Robust and systematic drug screening method using chemical arrays and the protein library: Identification of novel inhibitors of carbonic anhydrase II

Article abstract of DOI:10.1271/bbb.80383

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.

Full text of DOI:10.1271/bbb.80383

Biosci. Biotechnol. Biochem., 72 (10), 2739–2749, 2008
Robust and Systematic Drug Screening Method
Using Chemical Arrays and the Protein Library:
Identification of Novel Inhibitors of Carbonic Anhydrase II
y
Isao MIYAZAKI, Siro SIMIZU, Harumi ICHIMIYA, Makoto KAWATANI, and Hiroyuki OSADA
Antibiotics Laboratory and Chemical Biology Department, RIKEN, Wako, Saitama 351-0198, Japan
Received June 6, 2008; Accepted July 8, 2008; Online Publication, October 7, 2008
[doi:10.1271/bbb.80383]
The identification of specific interactions between
small molecules and human proteins of interest is a
fundamental step in chemical biology and drug develop-
ment. Here we describe an efficient method to obtain
novel binding ligands of human proteins by a chemical
array approach. Our method includes large-scale ligand
screening with two libraries, proteins and chemicals, the
use of cell lysates that express proteins of interest fused
with red fluorescent protein, and high-throughput
screening by merged display analysis, which removes
false positive signals from array experiments. Using our
systematic platform, we detected novel inhibitors of
carbonic anhydrase II. It is suggested that our system-
atic platform is a rapid and robust approach to screen
novel ligands for human proteins of interest.
of immobilization of small molecules have been
improved, but prior strategies rely on incubation with
a purified protein of interest. To resolve the problem,
Bradner and co-workers established a robust chemical
array platform with mammalian cell lysates.12) This
method made possible the detection of interactions with
a broad range of binding affinity using epitope-tagged or
chimeric fluorescent proteins without purification. The
use of cell lysates instead of purified proteins is
motivated by the facts that the mammalian proteins
expressed in mammalian cells are properly modi-
fied,13–15) are folded as active forms in the presence of
co-factors, and, are prepared easily.
Carbonic anhydrases (CAs) are ubiquitous zinc pro-
teins16) present in prokaryotes to eukaryotes.17) They are
involved in crucial physiological process connected with
respiration and the transport of CO2/bicarbonate. Among
them, CAII has high catalytic activity of CO2 hydra-
tion.16) CA inhibitors have been reported to serve as anti-
glaucoma drugs. Moreover, it has been reported that CA
inhibitors might serve as anti-cancer, anti-infective drugs
and as a novel therapy for Alzheimer’s disease.16,18) At
least 25 CA inhibitors have been reported to be clinically
used drugs. Hence the discovery of new CA inhibitors
and, establishment of novel uses of these inhibitors have
been disirable for clinical application.
In this report, we describe the establishment of a
systematic, large-scale chemical array method that can
detect specific interactions of small molecules with
human proteins of interest. As compared with our
previous technology, the immobilization of small mole-
cules in a functional group-independent manner, this
method has several improved points, including the use
of protein lysates of cells that express proteins fused
with red fluorescent protein (RFP) from a gene library
we constructed, the use of a public natural-products
depository system, and the high-throughput manner by
which the merged displaying analysis removes false
Key words: chemical array; carbonic anhydrase II;
small molecule inhibitor; protein library
Small molecules are used as tools in regulating
protein functions of interest. Discovery of them has
become a more important step due to increasing
information on the human genome and proteome.1,2)
Chemical arrays represent one of the most promising
and high-throughput approaches to searching ligands
against proteins of interest.3–6) Several successful results
from chemical arrays have been reported.7,8) One of the
key steps in the technology is immobilization of small
molecules on a solid surface. Selective coupling ap-
proaches have been developed and, are used in the
immobilization of small molecules on glass slides, as
reported by several groups.3,9) On the other hand, we
have developed a nonselective immobilization method
that makes possible the introduction of a variety of small
molecules to solid surfaces.6,10) This platform depends
on photo-generated carbene species that can crosslink a
variety of small molecules onto a solid surface in a
functional group-independent manner.11) The methods
y
To whom correspondence should be addressed. Tel: +81-48-467-9542; Fax: +81-48-462-4669; E-mail: hisyo@riken.jp
Abbreviations: CA, carbonic anhydrase; RFP, red fluorescent protein derived from DsRed-Express; mRFP, red fluorescent protein derived from
DsRed-Monomer; ITC, isothermal calorimetry; PALC, photo-affinity-linker coated; GLORIA, Gene Library of Osada Laboratory at RIKEN for
chemical array analysis; NPDepo, RIKEN Natural Products Depository

2740
I. MIYAZAKI et al.
positive signals on array experiments. Using the se-
quential screening strategy, we found novel inhibitors
for carbonic anhydrase II, suggesting that the method
should yield a method of searching for ligands of human
proteins of interest.
DMSO, washed successively with DMSO, DMF, THF,
DMSO, and Milli-Q water (1 h each), and dried. The
slides were degassed and stored at ꢀ20 ꢁC until use.
Merged display analysis. Glass slides were incubated
for 1 h at 4 ꢁC with cell lysates that overexpressed RFP-
fused proteins of interest or cell lysates that overex-
pressed RFP (3–7 mg protein/ml). After incubation, the
slides were washed 3 times in PBS, dried, and scanned at
532 nm on a GenePix microarray scanner (Amersham
Biosciences). The fluorescence signals were quantified
with GenePix 5.0 software. The images from each slide,
one was treated with cell lysates that overexpressed RFP
as control and the other was incubated with cell lysates
that overexpressed target proteins fused with RFP, were
painted green and red respectively using Photoshop 5.5
software. Then, the colored figures were merged. The
false positive signals that were caused by binding of
ligands to RFP or by autofluorescent signals of the
ligand itself were detected as yellow signals.
Materials and Methods
Cloning of human cDNAs and establishment of
GLORIA. The target genes were cloned by PCR from a
cDNA library of cultured human cell lines, such as HeLa
cells, and the obtained fragments were inserted into
pGEM-T-Easy vector (Promega, Medison, Wl). The
vectors were digested by several restriction enzymes, and
the fragments were subcloned into the pDsRed-Express-
N1 or pDsRed-Monomer-N1 (Clontech, Mountain View,
CA) vector. The sequences were confirmed using the
dideoxynucleotide chain termination procedure with an
automated sequencer (Applied Biosystems, Foster City,
CA). This gene library was named GLORIA (Gene
Library of Osada Laboratory at RIKEN for chemical
array analysis). GLORIA contains 100 human genes
listed in Table 1. The vectors, pDsRed-Express-N1 and
pDsRed-Monomer-N1, encoded DsRed-Express and
DsRed-Monomer respectively.19,20) DsRed-Express, a
variant of Discosoma sp. red fluorescent protein (DsRed),
forms a homo-tetramer. On the other hand, DsRed-
Monomer is a monomeric mutant of DsRed. In this
report, the fused proteins expressed from pDsRed-
Express-N1 and from pDsRed-Monomer-N1 are desig-
nated as RFP-protein and mRFP-protein respectively.
Isothermal calorimetry and CAII enzyme assay.
Isothermal calorimetry (ITC) experiments were done
at 25 ꢁC using MicroCal VP-ITC (MicroCal, MA). A
solution of carbonic anhydrase (Sigma-Aldrich) was
degassed for 3 min and then loaded into a calorimeter
cell. A Tris–SO4 buffer (pH 8.4) containing DMSO 4%
(v/v) and ligand (5 mM) was degassed for 3 min before
use, and then titrated with the protein (50 mM) in the
same buffer. After an initial dummy injection of 2 ml, the
injection (10 ml) of ligand solution into the calorimeter
cell was done. The resulting titration curves were then
processed and fitted with Origin 7 software (MicroCal).
The inhibitory activity of the compounds for CAII was
measured with 4-nirtophenylacetate as substrate, which
made possible detection of the esterase activity of the
enzyme.21,22) Ligands and CAII solution (final, 50 nM)
were pre-incubated together for 10 min in 50 mM Tris–
SO4 buffer (pH 8.4). After the addition of a solution
containing 10 mM 4-nirtophenylacetate, the reaction was
started. It was monitored with a Wallac ARVOSX plate
reader at 405 nm (Perkin Elmer, Turku, Finland). The
rates of non-enzymatic hydrolysis were subtracted from
all observed data. The IC50 values were calculated from
independent triplicate experiments.
Preparation of mammalian cell lysates that express
RFP-fused proteins of interest. HEK293T cells were
cultured in RPMI-1640 medium supplemented with 10%
fetal bovine serum in a humidified atmosphere contain-
ing 5% CO2. RFP-fused protein-expressing cells were
created by transfecting the plasmids from GLORIA into
exponentially growing HEK293T cells using Effectene
Transfection Reagent (Qiagen, Tokyo, Japan). After
transient transfection for 24 h, the cells were washed,
harvested, suspended in PBS, and lysed by sonication.
The lysates were centrifuged at 15,000 rpm for 15 min,
and the amount of protein in each lysate was measured
with Coomassie Brilliant Blue G-250 (BioRad, Hercules,
CA). Overexpression of the RFP-fused protein was
confirmed using a fluorescence scanner (Amersham
Biosciences, Foster City, CA), or by Western blotting.
General synthesis. Compounds 5–6 and 8–17 were
synthesized per a previous report for the synthesis of
cinnamoylthiourea derivatives.23) A solution of ammo-
nium thiocyanate (1 eq.) in acetone (30 ml) was refluxed
for 15 min, and the appropriate chloride (e.g., cinnamoyl
chloride derivatives) (1.2 eq.) was added to the mixture.
After the mixture was refluxed for 15 min, the appro-
priate amine (e.g., sulfanilamide, 4-aminobenzamide or
sulfamethoxypyridazine) (1.2 eq.) was added, and the
mixture was stirred for 1 h, cooled, and poured into ice
water, and the separated solid was filtered and washed
Preparation of photo-crosslinked chemical arrays.
The slides were prepared according to our previous
reports.6) A solution of the compounds (10 mM in
DMSO) from NPDepo was immobilized onto PALC
(photo-affinity-linker coated) glass slides with a high-
precision arrayer that was loaded with 16 micro-spotting
pins. Then the slides were exposed to UV irradiation of
4 J cmꢀ2 at 365 nm using a CL-1000L UV crosslinker
(UVP, CA) with the compounds. They were rinsed with
1
with H2O, Et2O. The structures were obtained by H

Identification of Novel Inhibitors of Carbonic Anhydrase II
2741
NMR and mass spectra using NMR spectrometer JEOL
V4.0 (JEOL, Tokyo)(400 MHz) and mass spectrometer
JREOL SX-102A by fast bombardment ionization
(FAB). The spectra are presented in ‘‘Compound
characterization.’’ Compounds 4 and 7 were purchased
from ChemBridge (San Diego, CA) and 18 and 19 were
purchased from Sigma-Aldrich (St. Lous, MO).
[ppm] 3.85 (s, 6H), 7.10 (d, J ¼ 8:5 Hz, 1H), 7.36
(brs, 2H), 7.62 (d, J ¼ 2:1 Hz, 1H), 7.70 (dd, J ¼ 8:5,
2.1 Hz, 1H), 7.82–7.91 (m, 4H) 13C-NMR (100 MHz, d6-
DMSO) ꢀ [ppm] 55.7, 55.7, 110.9, 111.4, 122.9, 123.3,
124.1, 126.1, 140.7, 141.0, 147.9, 152.9, 167.1, 179.3
HRMS m=z calcd for C16H17N3O5S2 ([M + H]þ)
396.0688, found 396.0699.
(E)-3-[3,4-(methylenedioxy)phenyl]-N-(4-sulfamoyl-
phenylcarbamothioyl)acrylamide (12) 1H-NMR (400
MHz, d6-DMSO) ꢀ [ppm] 6.10 (s, 2H), 6.89 (d, J ¼
15:9 Hz, 1H), 7.01 (d, J ¼ 8:1 Hz, 1H), 7.16 (s, 1H),
7.19 (d, J ¼ 8:1 Hz, 1H), 7.38 (brs, 2H), 7.70 (d, J ¼
15:9 Hz, 1H), 7.82–7.90 (m, 4H), 11.6 (s, 1H), 12.9 (s,
1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ [ppm] 101.7,
106.4, 108.7, 117.3, 123.9, 124.7, 126.1, 128.2, 140.5,
141.0, 144.6, 147.9, 149.6, 166.2, 178.9 HRMS m=z
calcd for C17H15N3O5S2 ([M + H]þ) 406.0531, found
406.0538.
(E)-3-(3-methoxyphenyl)-N-(4-sulfamoylphenylcar-
bamothioyl)acrylamide (13) 1H-NMR (400 MHz, d6-
DMSO) ꢀ [ppm] 3.80 (s, 3H), 7.05 (d, J ¼ 15:6 Hz, 1H),
7.06–7.10 (m, 1H), 7.20 (m, 1H), 7.22 (d, J ¼ 8:0 Hz,
1H), 7.39 (brs, 2H), 7.37–7.40 (m, 1H), 7.75 (d, J ¼
15:9 Hz, 1H), 7.82–7.90 (m, 4H) 13C-NMR (100 MHz,
d6-DMSO) ꢀ [ppm] 55.1, 113.1, 116.6, 119.6, 120.5,
123.9, 126.1, 130.1, 135.2, 140.5, 141.1, 144.5, 159.4,
165.9, 178.9 HRMS m=z calcd for C17H17N3O4S2
([M + H]þ) 392.0738, found 392.0746.
Compound characterization. (E)-3-(3,4-dimethoxy-
phenyl)-N-(3-sulfamoylphenylcarbamothioyl) acrylamide
(5) 1H-NMR (400 MHz, d6-DMSO) ꢀ [ppm] 3.81 (s,
6H), 6.96 (d, J ¼ 15:9 Hz, 1H), 7.04–7.06 (m, 1H),
7.22–7.25 (m, 2H), 7.44 (s, 2H), 7.58–7.62 (m, 1H),
7.69–7.74 (m, 2H), 7.86 (d, J ¼ 7:8 Hz, 1H), 8.17
(s, 1H), 11.6 (s, 1H), 12.8 (s, 1H) 13C-NMR (100 MHz,
d6-DMSO) ꢀ [ppm] 55.4, 55.6, 110.3, 111.8, 117.1,
121.3, 122.8, 123.2, 126.8, 127.5, 129.4, 138.3, 144.5,
145.0, 148.9, 151.4, 166.6, 179.4 HRMS m=z calcd for
C18H19 N3O5S2 ([M + H]þ) 422.0844, found 422.0860.
(E)-N-(4-sulfamoylphenylcarbamothioyl)-3-[3-(trifluo-
romethyl)phenyl]acrylamide (6) 1H-NMR (400 MHz,
d6-DMSO) ꢀ [ppm] 7.21 (d, J ¼ 16:1 Hz, 1H), 7.40
(brs, 2H), 7.72 (t, J ¼ 7:7 Hz, 1H), 7.81–8.0 (m, 8H),
11.7 (s, 1H), 12.7 (s, 1H) 13C-NMR (100 MHz, d6-
DMSO) ꢀ [ppm] 121.8, 124.0, 124.5, 126.1, 126.8,
129.5, 129.8, 130.1, 131.6, 135.0, 140.4, 141.1, 142.5,
165.5, 178.8 HRMS m=z calcd for C17H14 F3N3O3S2
([M + H]þ) 430.0507, found 430.0509.
(E)-3-(3,4-dichlorophenyl)-N-(4-sulfamoylphenylcar-
bamothioyl)acrylamide (8) 1H-NMR (400 MHz, d6-
DMSO) ꢀ [ppm] 7.14 (d, J ¼ 15:9 Hz, 1H), 7.39 (brs,
2H), 7.62 (d, J ¼ 8:3 Hz, 1H), 7.74–7.77 (m, 2H), 7.82–
7.89 (m, 4H), 7.89–7.92 (m, 1H), 11.7 (s, 1H), 12.7
(s, 1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ [ppm] 121.9,
124.0, 126.1, 127.5, 130.1, 131.2, 131.7, 132.8, 134.7,
140.4, 141.1, 141.7, 168.3, 184.3 HRMS m=z calcd
for C16H13Cl2N3O3S2 ([M + H]þ) 429.9853, found
429.9844.
(E)-3-(2-methoxyphenyl)-N-(4-sulfamoylphenylcar-
bamothioyl)acrylamide (14) 1H-NMR (400 MHz, d6-
DMSO) ꢀ [ppm] 3.99 (s, 3H), 7.04 (t, J ¼ 7:5 Hz, 1H),
7.11–7.14 (m, 1H), 7.16 (d, J ¼ 15:9 Hz, 1H), 7.39 (brs,
2H), 7.43–7.47 (m, 1H), 7.55 (d, J ¼ 7:5 Hz, 1H), 7.82–
7.91 (m, 4H), 7.95 (d, J ¼ 15:9 Hz, 1H), 11.7 (s, 1H),
12.9 (s, 1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ [ppm]
55.6, 111.8, 119.9, 120.7, 122.1, 123.8, 126.1, 129.1,
132.3, 139.9, 140.5, 141.0, 158.1, 166.6, 178.9 HRMS
m=z calcd for C17H17N3O4S2 ([M + H]þ) 392.0738,
found 392.0755.
(E)-3-(3,4,5-trimethoxyphenyl)-N-(4-sulfamoylphenyl-
1
carbamothioyl)acrylamide (9) H-NMR (400 MHz, d6-
(E)-N-{4-[N-(6-methoxypyridazin-3-yl)sulfamoyl]-
1
DMSO) ꢀ [ppm] 3.71 (s, 3H), 3.83 (s, 6H), 6.99 (s, 2H),
7.05 (d, J ¼ 15:7 Hz, 1H), 7.38 (brs, 2H), 7.71 (d, J ¼
15:7 Hz, 1H), 7.82–7.90 (m, 4H), 11.6 (s, 1H), 12.8 (s,
1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ [ppm] 55.8,
60.1, 105.6, 118.9, 124, 126.1, 129.4, 139.7, 140.5,
141.1, 144.6, 152.9, 166, 178.9 HRMS m=z calcd for
C19H21N3O6S2 ([M + H]þ) 452.0950, found 452.0964.
2-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenylcar-
bamothioyl)acetamide (10) 1H-NMR (400 MHz, d6-
DMSO) ꢀ [ppm] 3.31 (s, 2H), 3.72 (s, 3H), 3.74 (s,
3H), 6.83–6.64 (m, 3H), 7.36 (brs, 2H), 7.73–7.80
(m, 4H), 11.7 (s, 1H), 12.5 (s, 1H) 13C-NMR (100 MHz,
d6-DMSO) ꢀ [ppm] 41.9, 55.4, 55.5, 111.7, 113.2, 121.4,
124.1, 126.0, 126.2, 140.4, 141.1, 147.7, 148.3, 173.1,
178.9 HRMS m=z calcd for C17H19N3O5S2 ([M + H]þ)
410.0844, found 410.0828.
phenylcarbamothioyl}cinnamamide (15) H-NMR (400
MHz, d6-DMSO) ꢀ [ppm] 3.82 (s, 3H), 7.07 (d, J ¼
15:9 Hz, 1H), 7.41 (brs, 1H), 7.46–7.47 (m, 3H), 7.63 (m,
2H), 7.77 (d, J ¼ 15:9 Hz, 1H), 7.84–7.9 (m, 6H), 11.7
(s, 1H), 12.8 (s, 1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ
[ppm] 54.5, 119.5, 123.8, 126.6, 128.1, 129.0, 130.7,
133.8, 140.6, 144.6, 166.0, 178.7 HRMS m=z calcd for
C21H19N5O4S2 ([M + H]þ) 470.0956, found 470.0959.
(E)-4-{3-[3-(3,4-dimethoxyphenyl)acryloyl]thiourei-
do}benzamide (16) 1H-NMR (400 MHz, d6-DMSO) ꢀ
[ppm] 3.81 (s, 3H), 3.85 (s, 3H), 6.97 (d, J ¼ 15:9 Hz,
1H), 7.22–7.24 (m, 2H), 7.38 (brs, 1H), 7.71 (d,
J ¼ 15:9 Hz, 1H), 7.79–7.91 (m, 4H), 7.98 (brs, 1H),
11.5 (s, 1H), 12.9 (s, 1H) 13C-NMR (100 MHz, d6-
DMSO) ꢀ [ppm] 55.3, 55.6, 110.1, 111.6, 117.0, 122.7,
123.0, 126.6, 127.9, 131.4, 140.1, 144.8, 148.7, 151.1,
166.4, 166.9, 178.6 HRMS m=z calcd for C19H19N3O4S
([M + H]þ) 386.1174, found 386.1173.
3,4-dimethoxy-N-(4-sulfamoylphenylcarbamothioyl)-
benzamide (11) 1H-NMR (400 MHz, d6-DMSO) ꢀ

2742
I. MIYAZAKI et al.
Human cDNA library
Cloning genes
of interest
P CMV IE
pUC
ori
MCS
HSV TK
poly A
RFP
pDsRed-
Express-N1
4.7 kb
Sequencing
and collection
Kanr/
Neor
SV40
poly A
GLORIA
f1
ori
P
Transfection
and expression
P SV40 e
SV40 ori
Transfection
and expression
pDsRed-
Express-N1
(control)
HEK293T
Expression of
RFP
Expression of
RFP-fused protein
Preparation of cell lysates
and incubation with
the slides
Ex. 532 nm
Em. 575 nm
Ex. 532 nm
Em. 575 nm
Merged display analysis
Fig. 1. Overview of Systematic Chemical Array Screening Method.
Human genes of interest are cloned and restriction enzyme-digested fragments are inserted into pDsRed-Express and sequenced. GLORIA
contains 100 genes, which can be expressed as RFP-fused proteins in mammalian cells. HEK 293T cells are transfected with vectors encoding
RFP fused-proteins or RFP for 24 h. After transfection, cell lysates are prepared for incubation with glass slides, where compounds from NPDepo
are immobilized in duplicate by a photo-crosslinking technique. Detection of ‘‘hit ligands’’ is done by merged display analysis. Spots that interact
with RFP or RFP-fused proteins are designated green and red respectively. After two slides are merged, true hit compounds are detected as red
signals, while false positive signals are yellow.
(E)-3-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)-
acrylamide (17) 1H-NMR (400 MHz, d6-DMSO) ꢀ [ppm]
3.79 (s, 3H), 3.81 (s, 3H), 6.70 (d, J ¼ 15:6 Hz, 1H), 7.01
(d, J ¼ 8:0 Hz, 1H), 7.21–7.24 (m, 1H), 7.25 (brs, 2H),
7.56 (d, J ¼ 15:6 Hz, 1H), 7.75–7.84 (m, 4H), 10.5 (s,
1H) 13C-NMR (100 MHz, d6-DMSO) ꢀ [ppm] 55.4, 55.5,
109.9, 111.6, 118.5, 119.2, 121.8, 126.6, 127.1, 138,
140.9, 142.1, 148.7, 150.3, 164.0 HRMS m=z calcd for
C17H18N2O5S ([M + H]þ) 363.1014, found 363.1021.
Results
Development of a systematic chemical array method
of searching novel ligands
To screen a tremendous number of combinations of
compounds and human proteins, we designed a system-
atic ligand-screening strategy using a chemical array that
relied on two original libraries, the GLORIA gene
library, and the NPDepo (RIKEN Natural Products

Identification of Novel Inhibitors of Carbonic Anhydrase II
2743
A
Duplicated spots
A
B
C
D
A
B
C
D
1
Rapamycin
2
3
4
Rapamycin
B
40
30
20
10
0
Fig. 2. Identification of Rapamycin as an FKBP12-Binding Ligand.
A, The 2011 compounds from NPDepo were immobilized on
glass slides in duplicate. HEK293T cells were transfected with
vectors encoding FKBP12-RFP or RFP. After 24 h the cells were
lysed, and the cell lysates were incubated with the glass slides; then
the slides were scanned. The two positions spotted with rapamycin
are indicated by white arrows. Two rhodamine spots in every 144
compounds were used as a position marker. B, Comparison of
signal-to-noise ratio (S/N ratio) in the fluorescence image of a
rapamycin-spotted area on glass slides treated with cell lysates
overexpressing FKBP12-RFP (closed bar) or FKBP12-mRFP (open
column). Data were calculated from averages by duplicate positions
on a slide.
B
A
29
24
19
14
9
1
1
4
0
1
5
10
15
20
25
30 33
2
(Gene no. of GLORIA)
C
15
13
11
9
2
7
5
3
1
0
1
5
10
15
20
25
30 33
(Gene no. of GLORIA)
Fig. 3. Identification of Novel Specific Ligands for CAII.
A, Fluorescent image by merged display analysis after treatment with cell lysates that express CAII-RFP and RFP. Two positions
(B and C) spotted with 1 and 2 are indicated as red arrows. The S/N ratio by merged display analysis at the positions spotted with 1 (B) and 2 (C).
The glass slides were treated with cell lysates that expressed 33 different RFP-fused proteins (Gene nos. 1–33 in GLORIA). The gene number of
CAII in GLORIA is 15 (red bar). These data were calculated from averages by duplicate positions on a slide.

2744
I. MIYAZAKI et al.
Table 1. List of GLORIA
ID no. Gene names UniProt no. ID no. Gene names UniProt no. ID no. Gene names UniProt no. ID no. Gene names UniProt no.
1
2
HPSE
Q9Y251
P62942
P61457
P01033
O95994
Q969K3
O00743
Q07817
Q9UI95
P31947
P26447
P60903
Q16543
Q96GD4
P00918
P22392
P01344
Q13541
P62993
P01127
P04792
P07237
Q02750
P49450
P13667
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
MMP3
ARF6
MOCS2
P08254
P62330
O96033
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
ERP27
TXNDC4
PER1
Q96DN0
Q9BS26
Q6IN51
Q6IAH4
O95298
Q71U36
P23258
Q9Y570
P30040
Q13867
P01135
P18846
Q6FGD7
Q9H0N5
O43504
P22894
P09238
P00749
76 PARK7
77 CDA
Q99497
P32320
Q9Y294
P84077
P60981
Q00987
Q8IXB1
P23219
Q9Y3C7
Q99417
P53999
FKBP1A
PCBD1
TIMP1
AGR2
3
78 ASF1A
79 ARF1
4
CDC42EP3 Q9UKI2
PSMG2
NDUFC2
TUBA1A
TUBG1
PPME1
ERP29
BLMH
TGFA
5
FDPS
P14324
P45452
P16035
O95749
P00374
Q07817-2
Q9Y5L2
P28482
Q58A44
P67775
O15392
Q86YL7
P15531
Q75NE6
P54577
P23381
P41252
P49591
Q9NP10
P30101
80 DSTN
81 MDM2
82 DNAJC10
83 PTGS1
84 MED31
85 MYCBP
86 SUB1
6
RNF34
PPP6C
BCL2L1
MAD2L2
SFN
MMP13
TIMP2
GGPS1
DHFR
BCL2L1ꢂ
HIG2
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
S100A4
S100A10
CDC37
AURKB
CA2
MAPK1
PCOTH
PPP2CA
BIRC5
PDPN
ATF1
TBCA
87 COX7A2L O14548
88 E2F6
89 IL1A
90 GARS
91 SKP1
92 RBX1
93 AES
O75461
P01583
P41250
P63208
P62877
Q08117
Q9BYZ8
P36639
Q99519
P07437
P14780
P62136
P05412
PCBD2
HBXIP
MMP8
MMP10
PLAU
NME2
IGF2
NME1
MIRH1
YARS
WARS
IARS
EIF4EBP1
GRB2
MAP2K1IP1 Q9UHA4
94 REG4
95 NUDT1
96 NEU1
97 TUBB
98 MMP9
99 PPP1CA
100 JUN
PDGFB
HSPB1
P4HB
TP53AP1
C2orf12
MAX
Q9Y2A0-4
Q8TDM3
P61244
SARS
IGF1
PDIA3
MAP2K1
CENPA
PDIA4
DEFA1
KARS
RAN
P59665
Q15046
P62826
TXNDC12 O95881
Gene names and UniProt nos. are based on the UniProt website (http://beta.uniprot.org/).
ꢂ, splicing variant.
Depository) chemical library (Fig. 1). To use GLORIA
in connection with the robust chemical array system, we
cloned 100 genes and designed them to be able to express
RFP-fused proteins of interest. HEK293T cells were
transfected with vectors that encoded RFP-fused proteins
of interest or RFP, and cell lysates were prepared. There
are several reasons to use cell lysates instead of purified
proteins for high-throughput screening: (i) cell lysates
are very easy and fast to prepare, and (ii) target proteins
must be folded in their natural conformation and
modified properly. Moreover, this chemical array screen-
ing method contains another property called merged
display analysis, which excludes false positive signals.
Merged display analysis is done by the following steps:
Positive signals on glass slides after incubation with cell
lysates that express RFP-conjugated proteins of interest
or RFP are colored red and green respectively on a
computer. Then the colored figures are merged onto one
map. It can highlight false positive signals as yellow,
caused by binding of ligands to RFP or by autofluor-
escent signal of the ligand itself. Hence it is suggested
that the results due to this method give only real-hit
compounds, without false positive signals.
array method can be used in the detection of known
interactions, we used 2,011 compounds from NPDepo.
Cell lysates that expressed RFP-fused FKBP12 protein
and RFP were prepared. After incubation of glass slides
with the cell lysates, the images treated with cell lysates
that expressed RFP-fused FKBP12 or RFP were colored
red and green respectively, and the two images were
merged onto a map. We detected rapamycin specifically
as an FKBP12-binding ligand from glass slides that
were spotted in duplicate with the 2,011 compounds
(Fig. 2A). To determine whether the signal:noise ration
was changed by tetrameric or monomeric RFP, the signal
intensities of the slides treated with FKBP12-RFP and
FKBP12-mRFP were compared. Although binding sig-
nals due to cell lysates expressing FKBP12-RFP and
-mRFP were clearly observed, the use of FKBP12-RFP
yielded stronger signals. The S/N ratio of a spot treated
by FKBP12-RFP was 2–3 fold higher than that of a spot
treated by FKBP12-mRFP (Fig. 2B). Hence we decided
to use RFP-fused proteins of interest with our screening
system.
Development of GLORIA to be applied to chemical
array screening with cell lysates
Identification of rapamycin as an FKBP12-binding
protein by this method
Next we sought to construct a human gene library,
GLORIA, for our platform. We selected many human
disease-associated genes, including cancer and glauco-
ma. We cloned 100 genes and inserted them into
pDsRed Express-N1 vector, because the interaction of
small molecules with fluorescent protein-fused proteins
is very easy to detect (Table 1). The proteins encoded in
NPDepo was established recently by our laboratory.24)
The compounds from NPDepo were spotted in duplicate
onto PALC glass slides6) so that binding signals and
noise could be easily distinguished by comparing
duplicate slides. To confirm whether the unique chemical

Identification of Novel Inhibitors of Carbonic Anhydrase II
2745
A
Compound
Structure
IC50 (nM)
24 2.9
F
H
N
H
N
1
O
S
SO2NH2
SO2NH2
O
O
H
N
H
N
2
50 12
5400 67
34 3.0
O
S
O
O
H
N
H
N
3
N
N
O
O
S
S
O
N
H
N
N
acetazolamide
SO2NH2
H3COCHN
S
B
C
2
1
Kd = 508 nM
Kd = 159 nM
Fig. 4. Effects of 1 and 2 on CAII Inhibitory Activity and Binding to CA.
A, Effects of several compounds on CAII enzyme activity. The 50% inhibitory concentrations (IC50) of compounds were calculated from three
independent assays. B and C, Raw data were obtained after injection of a carbonic anhydrase solution (50 mM) into a solution (5 mM) of 1 (B) and
of 2 (C) in PBS at 25 ꢁC (top), and molar heat values were plotted as a function of the molar ratio (bottom). The solid line represents the best-fit
binding isotherm obtained by use of a single-site model.
GLORIA have various functions, such as transferase
activity (7%), hydrolase activity, (15%), lyase activity
(2%), and non-enzyme proteins (60%). For example,
transferases contain protein kinases, which include some
clinical therapeutic targets (e.g., aurora kinase B and
MEK1). Although many proteins are expressed in full-
length, some were designed to be expressed in truncated
forms, such as membrane proteins (e.g., Bcl-xL and
Aggrus). Furthermore, many kinds of post-translational
modifications occur in the proteins encoded in GLORIA,
but if these proteins were expressed by E. coli, they
would not be modified. In sum, it appears that the
proteins encoded in GLORIA might be useful for our
chemical array method.

2746
I. MIYAZAKI et al.
A
H
N
R1
R2
B
IC50
(nM)
IC50
(nM)
Compound
R1
R2
Compound
R1
R2
O
H
H
N
N
12
13
14
15
16
17
18
(p-)-SO2NH2
(p-)-SO2NH2
O
43 6.0
4
31 2.3
O
S
O
S
O
H
H
N
N
O
5
6
(m-)-SO2NH2280 29
(p-)-SO2NH2
(p-)-SO2NH2
O
36 2.1
54 11
O
S
O
S
H
H
N
N
(p-)-SO2NH2
F3C
60 2.9
O
S
O
O
S
H
O
(p-)
H
N
O
S
O
N
7
(p-)-SO2NH2
>10000
30 4.2
N
O
S
N
N
O
S
H
Cl
Cl
O
O
H
H
N
(p-)-CONH2
N
8
>10000
(p-)-SO2NH2
(p-)-SO2NH2
44 12
52 22
47 4.7
39 2.5
O
S
O
S
O
O
O
O
O
H
N
9
(p-)-SO2NH2
(p-)-SO2NH2
6.8 0.79
320 98
1700 610
O
O
S
H
O
N
10
11
(p-)-SO2NH2
(p-)-SO2NH2
O
O
S
O
O
H
H
19
(p-)-SO2NH2
O
N
O
S
Fig. 5. Continued.
Identification of novel ligands for carbonic anhydrase
II by the chemical array platform
To evaluate the inhibitory activities and binding
abilities of 1 and 2 to CAII, we performed an enzyme
assay and ITC measurement. The enzyme assay was
done in buffer with CAII and 4-nitrophenylacetate as
substrates.21,22) 1 and 2 showed strong inhibitory
activity, the same as acetazolamide, a well-known CAII
inhibitor (Fig. 4A). Moreover, ITC measurements dem-
onstrated that the Kd values of 1 and 2 for CA were
159 nM and 508 nM, respectively (Fig. 4B and C). On
the other hand, compound 3, which lacks the sulfona-
mide moiety changed to 4,6-dimethylpyrimidine, was
not detected by the array analysis. 3 shows very weak
inhibitory activity (Fig. 4A) and has undetectable Kd
value (data not shown). It has been found that the
To obtain novel ligands for CAII, we spotted about
10,000 compounds from NPDepo, and then carried out
screening with cell lysates from HEK293T-expressed
CAII-RFP. After merged display analysis, we identified
two compounds as CAII-binding compounds, 1 and 2,
out of 10,000 compounds (Fig. 3A). To investigate the
specificity of 1 and 2 to CAII, we prepared cell lysates
of HEK293T cells that expressed 33 different RFP-
fused proteins (gene numbers 1–33 of GLORIA,
Table 1), including CAII (no. 15 of GLORIA). Among
the 33 proteins tested, 1 (Fig. 3B) and 2 (Fig. 3C) were
bound specifically to cell lysates expressed CAII-RFP.

Identification of Novel Inhibitors of Carbonic Anhydrase II
2747
C
O
O
H
N
O
SO2NH2
17
Kd = 66 nM
Fig. 5. Synthesis and Identification of CAII Inhibitors.
A, Core chemical structure of the CAII inhibitors. B, Structure and CAII inhibitory activity by the compounds 4–19. Compounds 5–6 and
8–17 were synthesized, and 4, 7, 18, and 19 were purchased. C, Structure of 17 and ITC profiling of 17 for CA. A solution of ligand (6.7 mM)
in Tris-SO4 buffer containing 4% (v/v) DMSO was titrated with a solution of the protein (67 mM) in the same buffer, and molar heat values
were plotted as a function of the molar ratio (bottom). The solid line represents the best-fit binding isotherm obtained by use of a single-site
model.
sulfonamide moiety is very important in inhibiting and
interacting with CAII. Taken together with previous CA
inhibitor reports,16,25) these data strongly suggest that 1
and 2 bound to active site of CAII, and chelated zinc
ion in the active site with the sulfonamide moiety. This
binding might have resulted in the enzymatic inhibitory
effect.
Discussion
Although chemical arrays represent one of the most
promising and high-throughput approaches to searching
ligands against proteins of interest, the discovery of
novel interactions between proteins and small molecules
using chemical arrays has been limited.7,8,26,27) It has
been reported that the ratio is one hit per 120,000
compounds for enzyme inhibitors.28) Hence efficient
screening of a tremendous number of combinations of
proteins and small molecules is necessary.
Matsuyama et al. have reported a large-scale study
of the fission yeast Schizosaccharomyces pombe based
on cloning of the ORFeome.29) Their system has made
it possible to screen for ligands to yeast proteins in a
large-scale manner, and it can be applied to the
chemical array screening method. However, some
proteins associated with human diseases have potential
as drug targets, and they are expressed only in
mammalian cells (cf. growth factors). In this study,
we established that GLORIA contained 100 genes
many of which play important roles in human diseases.
We also recognized that GLORIA and NPDepo are not
definitive. For example, the current number of com-
pounds and proteins in the libraries described here is
Structure-activity relationship study
To obtain more potent inhibitors of CAII, we
synthesized compounds 5–6 and 8–17, and purchased
4, 7, 18, and 19 (Fig. 5A and B). The meta-position of
sulfonamide moieties on the phenyl ring (5) resulted in a
loss of potency against CAII as compared with the para-
position (2). Compounds 15 and 16 lacked sulfonamide
moieties on R2, which are thought to be an important
group for interaction with the zinc ions of enzymes.25)
As expected, the inhibitory activities of 15 and 16 for
CAII dramatically decreased. Although modification of
R1 appeared to be tolerant of inhibitory activity, 18 and
19 showed weak inhibitory activities. When exploring
variations at the R1 position, we found that 17 showed
the strongest inhibitory activity among the derivatives
tested and had strong binding affinity, with a Kd value of
66 nM (Fig. 5C).

2748
I. MIYAZAKI et al.
Davenport, L., Desilets, R., Dietz, S., Dodson, K., Doup,
L., Ferriera, S., Garg, N., Gluecksmann, A., Hart, B.,
Haynes, J., Haynes, C., Heiner, C., Hladun, S., Hostin,
D., Houck, J., Howland, T., Ibegwam, C., Johnson, J.,
Kalush, F., Kline, L., Koduru, S., Love, A., Mann, F.,
May, D., McCawley, S., McIntosh, T., McMullen, I.,
Moy, M., Moy, L., Murphy, B., Nelson, K., Pfannkoch,
C., Pratts, E., Puri, V., Qureshi, H., Reardon, M.,
Rodriguez, R., Rogers, Y. H., Romblad, D., Ruhfel, B.,
Scott, R., Sitter, C., Smallwood, M., Stewart, E., Strong,
R., Suh, E., Thomas, R., Tint, N. N., Tse, S., Vech, C.,
Wang, G., Wetter, J., Williams, S., Williams, M.,
Windsor, S., Winn-Deen, E., Wolfe, K., Zaveri, J.,
not sufficient to cover the vast interactions between
proteins of interest and small molecules. Cloning of
100 genes is nothing extraordinary in modern molecu-
lar biology, and hence we are trying to increase the
number of genes and proteins in GLORIA and
NPDepo.
In summary, our screening method can perform
large-scale chemical array screening in combination
with a gene library, GLORIA, and a chemical library,
NPDepo. The development of CAII inhibitors indicates
the utility of our screening method in the identification
of useful ligands to human protein. To discover of
these ligands for human proteins, the numbers of genes
in GLORIA and of compounds in NPDepo must be
increased. This should help to remove one of the
bottlenecks to in the discovery of useful tools for
modern chemical biology studies and drug discovery
research.
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Allen, D., Basu, A., Baxendale, J., Blick, L., Caminha,
M., Carnes-Stine, J., Caulk, P., Chiang, Y. H., Coyne,
M., Dahlke, C., Mays, A., Dombroski, M., Donnelly, M.,
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Lopez, J., Ma, D., Majoros, W., Murphy, S., Newman,
M., Nguyen, T., Nguyen, N., Nodell, M., Pan, S., Peck,
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Simpson, M., Smith, T., Sprague, A., Stockwell, T.,
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M., Xia, A., Zandieh, A., and Zhu, X., The sequence of
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Acknowledgments
We would like to thank Y. Ito, I. Matsuo, A. Asami,
and Y. Kondoh for valuable suggestions, and R.
Nakazawa for DNA sequencing. This study was sup-
ported in part by a Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science, and Technology of
Japan, the Chemical Biology Project (RIKEN).
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