Resolving Binding Events on the Multifunctional Human Serum Albumin

Article abstract of DOI:10.1002/cmdc.202000069

Physiological processes rely on initial recognition events between cellular components and other molecules or modalities. Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding- and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X-ray crystallography and time-resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin-binding drugs and is therefore well-suited for prediction of drug-drug and drug-food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.

Full text of DOI:10.1002/cmdc.202000069

Accepted Article
Title: Resolving Binding Events on the Multifunctional Human Serum
Albumin
Authors: Stefan Petry, Lea Wenskowsky, Michael Wagner, Johannes
Reusch, Herman Schreuder, Hans Matter, and Till Opatz
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.202000069
Link to VoR: http://dx.doi.org/10.1002/cmdc.202000069
A Journal of
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ChemMedChem
10.1002/cmdc.202000069
COMMUNICATION
Resolving Binding Events on the Multifunctional Human Serum
Albumin
[
b]
[a]
[c]
[a]
[a]
Lea Wenskowsky, Michael Wagner, Johannes Reusch, Herman Schreuder, Hans Matter, Till
Opatz,[b]* and Stefan Matthias Petry*[a]
Abstract: Physiological processes rely on initial recognition events
between cellular components and other molecules or modalities.
Biomolecules can have multiple sites or mode of interaction with
other molecular entities, so that a resolution of the individual binding
events in terms of spatial localization as well as association and
dissociation kinetics is required for a meaningful description. Here
we describe a trichromatic fluorescent binding- and displacement
assay for simultaneous monitoring and localization of three individual
binding sites in the important transporter and binding protein human
serum albumin. Independent investigations of binding events by X-
ray crystallography and time-resolved dynamics measurements
sites in albumin as well as their relevance to physiology and
pharmacokinetics, we then sought to develop a rapid assay
system with optical readout. This system should allow the
simultaneous monitoring of different albumin binding sites, when
interacting with ligands. Therefore, it provides significantly more
information for understanding and optimizing these interactions
than the current “general albumin binding” assay-readout.
In addition to the previously reported 7-nitrobenzo-2-oxa-1,3-
diazole (NBD) labelled fatty acid (NBD-FA) as molecular probe,
more site-specific fluorescent ligands are then needed as
additional probes to selectively label the ibuprofen binding site in
the albumin “Sudlow-site II” and the warfarin binding site
(Sudlow-site-I) respectively. These new labels should exhibit
only minimal spectral overlap with the existing probe NBD-FA to
allow for selective optical detection and possess a similar
binding affinity. Their saturation curve should show a clear
plateau region, thus indicating specific binding to the
corresponding albumin binding site. Moreover a clear 1:1
binding stoichiometry must be revealed by Job-plots[5] (see
supplemental).
While such behavior is indicative for specific binding and
release, effects like quenching, reorganization or rebinding
cannot be ultimately ruled out.
Finally, competition experiments of newly identified
molecular probes with known albumin site-specific binders need
to be performed to clearly assign these probes to known albumin
binding sites. These new probe molecules should be able to
release the known fluorescent ligands in a concentration-
dependent fashion.
(switchSENSE technology) confirm the validity of the assay, the
localization of binding sites and furthermore reveal conformational
changes associated with ligand binding. The described assay
system allows for the detailed characterization of albumin-binding
drugs and is therefore well-suited for prediction of drug-drug and
drug-food interactions. Moreover, conformational changes, usually
associated with binding events, can also be analyzed.
Human serum albumin (HSA) is an abundant plasma protein,
which binds and transports hydrophobic endogenous ligands like
fatty acids. It also interacts with a wide range of drug and drug-
like molecules.[1] Several X-ray crystal structures of ligands in
complex with HSA revealed seven individual fatty acid (FA)
binding sites in this biomolecule.[2]
Earlier, we addressed the specificity of these HSA binding
sites with respect to ligand binding. This lead to the identification
of a single high affinity fatty acid binding site by introducing and
applying the novel molecular probe NDB-FA.[3]
Under physiological conditions, an albumin molecule
[
4]
accommodates only 0.1–2 molecules of fatty acids. In contrast
to standard crystallization conditions using an excess of free
fatty acids to stabilize the albumin conformation, we obtained an
X-ray crystal structure (PDB 6ezq, resolution 2.4 Å) under
Recently, several publications reported syntheses and
applications of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene) dyes exhibiting similar “switch-on” characteristics
conditions close to those employed for K
D
determination for
[6]
upon binding as observed for NBD-FA and other NBD-labelled
albumin complexes (NBD-FA:HSA 6:1) in our previous study.[3]
To obtain more insight into the gradual occupancy of binding
[3, 7]
ligands.
BODIPY derivatives cover a wide range of
wavelengths of their fluorescence maxima which strongly
depend on their chemical substitution patterns. They have been
[
6]
described as polarity-based fluorescence sensors, as sensors
for hydrophobic protein surfaces,[ as products of diversity-
8]
[
[
[
a]
b]
c]
Dr. M. Wagner, Dr. H. Schreuder, Dr. H. Matter, Dr. S. M. Petry
Sanofi-Aventis Deutschland GmbH,
Industriepark Höchst, 65926 Frankfurt am Main, Germany
E-mail: StefanMatthias.Petry@sanofi.com
Dr. L. Wenskowsky, Prof. Dr. T. Opatz
Insitute of Organic Chemistry, Johannes Gutenberg-University
Duesbergweg 10–14, 55128 Mainz, Germany
E-mail: opatz@uni-mainz.de
oriented library synthesis for unbiased screening[9] of
hydrophobic protein surfaces or in combination with click-
chemistry.[10] Kim et al.[11] recently described NIR-labelling with
8-amino BODIPY derivatives, further increasing the application
range of these dyes.
In several reports,[6, 8-11] the affinity of BODIPY derivatives to
J. Reusch
bovine serum albumin (BSA) has been described. Er et al.[12]
have published a mixture of two fluorescent dyes for dual drug
site mapping on HSA using a BODIPY derivative selected from a
Dynamic Biosensors GmbH
Lochhamer Straße 15, 82152 Martinsried/Planegg, Germany
Supporting information for this article is given via a link at the end of
the document
product library as
a label for Sudlow-site II. Competition
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ChemMedChem
10.1002/cmdc.202000069
COMMUNICATION
6
15 nm. λem = 690 nm. D) Competition experiment of ibuprofen (1) against
experiments with ibuprofen proved the identity of the binding site.
However, these authors chose dansylamide as the second label
which we found to exhibit a 4:1[3] instead of the required 1:1
binding stoichiometry, which is also in agreement with results
from X-ray crystallography by Curry[13] et al. These studies led
us to the design and synthesis[12, 14] of a small, directed library of
BODIPY derivatives 5a–c for targeting Sudlow-site II (Scheme 1,
see Supplemental Experimental Procedures).
dimethylamino-BODIPY 5a on HSA (12.5 µM each). λexc = 615 nm. λem = 690
nm.
We then intended to use a third fluorescent ligand in order to
directly label the warfarin binding site (Sudlow-site I) as well.
Again, binding affinity in the same range as for NBD-FA and 5a
as well as a 1:1 binding stoichiometry were the selection criteria
for the optimal ligand.
Scheme 1. Synthesis of BODIPY derivatives 5a–c.
First, we investigated these compounds for unbiased HSA
Scheme 2. Synthesis of warfarin derivatives 8a–c.
binding. Saturation curves were recorded and the respective K
values were determined as described earlier.[3]
D
Compounds with suitable fluorescence emission wavelength
that showed clear saturation in a 1:1 HSA:BODIPY stoichiometry
were further investigated in competition experiments with
ibuprofen (1). BODIPY derivative 5a was finally selected as an
ibuprofen (1) competitive probe showing the desired 1:1 release
stoichiometry (Figure 1C).
Warfarin (2) itself showed suitable fluorescence intensity but
unfortunately shows absorption at the excitation wavelength of
dimethylamino-BODIPY 5a (Figure S5). Thus, a set of coumarin
derivatives 8a–c was synthesized[15] and screened as described
for compound 5a (Scheme 2).The warfarin derivative 8a fulfilled
the aforementioned criteria (Figure 2A–D). Furthermore, its
fluorescence emission was substantially different from those of
NBD-FA and BODIPY 5a, so that the simultaneous selective
quantification of all three ligands should be possible in an
unprecedented three-color fluorescent assay.
A
B
1.0
0.5
0.0
24
1
6
8
0
A
B
1.0
45
3
1
0
5
0
0
.5
450
550
650
0
25
50

(nm)
c (µM)
C
0.0
300
1
2
8
4
0
D
2
1
4
6
8
0
400
500
600
0
100
200
300
400

(nm)
c (µM)
C
D
1
2
2
1
4
6
8
0
8
4
0
0.0
0.5
1.0
0
50
100
150
200

HSA
c (µM)
0.0
0.5
1.0
Figure 1. A) Absorption and emission spectra (both normalized) of
dimethylamino-BODIPY 5a (40 µM ethanol). λexc = 625 nm. B) Titration of
0
50
100
150
200

HSA
c (µM)
dimethylamino-BODIPY 5a (0–50 µM) to HSA (12.5 µM) (K
0.9176). λexc = 615 nm. λem = 690 nm. C) Determination of the binding
stoichiometry of dimethylamino-BODIPY 5a to HSA (12.5 µM, Job plot). λexc
D
= 4.6 ± 0.8 µM. R²
=
Figure 2. A) Absorption and emission spectra (both normalized) of warfarin
derivative 8a (40 µM ethanol). λexc = 340 nm. B) Titration of warfarin derivative
=
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ChemMedChem
10.1002/cmdc.202000069
COMMUNICATION
D
8a (0–400 µM) to HSA (25 µM) (K = 26.5 ± 3.0 µM. R² = 0.9607). λexc = 330
nm. λem = 384 nm. C) Determination of the binding stoichiometry of warfarin
derivative 8a to HSA (25 µM, Job plot). λexc = 330 nm. λem = 384 nm. D)
Competition experiment of iophenoxic acid (3) against warfarin derivative 8a
on HSA (25 µM each). λexc = 330 nm. λem = 384 nm.
6
15
435
1.0
0
.5 330
D
Since we have determined the K values of our probes
0.0
exclusively by titration experiments, we considered the
independent validation of these values with a different method.
300
400
500
600
700
 (nm)
To this end, independent characterization of all three ligands
has been performed using the switchSENSE technology[16]
Figure 3. Absorption spectra (normalized) of warfarin derivative 8a (blue),
NBD-FA (green) and dimethylamino-BODIPY 5a (red). Excitation wavelengths
for each dye were drawn.
(Dynamic Biosensors, Munich). This fluorescence-based
biosensor allows for the biophysical characterization of
biomolecules determining kinetic rate constants of association
and dissociation (kON and kOFF), and the dissociation constant
(
K
D
)
via fluorescence proximity sensing. Furthermore,
Gratifyingly, all three labels also behaved well in the chosen
combination and in each case permitted the selective
competition in the respective binding site. Figures 4–5 show
typical displacement
switchSENSE enables the user to detect intramolecular
conformational changes by determination of the hydrodynamic
friction of surface-bound complexes using a complementary
measurement mode (molecular dynamics mode).[17]
A
B
4
5
42
Here, switchSENSE was used for K
D
determination via
kinetic rate constants and via binding equilibrium as well as
analyzing conformational changes of HSA upon binding of
different compounds (induced fit). For interaction analysis, HSA
was immobilized on a gold microelectrode via short, single
stranded DNA nanolevers using standard thiol coupling
chemistry. The complementary DNA strand, covalently attached
to the gold microelectrodes via Au-S bonds, carries a fluorescent
dye (oxazine derivative) on the top end for signal detection. For
the measurements of HSA with NBD-FA, dimethylamino-
3
0
2
7
15
1
2
12
10
0
0
0
50
100
150
200
0
50
100
150
200
c (µM)
c (µM)
C
D
47
65
40
BODIPY 5a and warfarin derivative 8a, K
micromolar range were determined (NBD-FA: K
BODIPY 5a: K = 5.2 µM, warfarin derivative 8a: K
D
values in the
D
= 27.5 µM,
D
= 25.5 µM)
2
7
D
(
Figures S8–11). In the molecular dynamics mode, HSA showed
7
6
15
8
a significant expansion upon NBD-FA binding. Due to a higher
hydrodynamic radius in solution (measured in dynamic response
units), the molecular dynamics of HSA slows down (Figure S10).
This effect was neither observed for BODIPY 5a nor for warfarin
derivative 8a (for further details see supplemental).
3
0
4
0
0
50
100
150
200
0
50
100
150
200
c (µM)
c (µM)
Figure 4. Competition experiments against NBD-FA/BODIPY 5a/coumarin 8a
(8 µM each) on HSA.  NBD-FA.  BODIPY 5a. ▪ warfarin derivative 8a. A)
ibuprofen (1). B) ketoprofen (4). C) indomethacin (5). D) iophenoxic acid (3).
To check the suitability of the chosen trichromatic dye
cocktail for simultaneous detection (for
a
graphical
representation of the fluorescent properties of all three labels,
see Figure 3), finally a mixture of 0.3 equivalents of each label
was added to a solution of HSA (25 µM). This stoichiometry
does not address differences in KD values, since this setup is
used for the localization of binding. For determination of KD we
generally use and propose independent measurements with a
single dye. Since the affinity of our labels is high enough to
ensure specific binding we could reduce the equivalents added.
We found that 0.3 equivalents of each label delivered the
highest assay sensitivity and reduces the possibility of unspecific
rebinding.
experiments. Commercial drugs were added to HSA loaded with
the trichromatic cocktail at the concentrations given. The assay
was performed in parallel in 96-well-microtiter plates, which were
analyzed with
wavelengths characteristic for the three reporter dyes (NBD-FA:
exc = 435 nm. λem = 535 nm. BODIPY 5a: λexc = 615 nm. λem
90 nm. warfarin derivative 8a: λexc = 330 nm. λem = 384 nm).
a fluorescent plate reader preset to the
λ
=
6
Ibuprofen (1), ketoprofen (4) and indomethacin (5) selectively
displaced dimethylamino-BODIPY 5a indicating selective binding
to the albumin ibuprofen binding site (Sudlow-site II) (Figures
4
A–C), Indomethacin (5) in addition to 5a also displaces the
warfarin derivative 8a.[ At high concentrations, both NSAIDs
18]
(non-steroidal anti-inflammatory drugs) 1 and 4 lead to
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ChemMedChem
10.1002/cmdc.202000069
COMMUNICATION
decreased fluorescence, indicating secondary binding sites.[19]
Iophenoxic acid (3) is competitive to coumarin derivative 8a[20] as
well as to dimethylamino-BODIPY 5a.[21]
additional interactions might be attributed to high sulfasalazine
(6) concentration during crystallization (16 mM 6, 2 mM HSA).
K
D
values for the two additional sites were estimated from the
fractional occupancies to be 0.9 mM and 3.4 mM, respectively
see Supporting Information).
A
B
4
3
1
5
45
(
0
30
5
8
15
8
4
0
4
0
0
50
100
150
200
0
50
100
150
200
c (µM)
c (µM)
Figure 5. Competition experiments against NBD-FA/BODIPY 5a/coumarin 8a
8 µM each) on HSA.  NBD-FA.  BODIPY 5a. ▪ warfarin derivative 8a. A)
(
sulfasalazine (6). B) balsalazide (7).
For sulfasalazine (6), a marketed drug for treatment of e.g.
rheumatoid arthritis, ulcerative colitis, and Crohn's disease, the
[
3,
fluorescence signal decreases for NBD-FA and coumarin 8a,
22]
which indicates release of the site-specific probe-ligands
while BODIPY 5a in (Sudlow-site II) is not released (Figure 5A).
The same behavior is observed for the structurally related drug
balsalazide (7) (Figure 5B).[3]
Thus, NBD-FA as well as coumarin 8a are effectively
competed by sulfasalazine (6). This interesting observation can
be explained by direct competition between ligand and
molecular probes in two separate albumin binding sites, by
neighboring sites, or by an allosteric binding event. The similar
shape of displacement curves for NBD-FA and warfarin
derivative 8a suggests a simultaneous release of both probes by
only a single sulfasalazine (6) molecule rather than binding of
two sulfasalazine molecules to these sites. Independent kinetic
Figure 6. X-ray crystal structure of HSA in complex with sulfasalazine (6)
PDB ID 6R7S resolution 2.2 Å). A) Binding of sulfasalazine (6) (orange
(
carbon atoms) to Sudlow-site I plus Fo-Fc omit map contoured at 3σ. Key
amino acids and water oxygen atoms are indicated. B) Binding site analysis of
the HSA-6 complex using SiteMap. Red regions indicate favorable potential H-
bond acceptor interactions; blue regions indicate favorable H-bond donor
interactions and yellow regions indicate favorable hydrophobic interactions. C)
Superposition of sulfasalazine (6) with the enantiomers of warfarin (2) from
PDB 1ha2 (S-warfarin, dark-blue) and 1h9z (R-warfarin, light-blue), both at 2.5
Å resolution.[24] D) Superposition of sulfasalazine with NBD-FA (green carbon
[
3]
analysis using switchSENSE technology revealed a K
D
of 1.31
atoms) from PDB 6ezq at 2.5 Å resolution.
µM, with a remarkably fast KON (5.76 10-1 M *s ). As expected,
-1
-1
only one binding event could be observed.
A comparable situation was described by F. Yang et al.[23]
They have observed independent binding of three different
compounds (cinnamic acid, lamivudine, indomethacin) within
this hydrophobic binding site, while binding is associated with a
conformational change of albumin.
To further investigate this interesting binding behavior, we
were able to solve an X-ray crystal structure of sulfasalazine (6)
in complex with HSA, obtained by co-crystallization. The
anisotropic crystal diffracted to 2.2 Å in the best and to 2.8 Å in
the worst direction. The structure was solved by molecular
replacement and the resulting electron density maps were clear.
Figure 6A shows experimentally determined interactions of
sulfasalazine bound to Sudlow-site I. While its location in this
binding site agrees with previous docking studies, the molecule
turned out to be flipped by 180°.[3] The salicylate carboxylate
group is engaged in hydrogen-bond interactions and a salt-
bridge to Arg257 and Ser287 side chains. The central
sulfasalazine aromatic ring is involved in a cation-π interaction to
Arg222. Both attached sulfonamide oxygen atoms interact via
bridging water molecules with either Trp214 or Lys199 side
chains. The distal pyridine aromatic ring is stacked on top of the
indole ring of Trp214 with both nitrogen atoms likely arranged
close together (distance 3.2 Å). The indole NH and the pyridine
N are interacting with bridging water molecules.
The binding site was then analyzed using SiteMap[25] (Figure
6B) to identify favorable hydrophobic (yellow), hydrogen-bond
donor (blue) and acceptor interactions (red), which are in
agreement to ligand interactions. A comparison with HSA X-ray
crystal structures with (R)- or (S)-warfarin (Figure 6C, PDB 1ha2
for (S)-warfarin and 1h9z for (R)-warfarin, both 2.5 Å resolution)
shows that the sulfasalazine salicylate and its central aromatic
ring occupy parts of the warfarin binding site. This finding
The conformation of HSA is more similar to its fatty-acid free
conformation[
18d]
(RMSD 2.5 Å to the PDB structure 1uor) than to
the fatty-acid bound structure[2a] (RMSD 5.5 Å to the myristate-
bound structure 1e7g). This might be due to our crystallization
conditions in the absence of fatty acids. In total, three molecules
of sulfasalazine (6) are bound to different HSA binding sites. As
expected, one of them is located in the Sudlow-site I with clear
electron density. Two other molecules showing lower occupancy
are bound to either HSA domain IIIb or to the interface between
domains IIIa and IIIb (see the Supporting Information). These
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ChemMedChem
10.1002/cmdc.202000069
COMMUNICATION
explains the competitive binding observed for sulfasalazine (6) Experimental Section
and warfarin (2).
A comparison with the HSA-NBD-FA complex (Figure 6D)
reveals that the pyrimidine ring overlaps with the NBD group,
both being stacked on top of the indole ring of Trp214. Also the
remaining parts of both ligands overlap, again explaining the
strong competition of both ligands for binding to HSA. Although
For experimental details, crystallographic data and further biochemical
data, see the Supporting Information.
Acknowledgements
a
significant excess of sulfasalazine (6) was used for
crystallization (vide supra), the X-ray crystal structure of its HSA
complex revealed the bridging of the fatty acid binding site and
Sudlow-site I by only a single sulfasalazine molecule.
The trichromatic ligand cocktail was then employed for the
analysis of the site-specific binding behaviour of a series of
commercial drugs. We also applied it for the analysis of
biomolecules which carry albumin-binding moieties, such as
fatty acids. As an example we describe the kinetic analysis of
We thank Dr. Michael Kurz (Frankfurt) and Dr. J. Liermann
(Mainz) for NMR spectroscopy, Dr. N. Hanold (Mainz, deceased)
for mass spectrometry, Dr. Dieter Schollmeyer (Mainz) for X-Ray
crystal structure analysis of BODIPY derivative 5a, Alexander
Liesum (Frankfurt) for help with crystallization and mounting of
the HSA crystal, Joachim Diez (Frankfurt) for data collection,
Petra Loenze (Frankfurt) for help with data processing, and Pia
Freisewinkel (Frankfurt) for competition experiments with fatty
acid modified GLP1 agonists.
HSA-binding of two new antidiabetic peptides. Table
1
summarizes the results, while respective titration curves are
given in the Supporting Information.
Keywords: Multicolor Assay • switchSENSE Technology •
kinetic investigation half-life extension • Drug interaction •
albumin binding
D
Table 1. Localization of binding site and determination of K of
fatty acid modified GLP1 agonists.
[
1]
a) K. Oettl, R. E. Stauber, Brit. J. Pharmacol. 2007, 151, 580-590; b) K.
J. Fehske, W. E. Müller, U. Wollert, Biochem. Pharmacol. 1981, 30,
[
b]
Compound
Liraglutide
Semaglutide
Duration
of action
Albumin binding site
NBD-FA[b] binding site
K
D
687-692; c) A. A. Spector, E. C. Santos, J. D. Ashbrook, J. E. Fletcher,
Ann. N. Y. Acad. Sci. 1973, 226, 247-258; d) A. A. Spector, J. Lipid Res.
1975, 16, 165-179; e) M. Fasano, S. Curry, E. Terreno, M. Galliano, G.
Fanali, P. Narciso, S. Notari, P. Ascenzi, IUBMB Life 2005, 57, 787-
796; f) G. Fanali, A. di Masi, V. Trezza, M. Marino, M. Fasano, P.
Ascenzi, Mol. Aspects Med. 2012, 33, 209-290.
Once
daily
8.9 µM
1.9 µM
Once
weekly
NBD-FA binding site
and FA3
[
2]
a) A. A. Bhattacharya, T. Grüne, S. Curry, J. Mol. Biol. 2000, 303, 721-
732; b) S. Curry, H. Mandelkow, P. Brick, N. Franks, Nat. Struct. Biol.
1998, 5, 827-835; c) I. Petitpas, T. Grüne, A. A. Bhattacharya, S. Curry,
J. Mol. Biol. 2001, 314, 955-960; d) S. Curry, Drug Metab.
[
[
a] NBD-FA: (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12 fatty acid.
b] K : dissociation constant.
D
Pharmacokinet. 2009, 24, 342-357.
[
[
3]
4]
L. Wenskowsky, H. Schreuder, V. Derdau, H. Matter, J. Volkmar, M.
Nazaré, T. Opatz, S. Petry, Angew. Chem. 2018, 130, 1056-1060.
a) D. S. Fredrickson, R. S. Gordon, J. Clin. Invest. 1958, 37, 1504-
Interestingly, these binding events were also associated with
changes in HSA’s hydrodynamic diameter.
1515; b) T. Peters Jr., All about albumin: biochemistry, genetics, and
The results obtained with the trichromatic assay are in
accordance to literature[3, 18b, 18c, 19-20, 22] and to X-ray crystal
structures of HSA-ligand complexes.[18a, 18d, 21] The advantage of
the present method over individual analysis of binding sites with
only a single fluorescent ligand at a time is the reduction of the
number of experiments required as well as of the overall protein
consumption. The simultaneous analysis of displacement
patterns also permits the rapid characterization of HSA binding
behaviour for larger (bio)-molecules displaying polyvalent
binding characteristics.
We propose this general concept for the investigation of
further biomolecules containing different binding sites. In the
case of HSA, the developed assay system is useful for the initial
characterization and differentiation of albumin-binding drugs and
is of potential relevance for the prediction of drug-drug and drug-
food interactions (in particular involving fatty acids and
cholesterol). It can also be used for nanobodies or the newly
described small molecule-based HSA-binders[26] employed to
prolong plasma half-life.
medical applications, Academic press, New York, 1995.
P. Job, Ann. Chim. Appl. 1928, 9, 113-203.
[
[
5]
6]
H. Sunahara, Y. Urano, H. Kojima, T. Nagano, J. Am. Chem. Soc. 2007,
129, 5597-5604.
[
7]
a) J. Rohacova, M. L. Marin, M. A. Miranda, J. Phys. Chem. B 2010,
114, 4710-4716; b) K. L. Diehl, M. A. Ivy, S. Rabidoux, S. M. Petry, G.
Müller, E. V. Anslyn, Proc. Natl. Acad. Sci. U.S.A. 2015, 112, E3977-
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A trichromatic fluorescent binding and
displacement assay for simultaneous
monitoring and localization of three
individual binding sites on HSA has
been developed using highly specific
binding molecules. Whereas localiza-
tion is confirmed by X-ray, kinetic data
are independently confirmed by
switchSENSE technology, revealing
important data on conformational
changes. The novel tool permits the
detailed characterization of albumin-
binding drugs and prediction of drug-
drug and drug-food interactions.
Lea Wenskowsky, Michael Wagner,
Johannes Reusch, Herman Schreuder,
Hans Matter, Till Opatz*, and Stefan
Matthias Petry*
Page No. – Page No.
Resolving Binding Events on the
Multifunctional Human Serum
Albumin
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