Identifying modulators of protein-protein interactions using photonic crystal biosensors

Article abstract of DOI:10.1021/ja907066r

(Figure Presented) Inhibitors and activators of protein-protein interactions are valuable as biological probes and medicinal agents but are often difficult to identify. Herein we describe a high-throughput assay, based upon photonic crystal (PC) biosensor

Full text of DOI:10.1021/ja907066r

Published on Web 12/07/2009
Identifying Modulators of Protein-Protein Interactions Using Photonic Crystal
Biosensors
James T. Heeres,† Seok-Ho Kim,‡ Benjamin J. Leslie,‡ Erich A. Lidstone,§ Brian T. Cunningham,*,§,|
and Paul J. Hergenrother*,†,‡
Departments of Biochemistry, Chemistry, Bioengineering, and Electrical and Computer Engineering, UniVersity of
Illinois, Urbana, Illinois 61801
Received August 20, 2009; E-mail: bcunning@illinois.edu; hergenro@uiuc.edu
While the value of small molecules that inhibit or enhance
proteinsprotein interactions is widely recognized,1 the identification
of such compounds remains problematic.2 This is partly due to the
wide and shallow nature of many proteinsprotein interfaces but
also because of the inherent difficulties associated with detecting
multiple proteinsprotein binding events en masse. Assays utilized
for the detection of proteinsprotein interactions include enzyme-
linked immunosorbent assays (ELISAs),3 fluorescence polarization
(FP),4 and surface plasmon resonance (SPR).5 ELISAs and FP have
been applied in a high-throughput screening (HTS) mode for the
identification of small molecule inhibitors of proteinsprotein
interactions,1b,6 while SPR can be multiplexed for low-to-medium
throughput applications.7 To develop an assay for the high-
throughput identification of modulators of proteinsprotein interac-
tions, we have devised a strategy that requires only a His6-tagged
protein and an alternately tagged or untagged version of the cognate
partner. This assay, based on photonic crystal (PC) biosensor
technology, is able to measure proteinsprotein interactions in real
time and is independent of antibodies or fluorescence measurements.
PC biosensors are surface structures comprised of a subwave-
length polymer grating coated with a high refractive index material
(TiO2).8 The PC surface resonantly reflects a single wavelength
that is modulated by the adsorption of biomaterial to the sensor
surface.8 Sheets of PC biosensors can be attached to bottomless
384-well plates for HTS and coated with streptavidin (SA). As
demonstrated previously,9 PC biosensor technology can be used
for measuring proteinsDNA interactions and HTS. As modulators
of proteinsprotein interactions are valuable as medicinal agents
and biological probes, but difficult to identify, we sought to adapt
PC biosensor technology to the detection of proteinsprotein
interactions. As diagrammed in Figure 1, we envisioned a D-biotin-
tris-NTA hybrid compound (BTN) as the linkage between the
biosensor surface and a His6-tagged protein of interest. As
demonstrated herein, after charging the sensor with Ni2+ a His6-
tagged protein can bind to the functionalized sensor, and the
interaction between two proteins of interest can readily be detected
by a peak wavelength value (PWV) shift, a change in the peak
reflectance of the sensor.
Figure 1. Structure of D-Biotin-tris-NTA (BTN) and a schematic of a His6-
tagged protein binding to the BTN-coupled PC biosensors, followed by
cognate protein binding. Each binding event results in a shift in the peak
wavelength value of the sensor.
as indicated by PWV shifts; this binding was not observed in the
absence of NiCl2 (see Figures S1, S2).
Next, three well-studied proteinsprotein pairs with known small
molecule modulators were chosen for proof-of-principle experi-
ments: XIAPscaspase-9,11 XIAPscaspase-7,11 and FKBP12smTOR
(FRB domain).12 XIAP binds to caspase-7 (KD ) 0.5 nM)13a and
caspase-913b (KD ) 0.5 µM) with KD values in the range typical of
many protein-protein interactions. As shown, immobilized His6-
caspase-9 (Figure 2a) and His6-caspase-7 (Figure 2c) bind to their
cognate protein (GST-XIAP), while His6-FKBP12 does not bind
GST-FRB in the absence of rapamycin (Figure 2e). The binding
of GST-XIAP to His6-caspase-3 was also readily detected using
this technology (Figure S3).
The XIAPscaspase-9 and XIAPscaspase-7 interactions can be
inhibited by SM-164,11 while FKBP12 will bind to FRB only in
the presence of rapamycin. The inhibitory properties of SM-164
were readily apparent using the BTN-conjugated biosensor. As
shown in Figures 2b and d, preincubation of XIAP with SM-164
resulted in a dramatic reduction in proteinsprotein binding. The
greater potency of SM-164 in the caspase-9 system is supported
by the differential binding affinity of XIAP for members of the
For these experiments, BTN was synthesized in a manner similar
to that for analogous compounds (see Supporting Information).10
After incubating individual wells of the SA-coated 384-well
biosensor plate with BTN and then charging with 500 µM NiCl2,
four different His6-tagged proteins (caspase-3, -7, -9, and FKBP12,
ranging in size from 12 to 37 kDa) were shown to bind the BTN-
functionalized PC biosensor surface in a dose-dependent manner
caspase family13a and the synthetic design of SM-164.13c In an
,b
analogous fashion, enhancement of proteinsprotein interactions was
demonstrated by the binding of GST-FRB to His6-FKBP12 in the
presence of rapamycin (Figure 2f). Disruption of the on-sensor His6-
caspase-9sGST-XIAP complex through addition of SM-164 could
also readily be detected (Figure S4).
Finally, to demonstrate the HTS capability of the PC biosensor
assay, we performed a “needle-in-a-haystack” experiment with His6-
FKBP12 and GST-FRB (Figure 3). Compounds (320) from an in-
house screening library were added to different wells of a 384-well
† Department of Biochemistry.
‡ Department of Chemistry.
§ Department of Bioengineering.
| Department of Electrical and Computer Engineering.
9
18202 J. AM. CHEM. SOC. 2009, 131, 18202–18203
10.1021/ja907066r  2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Proteinsprotein interactions and their inhibition or enhancement by small molecules. (a) The association of GST-XIAP and His6-caspase-9 and
(b) their inhibition by SM-164. (c) The association of GST-XIAP and His6-caspase-7 and (d) their inhibition by SM-164. (e) The inability of GST-FRB to
bind to His6-FKBP12 and (f) the enhancement of this interaction by rapamycin. All experiments were conducted at 25 °C, and preincubation times and other
experimental details are described in the Supporting Information. All error bars represent the range (n ) 2).
SA biosensor plate that had been charged with BTN, NiCl2, and His6-
FKBP12. Rapamycin was then spiked into a well; the final concentra-
tion of screening compounds was 50 µM, whereas rapamycin was at
1 µM. As shown in Figure 3, after addition of GST-FRB the
rapamycin-containing well is easily identified among the screening
compounds, demonstrating that this assay is capable of being performed
in an HTS mode for identifying modulators of protein-protein
interactions. In an analogous HTS experiment, SM-164 was readily
detected as an inhibitor of caspase-9sXIAP binding (Figure S5).
herein for their identification is general (interfacing with widely
utilized His6 fusion proteins), does not require antibodies or
fluorescent tags, and utilizes commercially available biosensor
readers and plates. Applications of this technique for the high-
throughput identification of novel proteinsprotein modulators are
ongoing and will be reported in due course.
Acknowledgment. We thank Prof. Colin Duckett (U. Michigan)
for pGEX-XIAP, Prof. Shaomeng Wang (U. Michigan) for SM-
164 and SM-122, Prof Qian Tin (Cornell University) for pET28a-
caspase-9, and Prof. Jie Chen (U. Illinois) for pGEX-FKBP12 and
pGEX-FRB. This work was supported by the NIH (R01CA118562)
and the Korea Research Foundation Grant (KRF-2008-357-E00064
to S.-H. K.).
Supporting Information Available: Materials and Methods, sup-
porting figures, and NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Small molecule modulators of proteinsprotein interactions have
tremendous potential in biology and medicine. The system described
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