Human serum albumin (HSA) plays a fundamental
role in the transport of drugs, metabolites, and endogenous
ligands. Binding to HSA controls the free,
active concentration of a drug; provides a reservoir for
a long duration of action; and ultimately affects drug
absorption, distribution, metabolism, and excretion
(ADME), making HSA binding a key parameter in
evaluating candidate lead compounds during drug
design.1 A broad range of approaches has been used
to study drug–protein binding, including equilibrium
dialysis, filtration, centrifugation, and chromatography.
However, the resolution, reproducibility, and
throughput of these methods are limiting.
Surface plasmon resonance (SPR) technology offers
several advantages over traditional methods for
analyzing drug interactions with proteins. Neither
radiochemical nor fluorescent labels are required to
provide real-time data on the affinity, specificity, and
kinetics of drug–protein interactions. This means that
all compounds can be analyzed, and binding measurements
are not limited only to those proteins that can
be derivatized with label moieties. SPR is also a highly
sensitive technique that requires much less sample
than traditional techniques, while enabling the detection
of picomolar levels of proteins and molecules
as small as a few hundred daltons, thus providing
detailed information about drug–protein interactions.
Since SPR provides real-time measurements, it delivers
kinetic data on both complex formation and dissociation,
unlike equilibrium-based methods. Finally,
SPR can be highly automated for high throughput.
While SPR has been used to provide kinetic data on
HSA–drug interactions,2,3 until recently, most SPR
experiments for the determination of kinetic rate
constants could only be run sequentially, limiting
the throughput in the drug development process. An
alternative approach utilizes the ProteOn™ XPR36
protein interaction array system and a One-shot
Kinetics™ approach4 (Bio-Rad Laboratories, Hercules,
CA). The system is a multiplexed SPR device
that integrates a 6 × 6 interaction array for the analysis
of up to six ligands with panels of up to six analytes,
producing 36 data points in a single injection
(Figure 1). Multiplexing improves and expands the
capabilities of traditional SPR technology and work
flow by enabling multiple quantitative protein binding
experiments in parallel. Because multiple conditions
can be tested in parallel, robust kinetic analysis
of an analyte concentration series can be handled
in one experiment. This one-shot parallel approach
generates a complete kinetic profile of a biomolecular
interaction without the need for regeneration, in
one experiment, using a single sensor chip.
Figure 1 - ProteOn XPR36 system 6 × 6 ligand–analyte
interaction array. a) HSA is immobilized in all six vertical
channels. b) Six different concentrations of a drug are injected
into the six horizontal channels. c) Detail of a single HSA–drug
interaction spot (green).
This article describes a study designed to demonstrate
the utility of the ProteOn XPR36 system for
the rapid and accurate determination of binding
parameters of small-molecule drugs to HSA. The
binding of the following drugs, which have different
molecular masses and affinities for HSA,
was studied: corticosterone, a steroid hormone;
naproxen and phenylbutazone, nonsteroidal antiinflammatory
drugs (NSAIDs); quinine, used to
prevent and treat malaria; and warfarin, an anticoagulant
with well-characterized binding properties
to HSA. The binding kinetics of dansylamide, a
fluorescent probe used in traditional HSA binding
methods, were also measured.
Real-time, label-free kinetic analysis of HSA–drug interactions
For SPR analysis, the ligand—in this case HSA—is
covalently bound to a biosensor surface, and the
binding partner—in this case a small-molecule
drug—is injected across the surface of the sensor
chip. As a binding event occurs, SPR detectors monitor
the increase in refractive index near the sensor
surface, leading to a shift in the SPR angle. When
the injection solution is switched from analyte (drug)
to buffer, the protein complex on the sensor surface
dissociates, and the SPR angle shifts back. The shift
in the SPR angle is measured in response units (RU)
and recorded as a function of time in the form of
a sensorgram (Figure 2). These changes in refractive
index are proportional to mass changes at the
surface of the sensor chip. The sensorgram thus displays
the time course of binding of the drug to HSA.
Optimized association, dissociation, and equilibrium
constants are calculated by fitting response data for
a range of analyte concentrations under identical
reaction conditions to a computational model. In
this case, drugs bind and dissociate from HSA very
rapidly, as shown by the “square wave” shape of the
binding curves.
Figure 2 - Representative sensorgram from SPR analysis
of drug binding to HSA. Each of the six curves represents one
of the six horizontal channels on the ProteOn XPR36 system,
each of which contains a different concentration of the drug
corticosterone. This sensorgram has a “square wave" shape due to the rapid binding and dissociation kinetics.
Drug affinities for HSA
The kinetics of six drugs can be measured using a
single ProteOn GLM sensor chip to which HSA
is immobilized in six vertical channels (Figure 1).
Different concentrations of one drug can then be
injected into the six horizontal channels simultaneously,
thus obtaining a complete kinetic profile
of the interaction of this drug with HSA, in a
single injection. Six different concentrations of
the second drug are then injected in the horizontal
channels. No regeneration is required between drug
injections, since the dissociation is very rapid, and
the signal drops to zero baseline. Figure 3a shows the
sensorgrams for each of six drugs tested, each curve
(sensorgram) representing a different drug concentration.
For the data shown in Figure 3, some drugs
were tested at more than six concentrations for
comparison to data reported in the literature. The
signal at equilibrium was then plotted versus the
drug concentration in order to determine the equilibrium
constants (Figure 3b). From this plot, the
equilibrium constant, KD, is fitted using the following
equation:
where Req is the signal at equilibrium for each concentration,
Rmax is the maximal signal when all the albumin binding sites are populated, and A is the analyte concentration.
For the high-affinity compounds, a different
model that includes two binding sites was used.3
Figure 3 - Drug-binding affinities to human serum albumin. a) Sensorgrams from binding kinetics studies of six drugs, at various
concentrations, to HSA. b) Plot of signal at equilibrium versus the drug concentrations, to determine affinities.
The KD values calculated using the equations for the
fitting are listed in Table 1, which also includes the literature
values for these drugs determined by conventional
SPR methods. The affinities vary as much as 1000-fold
from a weakly binding drug such as quinine to a very
tightly bound drug such as naproxen. Equilibrium binding
data have traditionally been stated as percent bound
values, which were originally calculated from equilibrium
dialysis experiments. The KD values in Table 1 were
used to calculate percent bound values as described in
Ref. 2, and are also listed. It has been shown previously
that affinity values measured by SPR correlate well with
those determined using traditional techniques.3 While
the values determined using the ProteOn XPR36 system
agree quite well with those from the literature, the 6 × 6
interaction array and One-shot Kinetics approach of the
system produce the same amount of data in about one-tenth
the analysis time required by the SPR systems used
to produce the values found in the literature.
Conclusion
It is advantageous to the drug discovery process to
gather as much information about a candidate chemical compound as early in the process as possible,
driving the need for high-throughput methods for
analyzing HSA–drug interactions. While SPR is a
high-resolution method for studying drug binding to
HSA, until recently, its throughput was limited by the
sequential approach used to perform binding analyses.
These systems utilized a single injection needle, which
meant that each concentration of each drug had to be
injected sequentially. Since several concentrations of
each drug are required to measure binding affinities,
and at least 10–20 compounds may be tested in each
study, this sequential injection approach greatly limited
the throughput of SPR methods.
The ProteOn XPR36 system features six injection
needles and the ability to perform injections
in both the horizontal and vertical orientations of
the sensor chip. This means that HSA and other
plasma proteins can be bound to the chip in six vertical
channels, and the sensor chip then rotated, so
that different concentrations of a drug candidate
compound can be injected across each of the plasma
protein channels simultaneously, allowing robust
kinetic analysis in one injection (One-shot Kinetics
method). Through regeneration, multiple drug
candidates can be screened using the same chip. The
end result is that the ProteOn XPR36 system can
significantly reduce the experimental time from days
to hours, while generating the same amount of data
as other SPR systems. With this significant increase
in throughput, SPR is now a more valuable tool that
can better meet the needs of drug discovery for rapid
screening and accurate characterization of the affinity
of chemical compounds for plasma proteins.
References
- Bertucci, C.; Domenici, E. Reversible and covalent binding of drugs to human serum albumin: methodological approaches and physiological relevance. Curr. Med. Chem. 2002, 9, 1463–81.
- Rich, R.L.; Day, Y.S.N.; Morton, T.A.; Myszka, D.G. High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using Biacore. Anal. Biochem.2001, 296, 197–207.
- Day, Y.S.N.; Myszka, D.G. Characterizing a drug’s primary binding site on albumin. J. Pharm. Sci.2003, 92, 333–43.
- Bravman, T.; Bronner, V.; Lavie, K.; Notcovich, A.; Papalia, G.A.; Myszka, D.G. Exploring “One-shot” Kinetics and small molecule analysis using the ProteOn XPR36 array biosensor. Anal. Biochem.2006, 358, 281–8.
Ms. Bronner and Mr. Nahshol are ProteOn Applications Scientists,
and Dr. Bravman is a ProteOn Applications Manager,
Bio-Rad Laboratories, Life Science Group, 2000 Alfred Nobel
Dr., Hercules, CA 94547, U.S.A.; tel.: 800-424-6723; fax:
800-849-2289; e-mail: [email protected].