhibit fibrosis in the Unilateral Ureteral Obstruction (UUO) kidney disease model. Fibronectin fibrillogenesis assays, using human fibroblasts and human proximal tubular epithelial cultures, showed that PEG-FUD can inhibit fibronectin fibrillogenesis in vitro with an IC50 similar to unconjugated FUD, in the order of 20-35 nM. In contrast, a mutated FUD (mFUD) conjugated to PEG that lacked activity did not inhibit fibronectin assembly, even at 20 µM. The in vivo activity of PEG-FUD was tested in the murine UUO model by daily subcutaneous injection of 12.5 mg/kg for 7 days until harvest at day 10. Control treatments included saline, PEG, unconjugated FUD, and PEG-mFUD. Immunoblotting studies showed that fibronectin was enriched in the extracellular matrix fractions of extracted UUO kidneys, compared to contralateral untreated kidneys. In vivo, PEG-FUD significantly decreased fibronectin by ~70% in UUO kidneys as determined by both IHC and immunoblotting, respectively. In contrast, neither PEG-mFUD, PEG, nor saline had any significant effect. PEG-FUD also decreased collagens I and III and CD45-expressing cells (leukocytes) by ~60% and ~50%, as ascertained by picrosirius red staining and IHC, respectively. Immunoblotting studies also showed that the fibronectin remaining after PEG-FUD treatment was intact. Utilizing a custom-made polyclonal antibody generated against pUR4/FUD, intact PEG-FUD was detected by immunoblotting in both the ECM and lysate fractions of UUO kidneys. No adverse reaction or event was noted with any treatment. In summary, these studies suggest that PEG-FUD reached the kidneys without degradation, and decreased fibronectin incorporation into interstitial tissue. Decreased fibronectin was accompanied by a decrease in collagen and leukocyte infiltration. We propose that PEG-FUD, a specific inhibitor of fibronectin assembly, may be a candidate therapeutic for the treatment of fibrosis in kidney diseases.