A portable albumin binder from a DNA-encoded chemical library

Article abstract of DOI:10.1002/anie.200704936

(Figure Presented) Seeing eye to eye: Plasma-protein binding is effective in improving the pharmacokinetic properties of otherwise short-lived molecules. One compound in a class of small portable albumin binders can be used to improve the in vivo circulatory half-life of two widely used contrast agents. It improves the imaging performance of fluorescein in angiographic analysis of the retina of mice (see picture).

Full text of DOI:10.1002/anie.200704936

Communications
DOI: 10.1002/anie.200704936
Protein Binding
A Portable Albumin Binder from a DNA-Encoded Chemical Library**
Christoph E. Dumelin, Sabrina Trüssel, Fabian Buller, Eveline Trachsel, Frank Bootz,
Yixin Zhang, Luca Mannocci, Susanne C. Beck, Mihaela Drumea-Mirancea, Mathias W. Seeliger,
Christof Baltes, Thomas Müggler, Felicitas Kranz, Markus Rudin, Samu Melkko,
JörgScheuermann, and Dario Neri*
Albumin represents the most abundant protein in human
plasma, at a concentration of 45 mgmLÀ1. To keep physio-
logical production rates to a minimum, albumin displays a
long circulatory half-life in mammals thanks to its size above
the renal filtration threshold and its unique ability to interact
with the neonatal FcRn receptor.[1] Fusions of biopharma-
ceuticals to albumin[2] or to albumin-binding peptides[3,4] have
been devised to expose the body to adequate concentrations
of the therapeutic agent for a sufficiently long period of time,
thus improving efficacy and reducing the number of injec-
tions.
In principle, small organic albumin-binding molecules
could be used as functional analogues of albumin-binding
peptides. However, although many small molecules are
known to bind to albumin, the success in isolating small
molecules as portable albumin-binding moieties has been
limited,[5] mainly because most albumin binders (for example,
ibuprofen) lose binding affinity upon chemical modification.
Myristoylation of insulin has been shown to significantly
prolong the circulatory half-life,[6] but this modification is not
applicable to a broader set of molecules because of its
negative effect on solubility. In another example, a 4,4-
diphenylcyclohexyl moiety has been connected through a
phosphodiester bond to the metal chelator diethylenetriami-
nepentaacetic acid (DTPA) for magnetic resonance imaging
(MRI) applications[7] and to short peptides.[8] These com-
pounds display dissociation constants (Kd) from human serum
albumin in the 100 mm range[9] and are susceptible to
hydrolysis in vivo.
Thus, there is a considerable scientific and biotechnolog-
ical interest in the identification of small portable binders that
display a stable noncovalent interaction with serum albumin.
Herein, we report the discovery and characterization of a
class of 4-(p-iodophenyl)butyric acid derivatives from a
DNA-encoded chemical library,[10] which display a stable
noncovalent binding interaction with both mouse serum
albumin (MSA) and human serum albumin (HSA). One of
these portable albumin-binding moieties was used to improve
the performance of the contrast agents fluorescein and Gd-
DTPA.
[*] S. Trüssel, F. Buller, Dr. F. Bootz, Dr. Y. Zhang, L. Mannocci,
Dr. J. Scheuermann, Prof. Dr. D. Neri
Institut für Pharmazeutische Wissenschaften
Departement für Chemie und Angewandte Biowissenschaften
ETH Zürich
Wolfgang-Pauli-Strasse 10, 8093 Zürich (Switzerland)
Fax: (+41)44-633-1358
HSA-binding molecules were selected from a DNA-
encoded chemical library consisting of 619 oligonucleotide-
compound conjugates carrying a six-base-pair code for
identification.[11–13] After selection, the DNA sequences of
the enriched compounds were amplified by PCR and decoded
on oligonucleotide microarrays displaying the complemen-
tary sequences (Figure 1a), normalizing the signal intensities
after selection against the intensities of compounds selected
on empty resin (Figure 1b). Some of the identified binding
molecules were excluded from further evaluation based on
being promiscuous binders or because of the high standard
deviations of the signal intensities on the microarrays (64, 313,
453, 454, 619). Several of the selected molecules (428, 533,
535, 539) displayed striking structural similarities. The basic
structure featured a 4-phenylbutanoic acid moiety, with
different hydrophobic substituents on the phenyl ring.
To obtain further insights into structure–activity relation-
ships, DNA-modified analogues containing propanoyl or
pentanoyl skeletons, and/or carrying various substituents on
the phenyl ring (Figure 1c; 536, 622–632), were characterized
in a radioactivity-based chromatographic albumin-binding
assay,[12] which allowed a first classification of the potential
binders (Retention: 428 > 539 > 624 > 535 > 533 > 536 >
326 > others; see the Supporting Information). The absence
of retention of compounds with propanoyl (625) and penta-
E-mail: neri@pharma.ethz.ch
Dr. C. E. Dumelin, Dr. E. Trachsel, Dr. S. Melkko
Philochem AG, c/o ETH Zürich
Wolfgang-Pauli-Strasse 10, 8093 Zürich (Switzerland)
Dr. S. C. Beck, Dr. M. Drumea-Mirancea, Dr. M. W. Seeliger
Departement für Augenheilkunde
Forschungsinstitut für Augenheilkunde
AG Neurodegeneration des Auges
Universitätsklinikum Tübingen
Schleichstrasse 4/3, 72076 Tübingen (Germany)
Dr. C. Baltes, Dr. T. Müggler, F. Kranz, Prof. Dr. M. Rudin
Animal Imaging Center, Institut für Biomedizinische Technik
Departement für Elektrotechnik, Universität und ETH Zürich
Wolfgang-Pauli-Strasse 10, 8093 Zürich (Switzerland)
[**] We thank Miljen Martic for help with the HPLC analysis of
radioactive derivatives, Madalina Jaggi for help with the synthesis of
the DNA-encoded chemical library, and Dr. Jens Sobek and Dr.
Ralph Schlapbach (Functional Genomics Center Zurich) for the
microarrays. Financial support from ETH Zürich, Philochem AG, the
Swiss National Science Foundation, Bundesamt für Bildung und
Wissenschaft (EU Project STROMA), the EU FP6 Projects Immuno-
PDT and DIANA, the German Research Council DFG (Se837/4-1
and 5-1 toM.W.S.), and IP “EVI-GenoRet” LSHG-CT-512036 (to
M.W.S.) is gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Fluorescein and
several derivatives
(428-d-Lys-FAM,
622-d-Lys-FAM, and
phenethylamine-
FAM ; FAM = 5-car-
boxyfluorescein)
were studied to inves-
tigate the applicabil-
ity of 428-d-Lys as a
portable
albumin-
binding moiety and
with the aim to
develop an innovative
reagent for angio-
graphic procedures.
Fluorescein is widely
used in ophthalmol-
ogy as
a
contrast
agent in fluorescein
angiography of the
eye fundus in patients
with retinal disor-
ders.[14] However, the
short circulatory half-
life of fluorescein
requires injection of
high doses (200–
500 mg per patient)
Figure 1. Selection of albumin binders. a) Microarray readout of the selections performed against inactivated resin
(left panel) and resin displaying HSA (right panel). The spots corresponding to the enriched compounds 428 and
539 are enlarged (center). b) Enrichment of compounds in selections for HSA binding (compound numbers are
indicated). c) Structures of the molecules identified as potential binders.
with
nonnegligible
side effects[15] and
noyl skeletons (630) revealed the essential requirement of a
butanoyl moiety for albumin binding. Furthermore, hydro-
phobic groups in the para position of the phenyl ring were
found to be important determinants of binding activity, as
molecules lacking this feature (536, 622) or carrying hydro-
philic para substitutents (631, 632) displayed weak or no
retention.
impedes the comparative study of both eyes.
The Kd value of 428-d-Lys-FAM to albumin measured
with ITC, by titrating HSA with 428-d-Lys-FAM, was 330 nm
at 378C (Figure 3a), whereas fluorescein displayed no binding
to HSA (see the Supporting Information). The affinities of
fluorescein and its derivatives at 258C were determined by
fluorescence polarization (FP) at increasing concentrations of
HSA. The Kd values were 76 mm for fluorescein, 108 nm for
428-d-Lys-FAM, 6.6 mm for 622-d-Lys-FAM, and 17 mm for
phenethylamine-FAM (Figure 3b). The binding affinities of
428-d-Lys-FAM to HSA and MSA measured by FP were
comparable: Kd,MSA = 118 nm. Additional characterization of
the interaction of the FAM derivatives with albumin by band-
shift assay confirmed their ability to form a kinetically stable
complex with HSA in solution and in serum (Figure 3c). To
elucidate the site of interaction of 428-d-Lys-FAM on HSA its
binding was competed with several known ligands (site I,
site II, fatty acid, and metal ion), which suggested binding of
428-d-Lys-FAM at site II (see the Supporting Information).
The pharmacokinetic properties of fluorescein and its
derivatives were studied in mice after intravenous (i.v.)
injection, and exhibited strikingly different plasma concen-
tration time courses. Fluorescein was no longer detectable
30 min after injection, whereas the derivatives displayed a
biphasic pharmacokinetic profile which correlated well with
their affinities towards HSA. In particular, the half-life of 428-
d-Lys-FAM was substantially increased and the molecule was
still detectable in the bloodstream 24 h after injection
Although the DNA-tagged compound 428 showed the
highest retention in the chromatographic albumin-binding
assay, it exhibited only weak binding in isothermal titration
calorimetry (ITC) studies performed in solution. However,
stable albumin binding could be recovered upon introduction
of a carboxylate group (reminiscent of the terminal 5’ phos-
phate group connecting the compound to the oligonucleotide)
by conjugation of d-lysine to 428 through the e-amino group
(Figure 2a). Modification of the a-amino group of d-lysine
with acetic (428-d-Lys-Ac, Kd = 3.2 mm) or butanoic acid
(Kd = 5.5 mm) was possible without any substantial change in
affinity. 428-d-Lys-Ac further bound to MSA with a similar
affinity (Kd = 3.6 mm). Figure 2b shows ITC profiles of several
HSA binders conjugated to acetylated d-lysine. All com-
pounds had affinities in the micromolar range, with a ranking
of Kd values well in line with the corresponding retention in
the chromatographic albumin-capture assay. Further insight
into the structure–activity relationship of the carboxylate was
obtained by ITC measurements of 428 derivatives with l-
lysine and l-ornithine (see the Supporting Information).
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Figure 2. Structure–activity relationship of albumin binders. a) Structures of 428 conjugated to an amino-tagged oligonucleotide and to acetylated
d-Lys (1, 7) and a general structure of the molecules measured by ITC (2–6; X=533, 535, 536, 539, and 624, respectively), which highlight the
similar positioning of the negative charge. b) ITC characterization of identified molecules binding to HSA (1–6) and MSA (7).
(Figure 4a; fluorescein: t1/2 = 4.6 min; 428-d-Lys-FAM: t1/2,a
=
To verify the versatility of the albumin-binding molecule,
a second contrast agent (Gd-DTPA (Magnevist)) was con-
jugated to 428-d-Lys (428-d-Lys-bAla-DTPA-Gd). DTPA is
commonly used to chelate GdIII and represents the most
widely used contrast agent in MRI. The dissociation constant
of 428-d-Lys-b-Ala-DTPA-Gd to HSA was determined by
ITC at 378C (Kd = 3.3 mm, Figure 5a), while Gd-DTPA
displayed only little binding to HSA (see the Supporting
Information). Pharmacokinetic profiles were studied in mice
by injecting DTPA and 428-d-Lys-bAla-DTPA complexed
with 177Lu, thus allowing quantification by g-counting. Similar
to the situation encountered with the fluorescein derivatives,
the plasma concentration of DTPA-177Lu decreased rapidly
and was no longer detectable 60 min after injection, whereas
428-d-Lys-bAla-DTPA-177Lu displayed a substantially slower
biphasic pharmacokinetic profile (Figure 5b; DTPA-177Lu: t1/
2,a = 8.6 min; 428-d-Lys-bAla-DTPA-177Lu: t1/2,a = 22 min, a-
phase = 90%, t1/2,b = 408 min).
27 min, a-phase = 90%, t1/2,b = 495 min; 622-d-Lys-FAM:
t1/2,a = 5.3 min, a-phase = 98%, t1/2,b = 100 min; phenethyl-
amine-FAM: t1/2,a = 5 min, a-phase = 99.5%, t1/2,b = 53 min).
To gain functional information on the performance of 428-
d-Lys-FAM as a contrast agent, a fluorescein angiographic
analysis of the retina was performed in mice. Identical molar
amounts of fluorescein and 428-d-Lys-FAM were applied by
i.v. injection and fluorescent images of the eye fundus were
recorded at different time points, which confirmed the results
of the pharmacokinetic studies. For fluorescein, the staining
of the blood vessels was weak after one minute, thus
indicating that a substantial reduction of plasma concentra-
tion had already taken place. One hour after injection, no
fluorescein was detectable in the retinal blood vessels. By
contrast, the staining of the blood vessels with 428-d-Lys-
FAM was longer lasting and allowed the visualization of
smaller blood vessels (Figure 4b).
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Figure 3. In vitro characterization of fluorescein derivatives. a) ITC profile of HSA titrated with 428-d-Lys-FAM todetermine the Kd value at 378C.
~
!
,
*
The structure of the compound is indicated. b) FP of fluorescein ( , Kd =70 mm), phenethylamine-FAM ( , Kd =17 mm), 622-d-Lys-FAM (
*
Kd =6.6 mm), and 428-d-Lys-FAM ( , Kd =108 nm) titrated with increasing amounts of HSA at 258C. c) Band-shift assay of fluorescein,
phenethylamine-FAM, 622-d-Lys-FAM, and 428-d-Lys-FAM in the absence (À) and presence of HSA (purified protein or in human serum).
Figure 4. In vivo characterization of fluorescein derivatives. a) Pharmacokinetic studies of fluorescein (black), 428-d-Lys-FAM (blue), 622-d-Lys-
FAM (red), and phenethylamine-FAM (green) after i.v. injection in two mice each. The plasma concentration time course of 177Lu-labeled MSA is
given for comparison (a). b) Fluorescein angiography in mice. Images were recorded over 1 h after i.v. injection of 50 nmol of fluorescein (top
row) and 428-d-Lys-FAM (bottom row).
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Figure 5. Characterization of DTPA derivatives. a) Kd value of 428-d-Lys-DTPA-Gd toHSA determined by ITC at 37 8C. The structure of the
compound is indicated. b) Pharmacokinetic studies of DTPA-177Lu (filled symbols) and 428-d-Lys-bAla-DTPA-177Lu (empty symbols) after i.v.
injection in two mice each. The plasma concentration time course of 177Lu-labeled MSA (a) is given for comparison. c) Transverse MR images
of the mouse head indicating the region of interest (ROI) used to select the blood vessel. Time course of the MR signal intensity in the ROI after
injection of Gd-DTPA (left panels) and 428-d-Lys-b-Ala-DTPA-Gd (right panels).
The rapid extravasation of DTPA-Gd in comparison to
428-d-Lys-bAla-DTPA-Gd was also observed by MRI pro-
cedures following i.v. injection of the contrast agents. MRI
analysis of major blood vessels of the head in mice revealed a
slower decrease of signal intensities in those injected with
428-d-Lys-bAla-DTPA-Gd (Figure 5c).
capable of forming kinetically stable complexes with both
HSA and MSA. The use of 428-d-Lys as a portable albumin
binder was shown to improve the in vivo circulatory half-life
of agents of pharmaceutical interest by more than 100-fold.
428-d-Lys derivatives of fluorescein and of metal ion–DTPA
complexes exhibited promising in vivo properties and are
likely to represent superior blood-pool contrast agents for
clinical applications by increasing the measurement time,
In summary, we have discovered a novel class of albumin-
binding derivatives using DNA-encoded chemical libraries
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Keywords: compound libraries · imaging agents ·
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