Chemical Biology - Drug Design and Discovery - page 72

protein was prepared by the ligation of four peptide fragments, two of which were modii ed with the
polymer and the EPO analog displayed improved properties in vivo compared to EPO.
In 1998, an extension of the NCL principles was introduced, called expressed protein ligation
(EPL). The technology applies the same reaction as in NCL, but in contrast to NCL, one of the
components is a protein, rather than a peptide (Figure 4.10). The protein is expressed as the so-
called intein construct, which allows the formation of a protein thioester, which subsequently can
be reacted with a peptide with an N-terminal cysteine in an NCL generating a full-length protein
(Figure 4.10). Thus, the EPL methodology combines the advantages of molecular biology with
chemical peptide synthesis, and enables the addition of unnatural functionality to a recombinant
protein framework.
EPL has been applied in studies of several proteins and here only a few noteworthy examples
are provided. Histone complexes are important for the storage of DNA and have l exible N-terminal
tails that are heavily modii ed by PTMs, and is of general importance for epigenetic gene regulation
(see Chapter 23). EPL has been applied to prepare full-length, ubiquilated H2B and subsequently
used to demonstrate a direct cross talk between PTMs on different histones. The list of proteins
prepared by EPL was extended to include integral membrane proteins, specii cally the potassium
channel KcsA, which is a tetrameric assembly of identical subunits (see also Chapter 13). EPL was
used to prepare KscA subunits (122 residues), which were then refolded and reconstituted into lipid
membranes. In subsequent studies, unnatural mutations, such as d-alanine and amide-to-ester muta-
tion, in the selectivity i lter of the channel have revealed the important information of the function of
this important segment of the potassium channel.
4.3.4 C HEMICAL M ODIFICATION OF P ROTEINS
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 modii cation
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 success-
fully carried out with either none or minimal changes to the parent protein. The advantages is that
the thiol of cysteine allows for selective modii cation, relative to the other proteinogenic amino
acids and the frequency by which cysteine occurs in proteins is relatively low, thus often allowing
the selective modii cation of specii c cysteine residues. Even if a protein contains more than one
cysteine residues, these might have different accessibility, which can allow the selective modii cation
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 modii cation 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 pro-
tein, 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 specii c residues has been exploited. An
example of this is the long-acting insulin analog, insulin detemir (Levemir ® ), where the N e -amino
group of a terminal lysine in the B-chain of insulin has been modii ed with tetradecanoic acid