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

Abstract

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.

Probing the subcutaneous absorption of a PEGylated FUD peptide nanomedicine via in vivo fluorescence imaging

“…The Kwon lab sought to increase the efficacy of this novel therapeutic through modification of FUD at the N-terminus with polyethylene glycol (PEG) of 10 kDa, 20 kDa, and 40 kDa MW, increasing the effective hydrodynamic size of FUD and thereby improving its delivery with a nanotechnology approach. All three variants of this PEG-FUD displayed preserved binding affinity (K d ) and in vitro FN matrix assembly inhibitory potency of the native FUD peptide [11]. These in vitro and biophysical results complement later successful application of PEG-FUD in a murine model of renal fibrosis.…”

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

“…In addition, using PEG-FUD to inhibit the deposition of fibronectin into the ECM did not cause any detectable levels of soluble fibronectin degradation, since intact fibronectin was detected in the kidney fractions, as well as in plasma. Indeed, the binding of PEG-FUD to fibronectin which, under in vitro conditions, is considerably tight (Kd~10 nM) [ 22 ], may actually protect at least the N-terminal 70 kDa fragment from degradation. That fibronectin is intact is of importance because fibronectin fragments are known to promote remodeling and pathological degeneration in some tissues [ 53 ].…”

“…FUD and a mutated version of FUD (mFUD, does not inhibit fibronectin fibrillogenesis) were generated and characterized as previously reported [ 23 , 24 ]. Both PEG-FUD and the control PEG-mFUD were generated and characterized as described [ 22 ]. The concentration of FUD and PEGylated FUD was obtained from absorbance measurements at 280 nm, using ε = 0.496, as described previously [ 23 ].…”

“…Molecular weight standards are depicted to the left of the gel. The molecular weights of FUD and of PEG-FUD are ~7 and ~ 27 kDa, respectively as determined by mass spectrometry [ 22 ]. However, on SDS-PAGE, both migrate close to the 50 kDa marker.…”

“…pUR4/FUD was reported to successfully reduce fibrosis in murine models of coronary artery disease, experimentally induced liver fibrosis [ 17 , 18 ], and more recently, heart failure [ 19 ]. In order to avoid the difficulties inherent in administering peptides in vivo, including rapid clearance in the kidney, as well as to take advantage of possible protection against peptide degradation and longer circulation time [ 20 , 21 ], we PEGylated pUR4/FUD specifically at the N-terminus with polyethylene glycol (PEG) of 20,000 g/mol and characterized its binding to fibronectin, determining that PEGylation did not affect affinity of pUR4/FUD towards fibronectin [ 22 ]. In this work, we investigated whether PEG-pUR4/FUD reaches the kidneys and inhibits fibronectin deposition and fibrosis in the unilateral ureteral obstruction (UUO) model of kidney disease.…”