A General Method for Screening of Protein-DNA

A General Method for Screening of Protein-DNA Interactions using SRU BIND Optical Biosensor Microplates
L. Chan, 2Maria F. Pineda, 2Sherine George, 2Erich Lidstone, 3James T. Heeres, 3,4Paul J. Hergenrother, and 1Brian T. Cunningham
[email protected]
University of Illinois at Urbana-Champaign
of Electrical and Computer Engineering, Nano Sensors Group
2Department of Bioengineering, 3Department of Biochemistry, 4Department of Chemistry
[email protected]
1Dept.
1. ABSTRACT
4. AIF MEDIATED CELL DEATH
Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA
damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be
powerful tools for investigation of these biological processes, and such compounds would have great
potential as therapeutics. Unfortunately, there are few methods available for the rapid identification
of compounds that disrupt protein-DNA interactions. Here we show that photonic crystal (PC)
technology can be utilized to detect protein-DNA interactions, and can be used in a high-throughput
screening mode to identify compounds that prevent protein-DNA binding. The PC technology is used
to detect binding between protein-DNA interactions that are DNA sequence-dependent (the bacterial
toxin-antitoxin system MazEF), and those that are DNA sequence-independent (the human
Apoptosis Inducing Factor (AIF)). This method was expanded into a high throughput screening
protocol that screened ~200,000 small molecules, where five chemical compounds were found to
inhibit the AIF-DNA interaction, and dose response curves were also obtained for the compounds.
The generality and simplicity of the photonic crystal method should enable this technology to find
broad utility for identification of other compounds that inhibit a variety of protein-DNA binding.
PAR = Poly-ADP Ribose
PARP = PAR Polymerase
PARG = PAR Glycohydrolase
Reflection Spectrum
Mitochondria
PWV shift
140
A Hit
O
OH
O
HO
HO
120
O
OH
100
% Inhibition
80
Positive
Control
(Free DNA)
100%
Inhibition
60
40
20
ML14K3
334I6
ML11C3
ML11D5
245A3
331I14
214G14
217G14
230M08
ML26N20
335P9
337P9
173C7
186D7
ATA
133B15
% Inhibition
9. DOSE RESPONSE OF HIT COMPOUNDS
120
110
100
90
80
70
60
2 5 10 19
50
40 DNA DNA + AIF
30
20
10
0
-3
-2
-1
5654435
%Inhibition
%Inhibition
1.  Automated liquid
handler was utilized
2.  200,000 compounds
were screened using
a single readout
instrument
3.  The screen used
approximately 110
384 well photonic
crystal microplates
4.  The duration of the
screen is very short,
an initial baseline
read and a 30 min
after incubation read
•  The 19 compounds
were screened
again using PC
biosensors
•  11 out of 19
compounds were
hits from the screen
•  Determining from
the 11 chemical
structures, only 5
are potential drug
candidates
•  ~0.0025% hit rate
-40
6. HIGH THROUGHPUT SCREENING OF 200,000 COMPOUNDS
Readout instrument can detect PWV shifts (above)
* * * 0
-20
PWV shift
PWV shift plot: Using the readout instrument, the amount of AIF binding to DNA can be analyzed. High PWV
shift (more binding) and low PWV (less binding) are compared to find potential inhibitors
% Inhibition plot: The PWV shift plot is transformed into % Inhibition using the negative and positive controls
3. INSTRUMENTATION & METHOD
* *
* * * * OH
Peak wavelength value (PWV)
= 3.5 µM AIF + DMSO
= 3.5 µM AIF + 6.2 µM Free DNA
= 3.5 µM AIF + 25 µM cmpds
* * 11 out of the 19 compounds were also
active in the PC biosensor assay
O
384H10
0.0
840 850 860 870 880 890 900
Wavelength (nm)
•  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
AIF-DNA inhibition assay protocol (right): (A) Streptavidin-coated
sensor. (B) Immobilize biotinylated DNA for overnight at 4ºC. (C)
Block surface for nonspecific binding. (D) AIF-DNA interaction and
(E) AIF premixed with 10 small molecule binding to DNA.
10 compounds 10 compounds
Hit Hit
Hit
Hit
DNA DNA + AIF Hit
•  Out of the 200,000 compounds, 20 hits wells were found,
corresponding to 200 compounds due to multiplexing
•  Gel electrophoresis confirmed 19 compounds out of 200 to be a hit
227J17
Negative
Control
(AIF Only)
0% Inhibition
0.2
•  Readout instrument
•  Uses infrared LED to illuminate through multiple
optical fibers to measure 8 wells at once
•  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 at user-specified time
intervals
•  The collected PWV is used to generate a kinetic plot
of biomolecular binding events
•  High throughput screen (HTS) for AIF-DNA inhibitors
•  Protocol automation and initial screening of 1000
small molecules
•  Multiplex method with 10 compounds/well
•  HTS of 200,000 compounds
•  Confirm results with gel electrophoresis
•  Dose response characterization of hits
10 compounds
8. PHOTONIC CRYSTAL BIOSENSOR CONFIRMATION
5. INITIAL SCREEN OF 1000 COMPOUNDS
0.4
Cross-section schematic of the sensor
19 active in gel shift assay
Heeres J.T. and Hergenrother, P.J. Curr Opin Chem Biol. 2007, 11, 644
0.6
Photonic crystal sensor incorporated
into multi-well microplate
Nucleus
10 compounds
138B15
0.8
200 compounds
Free DNA
Reflectance
1.0
20
primary hits
~200,000
compounds
DMSO
2. BACKGROUND
•  AIF mediated cell death process
1.  PARP attaches to dsDNA and flags damaged sites
2.  Induces the release of PAR into the cytoplasm
3.  The released PAR traverse across and attaches to the
mitochondria
4.  Induces the release of AIF into the cytoplasm
5.  The released AIF returns to the nucleus
6.  After returning to the nucleus, the AIF attaches to the
dsDNA in a non-sequence specific fashion
7.  AIF binds to the DNA phosphate backbone through AIF
surface lysine residues
8.  AIF then recruits other additional enzymes and dismantle
the DNA causing the cell to die
Cytoplasm
http://nano.ece.uiuc.edu
7. GEL ELECTROPHORESIS CONFIRMATION
177C5
1Leo
39 77 154 µM
0
1
2
3
Log{[SM]( µM)}
120
110
100
90
80
70
60
2 5 10 19
50
40 DNA DNA + AIF
30
20
10
0
-3
-2
-1
5541314
38 77 154 µM
0
1
2
3
Log{[SM]( µM)}
• Out of the 5 final hit compounds, 2 showed strong inhibition
• These 2 compounds were characterized with a dose response curve to calculate
the IC50 values, which were both less than 10 µM
•  IC50 values validates by gel shift assay
10. ACKNOWLEDGEMENTS
D
A
B
C
E
This work is supported by the NIH (PHS 1 R01 CA118562 A). Any opinions, findings,
and conclusions or recommendations expressed in this material are those of the author
(s) and do not necessarily reflect the views of the National Institute of Health.