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Modulating protein activity through ...

Decker, Caitlin Gayle.

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Modulating protein activity through polymer conjugation.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Modulating protein activity through polymer conjugation./
Author:	Decker, Caitlin Gayle.
Description:	145 p.
Notes:	Source: Dissertation Abstracts International, Volume: 77-02(E), Section: B.
Contained By:	Dissertation Abstracts International77-02B(E).
Subject:	Polymer chemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3725386
ISBN:	9781339096360

Modulating protein activity through polymer conjugation.
Decker, Caitlin Gayle.

Modulating protein activity through polymer conjugation. - 145 p.

Source: Dissertation Abstracts International, Volume: 77-02(E), Section: B.

Thesis (Ph.D.)--University of California, Los Angeles, 2015.

Protein therapeutics have become essential to the healthcare and pharmaceutical industries since the first recombinant proteins entered the clinic in the 1980s. Modification of proteins with polymers has traditionally been pursued as a means to improve protein stability and enhance pharmacokinetic properties. In addition to these benefits, polymer conjugation can also be utilized to control and modulate protein activity. The first polymer used for protein conjugation was poly(ethylene glycol) (PEG) in 1977. PEG is currently the only FDA-approved polymer for protein conjugation and 10 PEGylated protein drugs are currently on the market. This dissertation offers three modifications to traditional PEGylation, which allow for the modulation of protein activity.

ISBN: 9781339096360Subjects--Topical Terms:

3173488
Polymer chemistry.

Modulating protein activity through polymer conjugation.
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100 1 $a Decker, Caitlin Gayle. $3 3193107
245 1 0 $a Modulating protein activity through polymer conjugation.
300 $a 145 p.
500 $a Source: Dissertation Abstracts International, Volume: 77-02(E), Section: B.
500 $a Adviser: Heather D. Maynard.
502 $a Thesis (Ph.D.)--University of California, Los Angeles, 2015.
520 $a Protein therapeutics have become essential to the healthcare and pharmaceutical industries since the first recombinant proteins entered the clinic in the 1980s. Modification of proteins with polymers has traditionally been pursued as a means to improve protein stability and enhance pharmacokinetic properties. In addition to these benefits, polymer conjugation can also be utilized to control and modulate protein activity. The first polymer used for protein conjugation was poly(ethylene glycol) (PEG) in 1977. PEG is currently the only FDA-approved polymer for protein conjugation and 10 PEGylated protein drugs are currently on the market. This dissertation offers three modifications to traditional PEGylation, which allow for the modulation of protein activity.
520 $a In the first example, masking and unmasking the activity of a model protein, lysozyme, was achieved by incorporating both a reducible disulfide linkage between the polymer and the protein as well as incorporating degradable cyclic ketene acetal (CKA) moieties throughout the backbone of a PEG-like polymer (Chapter 2). Specifically 5,6-benzo-2-methylene-1,3-dioxepane and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were copolymerized by reversible addition-fragmentation chain transfer polymerization (RAFT) facilitated by a cysteine-reactive, pyridyl disulfide (PDS) modified chain transfer agent (CTA). Two polymers, a small (Mn GPC = 10.9 kDa) and a large (M n GPC = 20.9 kDa) PDS-pPEGMA-co-BMDO, were synthesized with reasonable control (dispersities (D) = 1.34 and 1.71, respectively). The polymers were then conjugated to a thiol-enriched hen egg white lysozyme by disulfide exchange. Conjugation with the 10.9 kDa polymer resulted in a conjugate, which exhibited high initial activity (63%) while the larger conjugate activity was highly attenuated (20%). Lysozyme release from both polymers by reduction of the disulfide linkage and by hydrolytic cleavage, in basic media, of the polymer backbone was visualized by gel electrophoresis. Reduction of the disulfide conjugation linkage with glutathione resulted in an increase in protein activity for both conjugates.
520 $a In the next example, site-specific chemical dimerization of fibroblast growth factor 2 (FGF2) with a PEG linker, of optimized length, resulted in a FGF2 homodimer with wound healing ability at exceptionally low concentrations (Chapter 3). Homodimers of FGF2 were synthesized through site-specific conjugation to both ends of poly(ethylene glycol) (PEG). FGF2 was conjugated to 2, 6, and 20 kDa vinyl sulfone bis-functionalized PEG, as well as to a small molecule and mono-functionalized PEG controls. The optimal linker length was determined by screening FGF2 dimer-induced proliferation of human dermal fibroblasts (HDF). The inter-cyteine distance was calculated to be approximately 70 A, which is similar in length to a 2 kDa PEG. FGF2-PEG2k-FGF2 induced greater fibroblast proliferation than FGF2 alone, all other dimers, and all monoconjugates, at each concentration tested, with the greatest difference observed at low (0.1 ng/mL) concentration. FGF2-PEG2k-FGF2 further exhibited superior activity compared to FGF2 for both proliferation and migration in human umbilical vein endothelial cells, as well as improved angiogenesis in vitro. Efficacy in an in vivo wound healing model was assessed in diabetic mice. FGF2-PEG2k-FGF2 increased granulation tissue and blood vessel density in the wound bed compared to FGF2. The results suggest that this rationally designed construct may be useful in chronic wound healing.
520 $a Lastly, a block copolymer capable of noncovalent and releasable conjugation to histidine-6 tagged proteins, consisting of a PEG-based block and a Ni(II) nitriolotriacetic acid (NTA)-based block was synthesized (Chapter 4). The first block was synthesized via RAFT polymerization of a NTA monomer. The resulting polymer was then utilized as a macro-CTA for the polymerization of PEGMA, resulting in a pNTAMA-b-PEGMA, containing 9 NTAMA repeats and 8 PEGMA repeats, with number average molecular weight (Mn) (GPC) = 9.9 kDa and dispersity (D) = of 4.5. The high dispersity indicates a lack of control, and disproportionation was further confirmed by 1H-NMR. Initial studies indicated that mono-NTA-His6 interactions are not sufficient for protein conjugation, therefore extension of this work towards a multi-valent approach may prove effective in the future.
590 $a School code: 0031.
650 4 $a Polymer chemistry. $3 3173488
650 4 $a Biochemistry. $3 518028
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710 2 $a University of California, Los Angeles. $b Chemistry 0153. $3 2096181
773 0 $t Dissertation Abstracts International $g 77-02B(E).
790 $a 0031
791 $a Ph.D.
792 $a 2015
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3725386