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

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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.

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Abbreviations

70 K:

70 kDa N-terminal region of fibronectin

ECM:

Extracellular Matrix

FN:

Fibronectin

FNI:

Fibronectin type I domain

FUD:

Functional Upstream Domain

ITC:

Isothermal Titration Calorimetry

MAA:

Matrix Assembly Assay

MALDI-TOF:

Matrix Assisted Laser Desorption/Ionization Time of Flight

PEG:

Polyethylene Glycol

TGF-β:

Transforming Growth Factor β

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ACKNOWLEDGMENTS AND DISCLOSURES

The MALDI-TOF MS and LC-MS analysis of FUD, mFUD, PEG-FUD, and PEG-mFUD was facilitated by Ian Miller, Molly Pellitteri Hahn, and Cameron Scarlett at the School of Pharmacy’s Analytical Instrumentation Center. We are grateful for the financial support of the University of Wisconsin-Madison School of Pharmacy and the National Institutes of Health Grant R01AI101157 to the Kwon lab and the National Eye Institute Grants EY017006 and EY0020490 (Peters lab), a Core grant to the Department of Ophthalmology and Visual Sciences P30EY016665 which provides support to the Peters lab.

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Authors and Affiliations

  1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Pawel Zbyszynski, Bianca R. Tomasini-Johansson & Glen S. Kwon

  2. Department of Pathology & Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Donna M. Peters

Authors
  1. Pawel Zbyszynski
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  2. Bianca R. Tomasini-Johansson
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  3. Donna M. Peters
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  4. Glen S. Kwon
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Corresponding author

Correspondence to Glen S. Kwon.

Electronic supplementary material

ESM 1

FPLC ion exchange chromatogram showing purification of the FUD and mFUD peptides. An anionic exchanger combined with a mobile phase gradient of 20 mM Tris A side and 1 M NaCl B side were used to elute and fractionate the peptides. (JPG 2018 kb)

ESM 2

FPLC ion exchange chromatogram showing isolation by fractionation of the 20 kDa PEG-FUD and 20 kDa PEG-mFUD constructs. An anionic exchanger combined with a mobile phase gradient of 20 mM Tris A side and 1 M NaCl B side were used to elute the peptide and separate it from unreacted FUD, PEG, and diPEGylated species. (JPG 2389 kb)

ESM 3

Reversed Phase High Performance Liquid Chromatography (RP-HPLC) chromatograph comparing retention times of FUD to mFUD and their respective PEGylates. The analysis was made using a C8 column combined with an A side H2O and B side acetonitrile mobile phase elution gradient. (JPG 2457 kb)

ESM 4

MALDI-TOF MS spectrum verifying the molecular weight of the 10 kDa PEG-FUD construct. A 6 kDa FUD reacted with a single 11.1 kDa nominal MW PEG yielded 16.9 kDa PEG-FUD. The analysis was made using α-cyano-4-hydroxycinnamic acid matrix. (JPG 2075 kb)

ESM 5

MALDI-TOF MS spectrum verifying the molecular weight of the 40 kDa PEG-FUD construct. A 6 kDa FUD reacted with a single 43.6 kDa nominal MW PEG yielded 49.6 kDa PEG-FUD. The analysis was made using α-cyano-4-hydroxycinnamic acid matrix. (JPG 2075 kb)

ESM 6

MALDI-TOF spectrum verifying the molecular weight of the 20 kDa PEG-mFUD construct. A 6 kDa mFUD reacted with a single 21.4 kDa nominal MW PEG yielded approximately 27.4 kDa PEG-mFUD. The analysis was made using α-cyano-4-hydroxycinnamic acid matrix. (JPG 1794 kb)

ESM 7

Determination of binding affinity (Kd) and other thermodynamic parameters using Isothermal Titration Calorimetry (ITC). All experiments were performed using pH 7.4 PBS, 25°C chamber conditions, and human plasma FN. A) 10 kDa PEG-FUD into FN and B) 40 kDa PEG-FUD into FN experiment sample isotherm and thermograph. Each experiment was repeated in triplicates. (JPG 6882 kb)

ESM 8

Matrix Assembly in vitro Assay (MAA) demonstrating inhibitory potency of FUD and 10 kDa, 20 kDa and 40 kDa PEG-FUD conjugates, and the 20 kDa PEG-mFUD control peptide. Human foreskin fibroblasts (AH1F) are grown in the presence of exogenous Alexa 488-labeled human plasma FN and in the presence or absence of an inhibitor. Results show parity of inhibitory potency between FUD and PEG-FUD conjugates. Extraction of IC50 values yielded A) 13 nM and 17 nM for FUD and 10 kDa PEG-FUD, respectively and B) 19 nM and 20 nM for FUD and 40 kDa PEG-FUD, respectively. For each data point, n = 4. (JPG 5221 kb)

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Zbyszynski, P., Tomasini-Johansson, B.R., Peters, D.M. et al. Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic. Pharm Res 35, 126 (2018). https://doi.org/10.1007/s11095-018-2412-7

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  • DOI: https://doi.org/10.1007/s11095-018-2412-7

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