Besides the two classes of technologies just described, which can be used to alter the very basic structure of proteins, there are a plethora of chemistry-based methods that allows the modification of the parent protein structure.
The endogenous protein structure can be exploited for selective derivatization. The most frequent way of modifying protein structure is by reacting cysteine residues; this can often be successfully carried out with either none or minimal changes to the parent protein. The advantages is that the thiol of cysteine allows for selective modification, relative to the other proteinogenic amino acids and the frequency by which cysteine occurs in proteins is relatively low, thus often allowing the selective modification of specific cysteine residues. Even if a protein contains more than one cysteine residues, these might have different accessibility, which can allow the selective modification of certain residues.
When proteins are being developed as drugs, the pharmacokinetic (PK) and pharmacodynamic (PD) properties of proteins can be improved by the chemical modification of the protein structure. A particularly promising strategy is the introduction of PEG moieties, known as PEGylation, which can help reducing immunogenicity, increasing the circulatory time by reducing renal clearance and also provide water solubility to hydrophobic drugs and proteins. PEGylation is generally performed by the reaction of a reactive derivative of PEG with the target protein, typically with side chains of amino acids such as lysine or cysteine, or by reaction at the C- or N-terminal of the protein or pep-tide. PEGylated proteins entered the market in the 1990s and today a number of therapeutic proteins are marketed as PEGylated derivatives including PEGylated a-interferons (see also Chapter 24), which are used in the treatment of hepatitis C; the PEGylated a-interferon is injected only once a week, compared to three times a week for conventional a-interferon.
An alternative way of improving protein and peptide therapeutics is by adding lipids to the protein, which can improve half-life. Adding lipids to a protein framework has been achieved by ligation strategies, but in a few cases the differential reactivity of specific residues has been exploited. An example of this is the long-acting insulin analog, insulin detemir (Levemir®), where the Ne -amino group of a terminal lysine in the B-chain of insulin has been modified with tetradecanoic acid
(myristic acid, C14 fatty acid chain). This modification increases self-association and binding to albumin, leading to stable insulin supply for up to 24 h. Similarly, liraglutide (Victoza®) is an analog of glucagon-like peptide-1 (GLP-1), where a lysine side chain has been modified; a palmitic acid (hexadecanoic acid, C16 fatty acid chain) was added through a glutamate linker. The modification lead to a substantial increase in half-life, due to increased binding to serum albumin, and the modification did not compromise the biological activity.
In some cases, the selective modification of proteins can be achieved by using simple chemical reactions similar to those used in conventional organic synthesis. A general requirement is that such reaction should be compatible with the aqueous (buffer) conditions, in which the protein is present and recently a number of robust and water-compatible reactions have evolved. However, such methods often require the introduction of selective handles, as previously described, in order to be sufficiently selective, but once a reactive handle is incorporated, a wealth of chemical reactions can be performed. The example of "click chemistry," that is, a 1,3-dipolar cycloaddition between an azide and an alkyne providing a 1,2,3-triazole, has already been mentioned. Another prominent example is the Staudinger reaction, which is a phosphine-mediated reduction of an azide to an amine, also known as an aza-Wittig reaction, that has been used particularly in protein glycosy-lation studies. Interestingly, the Staudinger reaction has recently been applied in the ligation of peptides and proteins.
Finally, enzymes can be used to selectively modify proteins. Enzymes have an inherent advantage that they efficiently add or remove groups to proteins and they are often highly specific for certain sequences (consensus motifs) of amino acids, so modifications are often site-specific. Enzymes are often also highly substrate-specific, that is, kinases add only phosphates groups to serine, theronine, and tyrosine; thus the modification of the enzyme is required if other groups have to be introduced. However, some enzymes, such as glycosyltransferases, which transfers carbohydrates to serines or asparagines, have broader substrate specificity, but in this case, it can be desirable to modify the enzyme to achieve increased reactivity for specific carbohydrates. A particularly powerful method to develop enzymes with desired properties is directed evolution, which basically consist of two steps: (1) the generation of a library of mutants of the enzymes and (2) rounds of screening/selection for the desired properties, which for example can be used to modify substrate specificity of enzymes.
Enzymes are particularly useful to furnish proteins with tailor-made PTMs, which are often essential for the regulation and dynamics of biological activity. For example, most proteins are gly-cosylated, and controlling glycosylation patterns of proteins is a key challenge. Glycosyltransferases are enzymes that can catalyze the transfer of a monosaccharide to a protein, and using directed evolution was possible to modify the transferases, so monosaccharides of interest could be selectively added to a protein framework. Another example is using transglutaminase (TGase) to obtain selective PEGylation. TGase catalyzes transfer reactions between the g-carboxamide group of glutamine residues and primary amines, resulting in the formation of g-amides of glutamic acid and ammonia. Thus, by using an aminoderivative of PEG (PEG-NH^) as substrate for the enzymatic reaction, it is possible to covalently bind the PEG polymer to a therapeutic protein.
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