Pawel Zbyszynski1, Bianca R Tomasini-Johansson1, Donna M Peters2, Glen S Kwon3. 1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA. 2. Department of Pathology & Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA. 3. Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA. gskwon@pharmacy.wisc.edu.
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.
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 FUDfibronectin 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.
Authors: Thomas R Cox; Demelza Bird; Ann-Marie Baker; Holly E Barker; Melisa W-Y Ho; Georgina Lang; Janine T Erler Journal: Cancer Res Date: 2013-01-23 Impact factor: 12.701
Authors: Hilary A Kenny; Chun-Yi Chiang; Erin A White; Elizabeth M Schryver; Mohammed Habis; Iris L Romero; Andras Ladanyi; Carla V Penicka; Joshy George; Karl Matlin; Anthony Montag; Kristen Wroblewski; S Diane Yamada; Andrew P Mazar; David Bowtell; Ernst Lengyel Journal: J Clin Invest Date: 2014-09-09 Impact factor: 14.808
Authors: Bianca R Tomasini-Johansson; Pawel W Zbyszynski; Inger Toraason; Donna M Peters; Glen S Kwon Journal: PLoS One Date: 2018-10-24 Impact factor: 3.240