21 research outputs found
Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted Ī²āSecretase Using Single-Molecule Imaging
Inhibition-in-solution assays (ISA)
employing surface-based biosensors
such as surface plasmon resonance (SPR) are an effective screening
approach in drug discovery. However, analysis of potent binders remains
a significant hurdle due to limited sensitivity and accompanied depletion
of the inhibiting compounds due to high protein concentrations needed
for detectable binding signals. To overcome this limitation, we explored
a microscopy-based single-molecule ISA compatible with liposome-reconstituted
membrane proteins. Using a set of validated small molecule inhibitors
against Ī²-secretase 1 (BACE1), the assay was benchmarked with
respect to sensitivity and dynamic range against SPR. We demonstrate
that the dynamic range of measurable affinities is greatly extended
by more than 2 orders of magnitude as compared to SPR, thus facilitating
measurements of highly potent (<i>K</i><sub>d</sub> <
nM) compounds
Time-Resolved Surface-Enhanced Ellipsometric Contrast Imaging for Label-Free Analysis of Biomolecular Recognition Reactions on Glycolipid Domains
We have applied surface-enhanced ellipsometry contrast
(SEEC) imaging
for time-resolved label-free visualization of biomolecular recognition
events on spatially heterogeneous supported lipid bilayers (SLB).
Using a conventional inverted microscope equipped with total internal
reflection (TIR) illumination, biomolecular binding events were monitored
with a lateral resolution near the optical diffraction limit at an
acquisition rate of ā¼1 Hz with a sensitivity in terms of surface
coverage of ā¼1 ng/cm<sup>2</sup>. Despite the significant improvement
in spatial resolution compared to alternative label-free surface-based
imaging technologies, the sensitivity remains competitive with surface
plasmon resonance (SPR) imaging and imaging ellipsometry. The potential
of the technique to discriminate local differences in protein binding
kinetics was demonstrated by time-resolved imaging of anti-GalCer
antibodies binding to phase-separated lipid bilayers consisting of
phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher
antibody binding capacity was observed on the GalCer-diluted fluid
region in comparison to the GalCer-rich gel phase domains. This observation
is tentatively attributed to differences in the presentation of the
GalCer epitope in the two phases, resulting in differences in availability
of the ligand for antibody binding. The complementary information
obtained by swiftly switching between SEEC and fluorescence (including
TIR fluorescence) imaging modes was used to support the data interpretation.
The simplicity and generic applicability of the concept is discussed
in terms of microfluidic applications
Time-Resolved Surface-Enhanced Ellipsometric Contrast Imaging for Label-Free Analysis of Biomolecular Recognition Reactions on Glycolipid Domains
We have applied surface-enhanced ellipsometry contrast
(SEEC) imaging
for time-resolved label-free visualization of biomolecular recognition
events on spatially heterogeneous supported lipid bilayers (SLB).
Using a conventional inverted microscope equipped with total internal
reflection (TIR) illumination, biomolecular binding events were monitored
with a lateral resolution near the optical diffraction limit at an
acquisition rate of ā¼1 Hz with a sensitivity in terms of surface
coverage of ā¼1 ng/cm<sup>2</sup>. Despite the significant improvement
in spatial resolution compared to alternative label-free surface-based
imaging technologies, the sensitivity remains competitive with surface
plasmon resonance (SPR) imaging and imaging ellipsometry. The potential
of the technique to discriminate local differences in protein binding
kinetics was demonstrated by time-resolved imaging of anti-GalCer
antibodies binding to phase-separated lipid bilayers consisting of
phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher
antibody binding capacity was observed on the GalCer-diluted fluid
region in comparison to the GalCer-rich gel phase domains. This observation
is tentatively attributed to differences in the presentation of the
GalCer epitope in the two phases, resulting in differences in availability
of the ligand for antibody binding. The complementary information
obtained by swiftly switching between SEEC and fluorescence (including
TIR fluorescence) imaging modes was used to support the data interpretation.
The simplicity and generic applicability of the concept is discussed
in terms of microfluidic applications
Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations
The
function of integral membrane proteins is critically dependent
on their naturally surrounding lipid membrane. Detergent-solubilized
and purified membrane proteins are therefore often reconstituted into
cell-membrane mimics and analyzed for their function with single-molecule
microscopy. Expansion of this approach toward a broad range of pharmaceutically
interesting drug targets and biomarkers however remains hampered by
the fact that these proteins have low expression levels, and that
detergent solubilization and reconstitution often cause protein conformational
changes and loss of membrane-specific cofactors, which may impair
protein function. To overcome this limitation, we here demonstrate
how antibody-modified nanoparticles can be used to achieve affinity
purification and enrichment of selected integral membrane proteins
directly from cell membrane preparations. Nanoparticles were first
bound to the ectodomain of Ī²-secretase 1 (BACE1) contained in
cell-derived membrane vesicles. In a subsequent step, these were merged
into a continuous supported membrane in a microfluidic channel. Through
the extended nanoparticle tag, a weak (ā¼fN) hydrodynamic force
could be applied, inducing directed in-membrane movement of targeted
BACE1 exclusively. This enabled selective thousand-fold enrichment
of the targeted membrane protein while preserving a natural lipid
environment. In addition, nanoparticle-targeting also enabled simultaneous
tracking analysis of each individual manipulated protein, revealing
how their mobility changed when moved from one lipid environment to
another. We therefore believe this approach will be particularly useful
for separation in-line with single-molecule analysis, eventually opening
up for membrane-protein sorting devices analogous to fluorescence-activated
cell sorting
Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations
The
function of integral membrane proteins is critically dependent
on their naturally surrounding lipid membrane. Detergent-solubilized
and purified membrane proteins are therefore often reconstituted into
cell-membrane mimics and analyzed for their function with single-molecule
microscopy. Expansion of this approach toward a broad range of pharmaceutically
interesting drug targets and biomarkers however remains hampered by
the fact that these proteins have low expression levels, and that
detergent solubilization and reconstitution often cause protein conformational
changes and loss of membrane-specific cofactors, which may impair
protein function. To overcome this limitation, we here demonstrate
how antibody-modified nanoparticles can be used to achieve affinity
purification and enrichment of selected integral membrane proteins
directly from cell membrane preparations. Nanoparticles were first
bound to the ectodomain of Ī²-secretase 1 (BACE1) contained in
cell-derived membrane vesicles. In a subsequent step, these were merged
into a continuous supported membrane in a microfluidic channel. Through
the extended nanoparticle tag, a weak (ā¼fN) hydrodynamic force
could be applied, inducing directed in-membrane movement of targeted
BACE1 exclusively. This enabled selective thousand-fold enrichment
of the targeted membrane protein while preserving a natural lipid
environment. In addition, nanoparticle-targeting also enabled simultaneous
tracking analysis of each individual manipulated protein, revealing
how their mobility changed when moved from one lipid environment to
another. We therefore believe this approach will be particularly useful
for separation in-line with single-molecule analysis, eventually opening
up for membrane-protein sorting devices analogous to fluorescence-activated
cell sorting
Simultaneous Imaging of AmyloidāĪ² and Lipids in Brain Tissue Using Antibody-Coupled Liposomes and Time-of-Flight Secondary Ion Mass Spectrometry
The
spatial localization of amyloid-Ī² peptide deposits, the
major component of senile plaques in Alzheimerās disease (AD),
was mapped in transgenic AD mouse brains using time-of-flight secondary
ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous
molecules that cannot be mapped using conventional immunohistochemistry
imaging, including phospholipids, cholesterol and sulfatides. Whereas
the endogenous lipids were detected directly, the amyloid-Ī²
deposits, which cannot be detected as intact entities with ToF-SIMS
because of extensive ion-induced fragmentation, were identified by
specific binding of deuterated liposomes to antibodies directed against
amyloid-Ī². Comparative investigation of the amyloid-Ī²
deposits using conventional immunohistochemistry and fluorescence
microscopy suggests similar sensitivity but a more surface-confined
identification due to the shallow penetration depth of the ToF-SIMS
signal. The recorded ToF-SIMS images thus display the localization
of lipids and amyloid-Ī² in a narrow (ā¼10 nm) two-dimensional
plane at the tissue surface. As compared to a frozen nontreated tissue
sample, the liposome preparation protocol generally increased the
signal intensity of endogenous lipids, likely caused by matrix effects
associated with the removal of salts, but no severe effects on the
tissue integrity and the spatial distribution of lipids were observed
with ToF-SIMS or scanning electron microscopy (SEM). This method may
provide an important extension to conventional tissue imaging techniques
to investigate the complex interplay of different kinds of molecules
in neurodegenerative diseases, in the same specimen. However, limitations
in target accessibility of the liposomes as well as unspecific binding
need further consideration
Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3Ļ
The interaction between the adapter protein 14-3-3Ļ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3Ļ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3Ļ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding
Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion
<div><p>Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 <i>in vitro</i> potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR <i>in vitro</i> potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na<sup>+</sup>/K<sup>+</sup> ratio, while eplerenone increased the Na<sup>+</sup>/K<sup>+</sup> ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.</p></div
Selective and Bioavailable HDAC6 2ā(Difluoromethyl)-1,3,4-oxadiazole Substrate Inhibitors and Modeling of Their Bioactivation Mechanism
Histone deacetylase 6 (HDAC6) is a unique member of the
HDAC family
mainly targeting cytosolic nonĀhistone substrates, such as Ī±-tubulin,
cortactin, and heat shock protein 90 to regulate cell proliferation,
metastasis, invasion, and mitosis in tumors. We describe the identification
and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles
(DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing
structureāactivity relationships and performing quantum mechanical
calculations of the HDAC6 catalytic mechanism, we show that potent
oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism
for the bioactivation. We also observe that the inherent electrophilicity
of the oxadiazoles makes them prone to degradation in water solution
and the generation of potentially toxic products cannot be ruled out,
limiting the developability for chronic diseases. However, the oxadiazoles
demonstrate high oral bioavailability and low in vivo clearance and
are excellent tools for studying the role of HDAC6 in vitro and in
vivo in rats and mice
AZD9977 and eplerenone activities in reporter gene assays.
<p>Concentration response curves of AZD9977 and eplerenone tested in a reporter gene assay in (a) presence or (b) absence of 0.1 nM aldosterone. <i>n</i> = 4, average Ā± SD.</p