Transcript Abstract - University of Illinois at Urbana–Champaign

http://nano.ece.uiuc.edu
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A Method for Identifying Small Molecule Aggregators
Using Photonic Crystal Biosensor Microplates
1Leo
[email protected]
L. Chan, 2Erich Lidstone, 3Kristin E. Finch, 3James T. Heeres, 3,4Paul J. Hergenrother, and 1Brian T. Cunningham
University of Illinois at Urbana-Champaign
1Dept. of Electrical and Computer Engineering, Nano Sensors Group, 2Department of Bioengineering, 3Department of Biochemistry, 4Department of Chemistry
4. SMALL MOLECULE LIBRARY
1. ABSTRACT
Small molecules identified through high-throughput screens are an essential element in
pharmaceutical discovery programs. It is now recognized that a substantial fraction of small
molecules exhibit aggregating behavior leading to false positive results in many screening assays,
typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify
compounds within a screening library that aggregate can streamline the screening process by
eliminating unsuitable molecules from further consideration. In this work we show that photonic
crystal (PC) optical biosensor microplate technology can be utilized to identify and quantify small
molecule aggregation. A group of aggregators and nonaggregators were tested using the PC
technology, and measurements were compared with those gathered by three alternative methods:
dynamic light scattering (DLS), an a-chymotrypsin colorimetric assay, and scanning electron
microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual
observation using SEM, and were in general agreement with the a-chymotrypsin assay. DLS
measurements, in contrast, demonstrated inconsistent readings for many compounds that are found
to form aggregates in shapes very different from the classical spherical particles assumed in DLS
modeling. As a label-free detection method, the PC biosensor aggregation assay is simple to
implement and provides a quantitative direct measurement of the mass density of material adsorbed
to the transducer surface, while the microplate-based sensor format enables compatibility with highthroughput automated liquid handling methods used in pharmaceutical screening.
2. BACKGROUND
1.0
• The compounds selected vary in mass, functional groups, and structure
• All molecules were selected from an in-house small molecule library.
5. DYNAMIC LIGHT SCATTERING (DLS) SURVEY OF COMPOUND LIBRARY
Reflection Spectrum
PWV shift
0.8
Reflectance
SM Aggregate
Drug-like compound library
• Congo Red serves as a control aggregator.
• Biotin is a control nonaggregator.
7. PHOTONIC CRYSTAL BIOSENSOR ASSAY
Particle Size
Fit Error
0.6
100 nm
bead
control
0.4
0.2
100 nm
bead
control
Wavelength (nm)
Cross-section schematic of the sensor
with small molecule aggregate
Peak wavelength value (PWV)
shift as a result of aggregation
• Label-free photonic crystal optical biosensors (SRU Biosystems) have recently been demonstrated
as a highly sensitive method for performing a wide variety of biochemical and cell-based assays
• The sensors are incorporated into SBS standard format 96, 384, and 1536-well microplates
• The device structure is designed to reflect only a narrow band of wavelengths when illuminated with
white light at normal incidence
• Positive shifts of the reflected Peak Wavelength Value (PWV) indicate the adsorption of detected
material on the sensor surface
• Readout instrument
• The sensor is illuminated at normal incidence and reflects a
narrow band wavelengths
• Reflected light is collected through a detection fiber, and
guided into a spectrometer
• Operation
• Reflected PWV is collected over a period of hours
• The collected PWV is used to generate a kinetic and
endpoint plots of biomolecular binding events
• High throughput screen (HTS) for aggregators
• Select 22 compound library including aggregators and
nonaggregators
• Screen for compound aggregation on sensor surface
• Confirm with existing aggregation detection assays
• Test compounds using dynamic light scattering (DLS)
• Confirm results with a-chymotrypsin inhibition assay
• Verify aggregation by visual inspection using scanning
electron microscopy (SEM)
•Particle size is determined using Mie Theory assumptions about the light scattered by sample particles in
solution. Particles are assumed to be spherical and uniform in size.
•Fit error is highly variable, as aggregates can, in fact, form in shapes including irregular non-uniform clumps, thin
sheets, and fibrous tendrils.
6. a-CHYMOTRYPSIN BASED INHIBITION ASSAY
3. INSTRUMENTATION & METHOD
The reflected narrow band of light is measured by a
spectrophotometer; shifts in the peak wavelength value (PWV )
indicate binding events on the sensor surface.
SM Addition
Wash
Step
SM Addition
Wash
Step
Non-Aggregator
SA-coated
biosensor
Aggregator
The streptavidin-coated biosensor is incubated with the suspected
aggregator for a period of hours. PWV is monitored throughout the
process.
Congo Red
Kinetic Profile
Biotin
Buffer
Endpoint Read
1. a-chymotrypsin cleaves
succinyl-AAPF-PNA to produce
an increase in absorbance at a
wavelength of 405 nm.
2. Reaction rate is determined by
examining the linear portion of
the data set (approx. 10 min)
3. Decreases in reaction rate
correspond with inhibitory
activity of compounds in
solution with reaction mixture
4. Increases in reaction rate may
be due to the presence of
spectral overlap in the sample
compound
5. Several compounds were
identified as promiscuous
inhibitors by this assay
Congo Red
Biotin
Buffer
8. PHOTONIC CRYSTAL BIOSENSOR VISUALIZATION and
SCANNING ELECTRON MICROSCOPY CONFIRMATION
The PWV shift data
shown above can be
visualized using another
detection instrument
SEM shows aggregate formation for compounds
indicated by photonic crystal assay
Compounds 8 and 19
were indicated as
aggregators by the PC
assay, and show
increased PWV across
the sensor surface
0.0
840 850 860 870 880 890 900
Photonic crystal sensor incorporated
into multi-well microplate
1. Samples are incubated in microplate wells over a
period of hours
2. During this time, peak wavelength (PWV) shift
data is collected using the BINDTM Reader (SRU
Biosystems, Woburn MA, USA)
3. After the incubation period, the sensor is washed
with buffer to reduce background noise
4. Endpoint data is collected after this wash, and is
useful for evaluating a number of compounds at a
glance
5. Kinetic data gives more information about the rate
at which the compound aggregates on the sensor
6. A steady rise in PWV indicates aggregator
characteristics, as does a sharp PWV increase
7. Results were comparable to those obtained using
the a-chymotrypsin inhibition assay
8. More time-efficient from a user standpoint
9. Direct assessment of the physical properties of
the small molecules, no dependence on an
enzymatic reaction
Kinetic
binding
profiles
mirrors that
of known
aggregator
control CR
The BINDTM imaging instrument (SRU
Biosystems, Woburn USA) uses free-space
optics to resolve binding events on the sensor
surface at a resolution of 22.6 mm/pixel
9. DETERGENT INHIBITION OF AGGREGATION
•Detergent has been shown to decrease the
amount of aggregation exhibited by known
promiscuous inhibitors
•The PC aggregation detection experiment was
repeated in the presence of detergent to confirm
that this decrease in aggregation could be
observed using the PC biosensor
•When the experiment was run with 0.05% (v/v)
Tween-20, marked decreases in PWV shift were
observed on the PC biosensor
•The concentration dependence of the detergent
effect is illustrated over a range of detergent
concentrations (0-5%, v/v) with Congo Red
10. ACKNOWLEDGEMENTS
We are grateful to the National Institutes of Health (R01 CA118562) for funding
this work. The authors thankfully acknowledge SRU Biosystems for providing
the photonic crystal biosensor microplates. One of the authors (BTC) is a
founder of SRU Biosystems. Co-authors L. L. Chan and E. A. Lidstone
contributed equally to this work.