Project

Discipline

protein; peptide; polymer; reversible; PEGylation

Lead
Lay summary
Approximately 25% of new drugs approved by the American Food and Drug Administration are peptides or proteins. Unfortunately, many of these therapeutic agents are rapidly eliminated by the body and/or are recognized by the immune system. For the past 30 years, these issues have been mainly addressed by grafting polymer chains to the peptide/protein, thus masking it from the immune system and preventing rapid elimination by increasing its size. One major obstacle to this approach lies in the loss of activity of the therapeutic agent, especially when specific binding events are involved. A recent direction taken in this field is to release the native peptide or protein from the polymer with time in the body, a process referred to as “reversible PEGylation” (rPEGylation). However, it is currently difficult to predictably and finely control the rate of release over a useful range in a biofluid, which would permit control over pharmacokinetics. We have recently reported coupling chemistry for selective conjugation of polymers to arginine residues on proteins. In this project, we will adapt our findings to achieve the selective, yet reversible, modification of arginine residues. Control of the rate of release of the polymer from the peptide or protein should be achievable through structural modifications to the reagent, and/or by altering the local micro-environment at the conjugation site. We will examine the stability of the prepared conjugates in model buffers and in biofluids for both a peptide containing a single arginine residue, and lysozyme, which contains multiple arginine residues. The research proposed herein is important because it addresses important needs in the field of rPEGylation. Such an innovation will be of great value for altering pharmacokinetics, in particular for well-defined mono-PEGylated peptides or proteins for which release kinetics cannot be altered by increasing the number of conjugated polymer chains. This research is of broad interest because conjugation can, a priori, be achieved between any peptide or protein containing at least one arginine residue, to entities such as polymers or small molecule ligands such as lipids or cell-penetrating peptides, etc. Applications of selective reversible conjugation to proteins can also be found in sensor and protein sorting applications.

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Last update: 21.02.2013

Active participation

Title	Year

Background and Problem. For over 30 years, grafting methoxy poly(ethylene glycol) (mPEG) to proteins, a process referred to as PEGylation, has been exploited to prevent their renal clearance and their recognition by the immune system. A recent direction taken in this field is to release the native protein from the polymer with time in the body (rPEGylation). However, despite great progress made in the field of permanent PEGylation, thus far only lysine and cysteine residues have been targeted as sites for rPEGylation. Thus, the possibilities for rPEGylating proteins at other amino acid residues is very limited. In addition, despite intriguing results from several important groups in the field, it is difficult to predictably and finely control the rate of release over a useful range in a biofluid. Pertinent findings in our group. We have recently reported coupling chemistry for selective conjugation of polymers to arginine residues on proteins. Our strategy relies on the selective glycation of arginine in vivo by a-oxo-aldehydes such as methylglyoxal. Interestingly, derivatives of phenylglyoxal (PGO), an aromatic a-oxo-aldehyde, have been extensively used for the selective, yet reversible, modification of arginine residues in a pH-dependent manner. Adapting our prior research to aromatic rather than aliphatic a-oxo-aldehydes should therefore permit reversible PEGylation at arginine, a residue for which rPEGylation has not yet been reported. Herein we will examine two possibilities for adjusting the rate of hydrolysis of the arginine-PGO adduct. Firstly, the influence of electron-withdrawing and donating groups on PGO will be examined. Secondly, based on our prior findings that the local environment surrounding labile bonds can be altered to tune their rate of reaction, we hypothesize that the local pH and thus the rate of hydrolysis of the arginine-PGO adduct can be controlled using a peptide platform. We will examine the stability of the prepared conjugates in model buffers and in biofluids for both a peptide (exendin-4), which contains a single arginine residue, and lysozyme, which contains multiple arginine residues. Importance. The research proposed herein is important because it addresses two important needs in the field of rPEGylation. Such an innovation will be of great value for altering pharmacokinetics, in particular for well-defined mono-PEGylated peptides/proteins for which release kinetics cannot be altered by increasing the number of conjugated mPEG chains. This research is of broad interest because conjugation can, a priori, be achieved between any peptide/protein containing at least one arginine residue, to entities such as polymers (other than mPEG) or small molecule ligands such as lipids or cell-penetrating peptides, etc. Applications of selective reversible conjugation to proteins can also be found in sensor and protein sorting applications.