Chemical Biology - Drug Design and Discovery - page 73

(myristic acid, C 14 fatty acid chain). This modii cation 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 modii ed; a palmitic acid
(hexadecanoic acid, C 16 fatty acid chain) was added through a glutamate linker. The modii cation
lead to a substantial increase in half-life, due to increased binding to serum albumin, and the modi-
i cation did not compromise the biological activity.
In some cases, the selective modii cation 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 pres-
ent 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 sufi ciently 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 efi ciently add or remove groups to proteins and they are often highly specii c for certain
sequences (consensus motifs) of amino acids, so modii cations are often site-specii c. Enzymes are
often also highly substrate-specii c, that is, kinases add only phosphates groups to serine, theronine,
and tyrosine; thus the modii cation 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 specii city, but in this case, it can be desirable to modify the
enzyme to achieve increased reactivity for specii c 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 specii city 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 evo-
lution 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 selec-
tive 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 2 ) as substrate for the enzymatic reaction, it is
possible to covalently bind the PEG polymer to a therapeutic protein.
4.4 CONCLUDINGREMARKS
In this chapter, we have focused on small molecules and how a systematic generation and applica-
tion of these can be expediently used to probe and discover biology, both in an academic setting and
in the initial drug design and development process. We also discussed the application of chemical
biology technologies in studies of proteins and how this has opened up new avenues in protein engi-
neering and paved the way for studies of proteins in that has previously not been possible. Similar
principles and technologies are applied in studies of nucleic acids and polysaccharides with great
benei t for basic research, but likewise in the development of biologicals.
Chemical biology is a scientii c discipline that has emerged primarily from chemical sciences to
apply chemical tools and principles in studies of biological phenomena. However, chemical biology