Small molecule:ligand interactions

Small molecule:ligand interactions
Application Note NT026
Small molecule screening unaffected by DMSO mismatch
between sample and running buffer
Anja Winter, Anna Sigurdardottir
Structural Biology Group of Prof. Sir Tom Blundell at the University of Cambridge, UK
Abstract
Fast and efficient screening of fragments and
small molecules is becoming increasingly
important in the drug-discovery process. The
detection and quantification of binding events
is hampered by the low affinity of compounds
in the early stages of drug development. In
addition, their low solubility in aqueous
buffers challenges biophysical interaction
methods,
since
fragments
and
small
molecules are often dissolved in DMSO whose
refractory index typically adversely affects
optical detection methods such as Surface
Plasmon Resonance (SPR). In this study, we
show that Surface Acoustic Wave (SAW) is
able to quantify interactions of compounds
with target proteins despite the DMSO
mismatch between sample and running buffer.
Thus, with SAW, more fragments can be
characterized accurately and further optimized
for binding, solubility and bioavailability.
In addition, their low solubility in aqueous buffers
challenges biophysical interaction methods, since
fragments and small molecules are often
dissolved in DMSO whose refractory index
typically adversely affects optical detection
methods such as Surface Plasmon Resonance
(SPR).
In contrast, the propagation of acoustic waves on
SAW biosensors is independent of the optical
properties of the solution. Therefore, the resulting
binding signals are less sensitive to DMSO
mismatches.
To test this, we measured the interaction of a
target protein and a small molecule dissolved in
up to 1 % DMSO whereas the running buffer did
not contain any DMSO.
The target protein we chose for this test is
involved in crucial protein-protein interactions
activating a trans-membrane receptor.
Introduction
Once a target protein has been identified and
qualified, huge libraries of fragments and small
molecules need to be screened to find suitable
pharmacophores and lead structures which will be
further developed and optimized in the drug
development process. Fast and efficient screening
of fragments and small molecules to a target
protein is therefore becoming increasingly
important. The detection and quantification of
binding events is hampered by the low affinity of
compounds in the early stages of drug
development.
Figure 1. Schematic representation of a protein-protein
interaction between proteins A and B. A small molecule
inhibitor binds to the protein binding interface of protein A
thereby disrupting the protein-protein interaction.
Fragment-based screening using SPR had
previously revealed a fragment hit with a steadystate binding affinity of 300 µM (SPR). This hit was
later confirmed by X-ray crystallography and
protein-observed NMR and its binding affinity was
determined at 110 µM using 2D 1H-15N
Heteronuclear Single Quantum Coherence
(HSQC) NMR spectroscopy.
Results
In contrast to other surface-based methods, no
DMSO correction was needed on Seismos NT.V*,
despite the DMSO mismatch between sample and
running buffer (Figure 2). At the beginning and at
the end of each injection, the difference in DMSO
concentration leads to blurs at the interfaces due
to the large differences in viscosity. Since SAW is
able to detect mass and conformation signals
separately, the data are not affected by this
DMSO mismatch.
Figure 2 Injections of DMSO-containing small molecules at
varying concentrations into running buffer PBS + 0.05 %
Tween. Sensograms were fitted directly using the 1:1 binding
model. Please note that at the beginning and at the end of
each injection, the difference in DMSO concentration leads to
blurs at the interfaces due to the large differences in viscosity.
The affinity constants we determined for the
protein-small molecule interaction with Seismos
NT.V are in good agreement with data obtained
from NMR and SPR measurements (see Fig. 3
and 4).
The interaction was tested with the protein being
immobilized on the chip surface. The small
molecule was present in different DMSO
concentrations and was flushed over the surface
in a running buffer without any DMSO. Despite
this mismatch, binding kinetics and the affinity
could be determined.
Figure 4: Plot of kobs vs Concentration using 1:1 fit binding
model. The 5 µM surface showed highest concentration
dependency of the kobs values and the koff and kon values were
calculated thereof with 80.76 M-1 s-1 and 0.01927 s-1,
respectively (Χ2 = 4.58 * 10-5; R = 0.912). The Kd values (Kd =
201 ± 49 µM) are in good agreement with the NMR and SPR
data. On- and off-rates could not be concluded from NMR and
SPR data.
Conclusions
The Seismos NT.V instrument is an easy to
handle device which allows for rapid determination
of affinity constants. In this study, the fragment
investigated yielded Kd values similar to results
obtained with NMR and SPR. Interestingly, the
resolution of small fragments is significantly higher
using the Seismos NT.V* instrument than with
SPR (BIAcore T100), since the DMSO mismatch
effects between sample and running buffer are
minimal using the SAW approach.
Figure 3: The Aeq vs Concentration plot is usually best suited
for analyses at high concentrations. The Kd values (Kd =
126 ± 35 µM) are in excellent agreement with the NMR data.
In contrast to optical surface-based methods,
SAW is working acoustically and is therefore not
affected by any optically active substance, such as
DMSO. Therefore, this technology also allows
interaction studies of drug fragments to membrane
receptors in close to native conditions such as
directly in membrane preparations or reconstituted
in vesicles, liposomes or micelles.
Materials and Methods
Protein immobilization
The target protein was immobilized at
concentrations of 5 µM (chip position 1), 1.25 µM
(chip position 2) and 2.5 µM (chip position 3) on a
3D CM-dextran surface of a Seismos NT.V* chip
using carboxylamide coupling. Measurements
were performed at 25 °C, at a flow rate of
30 µl/min and using PBS supplemented with
0.05 % Tween 20 as running buffer. Please note
that there was no DMSO in the running buffer.
Preparation of compounds and dilution series
Compounds were dissolved to 100 mM in DMSO
or water and tested in concentration series of
1 mM
(equivalent
to
1%
DMSO
final
concentration), 0.5 mM (0.5 % DMSO), 0.25 mM
(0.25 % DMSO), 0.125 mM (0.125 % DMSO) and
0.063 mM (0.063 % DMSO) by diluting into
running buffer.
In between injections the surface was regenerated
using 1 M NaCl ensuring that all remaining
compound was removed sufficiently.
Data analysis
Using the FitMaster® Origin-based software raw
data were cut and analyzed in three steps:
 Subtraction of a reference channel
(channel with no protein immobilized).
 Subtracting DMSO mass and subtracting
glycerol mass.
 Affinity constants and Kd values were
determined using the provided 1:1 binding
model.
Instrumentation
Surface acoustic wave biosensor, Seismos NT.V*
from NanoTemper Technologies GmbH.
*corresponding to the sam®5BLUE from SAW
Instruments GmbH
References
Jubb H., Higueruelo A. P., Winter A., Blundell T. L.
Structural biology and drug discovery for protein-protein
interactions. Trends Pharmacol. Sci. (2012) 33(5): 241 -248.
Winter A., Higueruelo A. P., Marsh M., Sigurdardottir A.,
Pitt W. R., Blundell T. L. Biophysical and computational
fragment-based approaches to targeting protein-protein
interactions: applications in structure-guided drug discovery. Q.
Rev. Biophys. (2012) 45 (4): 383 – 426.
© 2014 NanoTemper Technologies GmbH

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