close

Вход

Забыли?

вход по аккаунту

?

Organic Protein Chemistry Drug Discovery through the Chemical Modification of Proteins.

код для вставкиСкачать
Highlights
Medicinal Chemistry
Organic Protein Chemistry: Drug Discovery through the
Chemical Modification of Proteins
Jrg Rademann*
Keywords:
chemical ligation · drug discovery · medicinal
chemistry · proteins
F
or a long time the preparation and
transformation of proteins was considered a domain of molecular biology.
With modern ligation techniques, however, success has been achieved not only
in the de novo preparation of proteins
but also in their targeted chemical
modification.[1–3] In this way “organic
protein chemistry” was founded, which
has made an important contribution to
the understanding of proteins and to the
development of chemical biology. The
chemical modification of proteins appears to be a very promising route to
new protein-based drugs and low-molecular-weight protein ligands.
Ligation techniques are used preferentially in the chemical transformation
of proteins. Chemical ligation is understood to mean synthetic methods for the
selective attachment to partially unprotected biopolymers in aqueous buffers
under neutral, mild conditions (Figure 1). The starting point for modern
ligation techniques was the discovery by
Wieland et al. that partially unprotected
thioesters of amino acids and peptides
form peptide bonds selectively with Nterminal cysteine residues in neutral
aqueous solution.[1] In this way an intermediate cysteine thioester initially
formed by “thioester exchange” then
[*] Prof. Dr. J. Rademann
FMP Forschungsinstitut f!r
Molekulare Pharmakologie
Robert-R%ssle-Strasse 10
13125 Berlin (Germany)
and
Institut f!r Chemie
Freie Universit3t Berlin
Takustrasse 3
14195 Berlin (Germany)
Fax: (+ 49) 30-94793-159
E-mail: rademann@fmp-berlin.de
4554
Figure 1. Ligation techniques for de novo synthesis and modification of proteins.
rearranges into a stable amide bond by
means of an S N acyl shift. Even in the
original publication in 1953 it was proposed that a natural synthesis principle
for cysteine-containing peptides and
proteins had been discovered. In the
1990s this thioester ligation was named a
“natural chemical ligation” by Kent1s
group and was used successfully in the
de novo syntheses of proteins in aqueous buffers.[2] The success of thioester
ligation has inspired the development of
further ligation techniques which have
found many applications in the synthesis
of proteins and protein mimics (Figure 1).
The particular advantage of chemical protein synthesis is the access to
chemically modified biopolymers that
cannot be obtained with the aid of the
methods of molecular biology. With
organic synthesis it is possible to produce specifically proteins with new
property profiles. The activity, selectiv-
ity, and stability of biopolymers become
controllable, and proteins can be tailormade like small organic molecules.
A particularly rewarding target is
the construction of chemically modified
proteins with increased bioavailability
and stability in order to prepare new and
improved active peptides and proteins.
In this context an important milestone
was reached with the chemical preparation of a synthetic, polymer-stabilized
erythropoietin.[4] Human erythropoietin
(EPO), a glycoprotein hormone, is of
considerable interest since it stimulates
the growth, differentiation, and maturation of stem cells to erythrocytes. The
considerable clinical significance for the
therapy of anemia can be appreciated in
that the annual EPO turnover exceeds
one billion dollars. Unfortunately the
action of natural EPO is limited by its
short half-life. For this reason a chemical
EPO mutant has been prepared which is
protected against biodegradation by two
DOI: 10.1002/anie.200460075
Angew. Chem. Int. Ed. 2004, 43, 4554 –4556
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angewandte
Chemie
branched polyether structures without
compromising the stimulating activity
on blood cell precursor cells. In rats the
synthetic protein has a longer half-life
than natural EPO and leads to a greater
increase in the hematocrit value.
The chemically modified EPO was
prepared by a clever combination of
thioester and oxime ligation (Figure 2).
A peptide chain of 166 amino acids
derived from natural EPO was initially
divided into four fragments (A–D) and
built up by standard solid-phase methods. Since natural EPO does not contain
cysteine side chains in sufficient numbers for ligation, two glutamate residues
at the N terminus of fragments C and D
were replaced by cysteine. Following
ligation these cysteine residues were
alkylated with bromoacetic acid to obtain glutamate-like side chains. Fragments B, C, and D thus contained an Nterminal cysteine unit; in fragments B
and C this group was protected with an
S-acetamidomethyl group (Acm). In
addition an Ne-levulinyl-lysine was incorporated into both fragments A and
D. The keto function contained therein
served in each case to couple a branched
aminoxy polyether by oxime ligation.
The two polyethers were attached to
sites in two of the four oligosaccharides
present in the natural glycoprotein.
Finally the fragments were conjoined
by thioester ligation, and after folding
and purification a uniform product with
a mass of 50 825 Da was obtained.[5]
Ketoproteins as starting materials
for oxime ligation had previously been
prepared by Dixon in 1960 by coppercatalyzed transamination of proteins
with glyoxylate.[6] Forty years later this
reaction was developed further into a
valuable preparative method.[7] More
recently ketopeptides with variable side
chains were accessible by mild solidphase acetylation of a cyanophosphorane.[8]
It is even possible to introduce
levulinic acid residues into the lysine
side chains in defined positions in the
protein by de novo formation of the
peptide. Alternatively, dithiolane-protected levulinyl residues may be used.
The keto functions are released by
oxidative cleavage following protein
synthesis.[9]
Chemical protein engineering may
be used not only for the development of
protein-based drugs. The search for lowmolecular-weight active compounds is
also possible with chemically modified
proteins to discover suitable binding
pockets and high-affinity ligands for
Figure 2. Synthesis of a polymer-modified variant of human erythropoietin by a combination of
thioester and oxime ligation. The blue regions denote the polyether units.
Angew. Chem. Int. Ed. 2004, 43, 4554 –4556
www.angewandte.org
protein surfaces. For some time it has
been a standard approach to vary individual amino acids in proteins by targeted mutagenesis in order to understand enzyme mechanisms and to modify them systematically. By means of
targeted mutagenesis, attachment sites
for chemical ligation techniques can also
be produced on protein surfaces. For
example, an equilibrium with sulfide
exchange exists between cysteine residues and disulfides in aqueous solution
(Figure 1). If by specific mutagenesis a
cysteine residue is now brought into the
immediate proximity of the binding
pocket of an enzyme, the “hot spot” of
a protein–protein binding surface, preferentially binding residues can be selected from an ensemble of dissolved
disulfides and identified by mass spectrometry (LC-MS) of the synthetic protein product. In this way structures
active with interleukin-2 could be filtered out of 7000 disulfides.[10]
Ligation on protein surfaces can also
be used to discover new interaction
regions in addition to known binding
pockets that could be used to increase
the affinity or selectivity of a ligand.
This concept was applied successfully to
caspase-3.[11] The caspases are a class of
cysteine proteases that play a key role
(inter alia in the development of cancer)
because of their central function in
programmed cell death (apoptosis).
The cysteine residue of the active center
was chosen for the in situ synthesis of an
affinity ligand. (Figure 3). The sulfanyl
function of the active center was first
alkylated with a known irreversible
inhibitor that contained a masked SH
function. This second sulfanyl function
then reacted with a disulfide library as in
the aforementioned in situ ligation. A
ligand–linker combination that preferentially occupied a binding pocket outside the active position of the enzyme
was selected. For control the structure
found was converted into a soluble,
reversible inhibitor. However, changes
on the linker were necessary to some
extent in order to achieve Ki values
below 1 mm.
The two examples illustrate the
considerable advances in organic protein chemistry. Over and above de novo
syntheses of proteins it has become
possible to modify the properties of
proteins in a targeted manner with
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4555
Highlights
Figure 3. Disulfide ligation on the surface of proteins (in situ ligation) for the identification of
high-affinity ligands and new interaction regions outside of the catalytically active binding pocket.
chemical methods. The techniques described can simplify the development of
new active proteins, and also the search
for new low-molecular-weight active
compounds. At the same time it is also
evident how important analytical and
separation techniques are for the achievement of such demanding targets.
Only the extensive availability of highresolution mass spectrometric and liquid
chromatography techniques has made
possible the work described. The exact
characterization of ligation methods still
requires further research. It would be
4556
desirable in this context to learn through
systematic work more about by-products, the protective groups that may be
necessary, and the extent of racemization of the amino acid building blocks in
the ligation products.
Published Online: July 20, 2004
[1] T. Wieland, E. Bokelmann, L. Bauer,
H. U. Lang, H. Lau, Justus Liebigs Ann.
Chem. 1953, 586, 129 – 149.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
[2] P. E. Dawson, T. W. Muir, I. ClarkLewis, S. B. H. Kent, Science 1994, 266,
776 – 779.
[3] a) E. Saxon, C. R. Bertozzi, Science
2000, 287, 2007 – 2010; b) B. L. Nilsson,
L. L. Kiessling, R. T. Raines, Org. Lett.
2000, 2, 1939 – 1941.
[4] G. G. Kochendoerfer, S. Y. Chen, F.
Mao, S. Cressman, S. Traviglia, H. Shao,
C. L. Hunter, D. W. Low, E. N. Cagle, M.
Carnevali, V. Gueriguian, P. J. Keogh, H.
Porter, S. M. Stratton, M. C. Wiedeke, J.
Wilken, J. Tang, J. J. Levy, L. P. Miranda,
M. M. Crnogorac, S. Kalbag, P. Botti, J.
Schindler-Horvat, L. Savatski, J. W.
Adamson, A. Kung, S. B. H. Kent, J. A.
Bradburne, Science 2003, 299, 884 – 887.
[5] Yields of 15–30 % were reported for the
peptide synthesis and ligation, 30–50 %
for the oxime ligation, 40–70 % for the
thioester ligation, and 25–40 % for the
folding. This gives an overall yield of
between 0.072 and 2 % without consideration of losses in yield during peptide
synthesis.
[6] a) H. B. F. Dixon, Biochem. J. 1964, 90,
2c – 3c; b) H. B. F. Dixon, Biochem. J.
1964, 92, 661 – 666.
[7] A. Papanikos, J. Rademann, M. Meldal,
J. Am. Chem. Soc. 2001, 123, 2176 –
2181.
[8] S. Weik, J. Rademann, Angew. Chem.
2003, 115, 2595 – 2598; Angew. Chem.
Int. Ed. 2003, 42, 2491 – 2494.
[9] D. Tumelty, M. Carnevali, L. P. Miranda,
J. Am. Chem. Soc. 2003, 125, 14 238 –
14 239.
[10] M. R. Arkin, M. Randal, W. L. DeLano,
J. Hyde, T. N. Luong, J. D. Oslob, D. R.
Raphael, L. Taylor, J. Wang, R. S.
McDowell, J. A. Wells, A. C. Braisted,
Proc. Natl. Acad. Sci. USA 2003, 100,
1603 – 1608.
[11] D. A. Erlanson, J. W. Lam, C. Wiesmann, T. N. Luong, R. L. Simmons,
W. L. DeLano, I. C. Choong, M. T. Burdett, W. M. Flanagan, D. Lee, E. M.
Gordon, T. O1Brien, Nat. Biotechnol.
2003, 21, 308 – 314.
Angew. Chem. Int. Ed. 2004, 43, 4554 –4556
Документ
Категория
Без категории
Просмотров
2
Размер файла
144 Кб
Теги
chemistry, discovery, drug, chemical, organiz, protein, modification
1/--страниц
Пожаловаться на содержимое документа