Robust and Systematic Drug Screening Method Using Chemical Arrays and the Protein Library: Identiﬁcation of Novel Inhibitors of Carbonic Anhydrase II

Biosci. Biotechnol. Biochem., 72 (10), 2739–2749, 2008

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 of immobilization of small molecules have been small molecules and human proteins of interest is a improved, but prior strategies rely on incubation with fundamental step in chemical biology and drug develop- a purified protein of interest. To resolve the problem, ment. Here we describe an efficient method to obtain Bradner and co-workers established a robust chemical novel binding ligands of human proteins by a chemical array platform with mammalian cell lysates.12) This array approach. Our method includes large-scale ligand method made possible the detection of interactions with screening with two libraries, proteins and chemicals, the a broad range of binding affinity using epitope-tagged or use of cell lysates that express proteins of interest fused chimeric fluorescent proteins without purification. The with red fluorescent protein, and high-throughput use of cell lysates instead of purified proteins is screening by merged display analysis, which removes motivated by the facts that the mammalian proteins false positive signals from array experiments. Using our expressed in mammalian cells are properly modi- systematic platform, we detected novel inhibitors of fied,13–15) are folded as active forms in the presence of carbonic anhydrase II. It is suggested that our system- co-factors, and, are prepared easily. atic platform is a rapid and robust approach to screen Carbonic anhydrases (CAs) are ubiquitous zinc pro- novel ligands for human proteins of interest. teins16) present in prokaryotes to eukaryotes.17) They are involved in crucial physiological process connected with Key words: chemical array; carbonic anhydrase II; respiration and the transport of CO2/bicarbonate. Among small molecule inhibitor; protein library them, CAII has high catalytic activity of CO2 hydra- tion.16) CA inhibitors have been reported to serve as anti- Small molecules are used as tools in regulating glaucoma drugs. Moreover, it has been reported that CA protein functions of interest. Discovery of them has inhibitors might serve as anti-cancer, anti-infective drugs become a more important step due to increasing and as a novel therapy for Alzheimer’s disease.16,18) At information on the human genome and proteome.1,2) least 25 CA inhibitors have been reported to be clinically Chemical arrays represent one of the most promising used drugs. Hence the discovery of new CA inhibitors and high-throughput approaches to searching ligands and, establishment of novel uses of these inhibitors have against proteins of interest.3–6) Several successful results been disirable for clinical application. from chemical arrays have been reported.7,8) One of the In this report, we describe the establishment of a key steps in the technology is immobilization of small systematic, large-scale chemical array method that can molecules on a solid surface. Selective coupling ap- detect specific interactions of small molecules with proaches have been developed and, are used in the human proteins of interest. As compared with our immobilization of small molecules on glass slides, as previous technology, the immobilization of small mole- reported by several groups.3,9) On the other hand, we cules in a functional group-independent manner, this have developed a nonselective immobilization method method has several improved points, including the use that makes possible the introduction of a variety of small of protein lysates of cells that express proteins fused molecules to solid surfaces.6,10) This platform depends with red fluorescent protein (RFP) from a gene library on photo-generated carbene species that can crosslink a we constructed, the use of a public natural-products variety of small molecules onto a solid surface in a depository system, and the high-throughput manner by functional group-independent manner.11) The methods which the merged displaying analysis removes false

y To whom correspondence should be addressed. Tel: +81-48-467-9542; Fax: +81-48-462-4669; E-mail: [email protected] 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- DMSO, washed successively with DMSO, DMF, THF, quential screening strategy, we found novel inhibitors DMSO, and Milli-Q water (1 h each), and dried. The for carbonic anhydrase II, suggesting that the method slides were degassed and stored at 20 C until use. should yield a method of searching for ligands of human proteins of interest. Merged display analysis. Glass slides were incubated for 1 h at 4 C with cell lysates that overexpressed RFP- Materials and Methods fused proteins of interest or cell lysates that overexpressed RFP (3–7 mg protein/ml). After incubation, the Cloning of human cDNAs and establishment of slides were washed 3 times in PBS, dried, and scanned at GLORIA. The target genes were cloned by PCR from a 532 nm on a GenePix microarray scanner (Amersham cDNA library of cultured human cell lines, such as HeLa Biosciences). The fluorescence signals were quantified cells, and the obtained fragments were inserted into with GenePix 5.0 software. The images from each slide, pGEM-T-Easy vector (Promega, Medison, Wl). The one was treated with cell lysates that overexpressed RFP vectors were digested by several restriction enzymes, and as control and the other was incubated with cell lysates the fragments were subcloned into the pDsRed-Express- that overexpressed target proteins fused with RFP, were N1 or pDsRed-Monomer-N1 (Clontech, Mountain View, painted green and red respectively using Photoshop 5.5 CA) vector. The sequences were confirmed using the software. Then, the colored figures were merged. The dideoxynucleotide chain termination procedure with an false positive signals that were caused by binding of automated sequencer (Applied Biosystems, Foster City, ligands to RFP or by autofluorescent signals of the CA). This gene library was named GLORIA (Gene ligand itself were detected as yellow signals. Library of Osada Laboratory at RIKEN for chemical array analysis). GLORIA contains 100 human genes Isothermal calorimetry and CAII enzyme assay. listed in Table 1. The vectors, pDsRed-Express-N1 and Isothermal calorimetry (ITC) experiments were done pDsRed-Monomer-N1, encoded DsRed-Express and at 25 C using MicroCal VP-ITC (MicroCal, MA). A DsRed-Monomer respectively.19,20) DsRed-Express, a solution of carbonic anhydrase (Sigma-Aldrich) was variant of Discosoma sp. red fluorescent protein (DsRed), degassed for 3 min and then loaded into a calorimeter forms a homo-tetramer. On the other hand, DsRed- cell. A Tris–SO4 buffer (pH 8.4) containing DMSO 4% Monomer is a monomeric mutant of DsRed. In this (v/v) and ligand (5 mM) was degassed for 3 min before report, the fused proteins expressed from pDsRed- use, and then titrated with the protein (50 mM) in the Express-N1 and from pDsRed-Monomer-N1 are desig- same buffer. After an initial dummy injection of 2 ml, the nated as RFP-protein and mRFP-protein respectively. injection (10 ml) of ligand solution into the calorimeter cell was done. The resulting titration curves were then Preparation of mammalian cell lysates that express processed and fitted with Origin 7 software (MicroCal). RFP-fused proteins of interest. HEK293T cells were The inhibitory activity of the compounds for CAII was cultured in RPMI-1640 medium supplemented with 10% measured with 4-nirtophenylacetate as substrate, which fetal bovine serum in a humidified atmosphere contain- made possible detection of the esterase activity of the 21,22) ing 5% CO2. RFP-fused protein-expressing cells were enzyme. Ligands and CAII solution (final, 50 nM) created by transfecting the plasmids from GLORIA into were pre-incubated together for 10 min in 50 mM Tris– exponentially growing HEK293T cells using Effectene SO4 buffer (pH 8.4). After the addition of a solution Transfection Reagent (Qiagen, Tokyo, Japan). After containing 10 mM 4-nirtophenylacetate, the reaction was transient transfection for 24 h, the cells were washed, started. It was monitored with a Wallac ARVOSX plate harvested, suspended in PBS, and lysed by sonication. reader at 405 nm (Perkin Elmer, Turku, Finland). The The lysates were centrifuged at 15,000 rpm for 15 min, rates of non-enzymatic hydrolysis were subtracted from and the amount of protein in each lysate was measured all observed data. The IC50 values were calculated from with Coomassie Brilliant Blue G-250 (BioRad, Hercules, independent triplicate experiments. CA). Overexpression of the RFP-fused protein was confirmed using a fluorescence scanner (Amersham General synthesis. Compounds 5–6 and 8–17 were Biosciences, Foster City, CA), or by Western blotting. synthesized per a previous report for the synthesis of cinnamoylthiourea derivatives.23) A solution of ammo- Preparation of photo-crosslinked chemical arrays. nium thiocyanate (1 eq.) in acetone (30 ml) was refluxed The slides were prepared according to our previous for 15 min, and the appropriate chloride (e.g., cinnamoyl reports.6) A solution of the compounds (10 mM in chloride derivatives) (1.2 eq.) was added to the mixture. DMSO) from NPDepo was immobilized onto PALC After the mixture was refluxed for 15 min, the appro- (photo-affinity-linker coated) glass slides with a high- priate amine (e.g., sulfanilamide, 4-aminobenzamide or precision arrayer that was loaded with 16 micro-spotting sulfamethoxypyridazine) (1.2 eq.) was added, and the pins. Then the slides were exposed to UV irradiation of mixture was stirred for 1 h, cooled, and poured into ice 4Jcm2 at 365 nm using a CL-1000L UV crosslinker water, and the separated solid was filtered and washed 1 (UVP, CA) with the compounds. They were rinsed with 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 [ppm] 3.85 (s, 6H), 7.10 (d, J ¼ 8:5 Hz, 1H), 7.36 V4.0 (JEOL, Tokyo)(400 MHz) and mass spectrometer (brs, 2H), 7.62 (d, J ¼ 2:1 Hz, 1H), 7.70 (dd, J ¼ 8:5, 13 JREOL SX-102A by fast bombardment ionization 2.1 Hz, 1H), 7.82–7.91 (m, 4H) C-NMR (100 MHz, d6- (FAB). The spectra are presented in ‘‘Compound DMSO) [ppm] 55.7, 55.7, 110.9, 111.4, 122.9, 123.3, characterization.’’ Compounds 4 and 7 were purchased 124.1, 126.1, 140.7, 141.0, 147.9, 152.9, 167.1, 179.3 þ from ChemBridge (San Diego, CA) and 18 and 19 were HRMS m=z calcd for C16H17N3O5S2 ([M + H] ) purchased from Sigma-Aldrich (St. Lous, MO). 396.0688, found 396.0699. (E)-3-[3,4-(methylenedioxy)phenyl]-N-(4-sulfamoyl- Compound characterization. (E)-3-(3,4-dimethoxy- phenylcarbamothioyl)acrylamide (12) 1H-NMR (400 phenyl)-N-(3-sulfamoylphenylcarbamothioyl) acrylamide MHz, d6-DMSO) [ppm] 6.10 (s, 2H), 6.89 (d, J ¼ 1 (5) H-NMR (400 MHz, d6-DMSO) [ppm] 3.81 (s, 15:9 Hz, 1H), 7.01 (d, J ¼ 8:1 Hz, 1H), 7.16 (s, 1H), 6H), 6.96 (d, J ¼ 15:9 Hz, 1H), 7.04–7.06 (m, 1H), 7.19 (d, J ¼ 8:1 Hz, 1H), 7.38 (brs, 2H), 7.70 (d, J ¼ 7.22–7.25 (m, 2H), 7.44 (s, 2H), 7.58–7.62 (m, 1H), 15:9 Hz, 1H), 7.82–7.90 (m, 4H), 11.6 (s, 1H), 12.9 (s, 13 7.69–7.74 (m, 2H), 7.86 (d, J ¼ 7:8 Hz, 1H), 8.17 1H) C-NMR (100 MHz, d6-DMSO) [ppm] 101.7, (s, 1H), 11.6 (s, 1H), 12.8 (s, 1H) 13C-NMR (100 MHz, 106.4, 108.7, 117.3, 123.9, 124.7, 126.1, 128.2, 140.5, d6-DMSO) [ppm] 55.4, 55.6, 110.3, 111.8, 117.1, 141.0, 144.6, 147.9, 149.6, 166.2, 178.9 HRMS m=z þ 121.3, 122.8, 123.2, 126.8, 127.5, 129.4, 138.3, 144.5, calcd for C17H15N3O5S2 ([M + H] ) 406.0531, found 145.0, 148.9, 151.4, 166.6, 179.4 HRMS m=z calcd for 406.0538. þ C18H19 N3O5S2 ([M + H] ) 422.0844, found 422.0860. (E)-3-(3-methoxyphenyl)-N-(4-sulfamoylphenylcar- 1 (E)-N-(4-sulfamoylphenylcarbamothioyl)-3-[3-(trifluo- bamothioyl)acrylamide (13) H-NMR (400 MHz, d6- romethyl)phenyl]acrylamide (6) 1H-NMR (400 MHz, DMSO) [ppm] 3.80 (s, 3H), 7.05 (d, J ¼ 15:6 Hz, 1H), d6-DMSO) [ppm] 7.21 (d, J ¼ 16:1 Hz, 1H), 7.40 7.06–7.10 (m, 1H), 7.20 (m, 1H), 7.22 (d, J ¼ 8:0 Hz, (brs, 2H), 7.72 (t, J ¼ 7:7 Hz, 1H), 7.81–8.0 (m, 8H), 1H), 7.39 (brs, 2H), 7.37–7.40 (m, 1H), 7.75 (d, J ¼ 13 13 11.7 (s, 1H), 12.7 (s, 1H) C-NMR (100 MHz, d6- 15:9 Hz, 1H), 7.82–7.90 (m, 4H) C-NMR (100 MHz, DMSO) [ppm] 121.8, 124.0, 124.5, 126.1, 126.8, d6-DMSO) [ppm] 55.1, 113.1, 116.6, 119.6, 120.5, 129.5, 129.8, 130.1, 131.6, 135.0, 140.4, 141.1, 142.5, 123.9, 126.1, 130.1, 135.2, 140.5, 141.1, 144.5, 159.4, 165.5, 178.8 HRMS m=z calcd for C17H14 F3N3O3S2 165.9, 178.9 HRMS m=z calcd for C17H17N3O4S2 ([M + H]þ) 430.0507, found 430.0509. ([M + H]þ) 392.0738, found 392.0746. (E)-3-(3,4-dichlorophenyl)-N-(4-sulfamoylphenylcar- (E)-3-(2-methoxyphenyl)-N-(4-sulfamoylphenylcar- 1 1 bamothioyl)acrylamide (8) H-NMR (400 MHz, d6- bamothioyl)acrylamide (14) H-NMR (400 MHz, d6- DMSO) [ppm] 7.14 (d, J ¼ 15:9 Hz, 1H), 7.39 (brs, DMSO) [ppm] 3.99 (s, 3H), 7.04 (t, J ¼ 7:5 Hz, 1H), 2H), 7.62 (d, J ¼ 8:3 Hz, 1H), 7.74–7.77 (m, 2H), 7.82– 7.11–7.14 (m, 1H), 7.16 (d, J ¼ 15:9 Hz, 1H), 7.39 (brs, 7.89 (m, 4H), 7.89–7.92 (m, 1H), 11.7 (s, 1H), 12.7 2H), 7.43–7.47 (m, 1H), 7.55 (d, J ¼ 7:5 Hz, 1H), 7.82– 13 (s, 1H) C-NMR (100 MHz, d6-DMSO) [ppm] 121.9, 7.91 (m, 4H), 7.95 (d, J ¼ 15:9 Hz, 1H), 11.7 (s, 1H), 13 124.0, 126.1, 127.5, 130.1, 131.2, 131.7, 132.8, 134.7, 12.9 (s, 1H) C-NMR (100 MHz, d6-DMSO) [ppm] 140.4, 141.1, 141.7, 168.3, 184.3 HRMS m=z calcd 55.6, 111.8, 119.9, 120.7, 122.1, 123.8, 126.1, 129.1, þ for C16H13Cl2N3O3S2 ([M + H] ) 429.9853, found 132.3, 139.9, 140.5, 141.0, 158.1, 166.6, 178.9 HRMS þ 429.9844. m=z calcd for C17H17N3O4S2 ([M + H] ) 392.0738, (E)-3-(3,4,5-trimethoxyphenyl)-N-(4-sulfamoylphenyl- found 392.0755. 1 carbamothioyl)acrylamide (9) H-NMR (400 MHz, d6- (E)-N-{4-[N-(6-methoxypyridazin-3-yl)sulfamoyl]- DMSO) [ppm] 3.71 (s, 3H), 3.83 (s, 6H), 6.99 (s, 2H), phenylcarbamothioyl}cinnamamide (15) 1H-NMR (400 7.05 (d, J ¼ 15:7 Hz, 1H), 7.38 (brs, 2H), 7.71 (d, J ¼ MHz, d6-DMSO) [ppm] 3.82 (s, 3H), 7.07 (d, J ¼ 15:7 Hz, 1H), 7.82–7.90 (m, 4H), 11.6 (s, 1H), 12.8 (s, 15:9 Hz, 1H), 7.41 (brs, 1H), 7.46–7.47 (m, 3H), 7.63 (m, 13 1H) C-NMR (100 MHz, d6-DMSO) [ppm] 55.8, 2H), 7.77 (d, J ¼ 15:9 Hz, 1H), 7.84–7.9 (m, 6H), 11.7 13 60.1, 105.6, 118.9, 124, 126.1, 129.4, 139.7, 140.5, (s, 1H), 12.8 (s, 1H) C-NMR (100 MHz, d6-DMSO) 141.1, 144.6, 152.9, 166, 178.9 HRMS m=z calcd for [ppm] 54.5, 119.5, 123.8, 126.6, 128.1, 129.0, 130.7, þ C19H21N3O6S2 ([M + H] ) 452.0950, found 452.0964. 133.8, 140.6, 144.6, 166.0, 178.7 HRMS m=z calcd for þ 2-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenylcar- C21H19N5O4S2 ([M + H] ) 470.0956, found 470.0959. 1 bamothioyl)acetamide (10) H-NMR (400 MHz, d6- (E)-4-{3-[3-(3,4-dimethoxyphenyl)acryloyl]thiourei- 1 DMSO) [ppm] 3.31 (s, 2H), 3.72 (s, 3H), 3.74 (s, do}benzamide (16) H-NMR (400 MHz, d6-DMSO) 3H), 6.83–6.64 (m, 3H), 7.36 (brs, 2H), 7.73–7.80 [ppm] 3.81 (s, 3H), 3.85 (s, 3H), 6.97 (d, J ¼ 15:9 Hz, (m, 4H), 11.7 (s, 1H), 12.5 (s, 1H) 13C-NMR (100 MHz, 1H), 7.22–7.24 (m, 2H), 7.38 (brs, 1H), 7.71 (d, d6-DMSO) [ppm] 41.9, 55.4, 55.5, 111.7, 113.2, 121.4, J ¼ 15:9 Hz, 1H), 7.79–7.91 (m, 4H), 7.98 (brs, 1H), 13 124.1, 126.0, 126.2, 140.4, 141.1, 147.7, 148.3, 173.1, 11.5 (s, 1H), 12.9 (s, 1H) C-NMR (100 MHz, d6- þ 178.9 HRMS m=z calcd for C17H19N3O5S2 ([M + H] ) DMSO) [ppm] 55.3, 55.6, 110.1, 111.6, 117.0, 122.7, 410.0844, found 410.0828. 123.0, 126.6, 127.9, 131.4, 140.1, 144.8, 148.7, 151.1, 3,4-dimethoxy-N-(4-sulfamoylphenylcarbamothioyl)- 166.4, 166.9, 178.6 HRMS m=z calcd for C19H19N3O4S 1 þ benzamide (11) H-NMR (400 MHz, d6-DMSO) ([M + H] ) 386.1174, found 386.1173. 2742 I. MIYAZAKI et al.

Human cDNA library

Cloning genes P CMV IE pUC of interest ori MCS HSV TK poly A RFP pDsRed- Express-N1 4.7 kb Sequencing Kanr/ r SV40 and collection Neo poly A GLORIA f1 P ori P SV40 e Transfection SV40 ori and expression

Transfection pDsRed- and expression Express-N1 HEK293T (control)

Expression of Expression of RFP RFP-fused protein

Preparation of cell lysates and incubation with the slides

Ex. 532 nm Ex. 532 nm Em. 575 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)- Results 1 acrylamide (17) H-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 Development of a systematic chemical array method (d, J ¼ 8:0 Hz, 1H), 7.21–7.24 (m, 1H), 7.25 (brs, 2H), of searching novel ligands 7.56 (d, J ¼ 15:6 Hz, 1H), 7.75–7.84 (m, 4H), 10.5 (s, To screen a tremendous number of combinations of 13 1H) C-NMR (100 MHz, d6-DMSO) [ppm] 55.4, 55.5, compounds and human proteins, we designed a system- 109.9, 111.6, 118.5, 119.2, 121.8, 126.6, 127.1, 138, atic ligand-screening strategy using a chemical array that 140.9, 142.1, 148.7, 150.3, 164.0 HRMS m=z calcd for relied on two original libraries, the GLORIA gene þ C17H18N2O5S ([M + H] ) 363.1014, found 363.1021. library, and the NPDepo (RIKEN Natural Products i.3. Fig. BadC pte with spotted C) and (B AIi LRAi 5(e a) hs aawr acltdfo vrgsb ulct oiin naslide. a on positions duplicate of by number averages gene from The GLORIA). calculated in were 1–33 data nos. These (Gene proteins bar). RFP-fused (red different 15 33 expressed is that GLORIA lysates in cell with CAII treated were slides glass The ,Fursetiaeb egddslyaayi fe ramn ihcl yae htepesCI-F n F.Topositions Two RFP. and CAII-RFP express that lysates cell with treatment after analysis display merged by image Fluorescent A, A B A dnicto fNvlSeicLgnsfrCAII. for Ligands Specific Novel of Identification 4 3 2 1 BDBCD ABCDAB 2 1 and Signal/Noise ratio 10 20 30 40 2 0 r niae srdarw.TeSNrtob egddslyaayi ttepstossotdwith spotted positions the at analysis display merged by ratio S/N The arrows. red as indicated are

Rapamycin FKBP12-mRFP Duplicated spots dnicto fNvlIhbtr fCroi nyrs I2743 II Anhydrase Carbonic of Inhibitors Novel of Identiﬁcation

1 FKBP12-RFP C B i.2. Fig. oun.Dt eecluae rmaeae ydpiaepositions duplicate slide. lysates by a averages cell on from a calculated with (open were of FKBP12-mRFP Data treated column). or image of slides bar) ﬂuorescence (closed Comparison glass FKBP12-RFP the B, overexpressing on in marker. area ratio) position rapamycin-spotted 144 (S/N a every ratio in as spots signal-to-noise used rhodamine Two were rapamycin arrows. with compounds white spotted positions by then two indicated slides; were The glass are the scanned. cells with were with the incubated slides were h transfected the 24 lysates cell After were the RFP. and cells lysed, or HEK293T FKBP12-RFP encoding duplicate. vectors in slides glass

Signal/Noise ratio Signal/Noise ratio on immobilized were NPDepo from compounds 2011 The A, 11 13 15 14 19 24 29 0 1 3 5 7 9 0 4 9 dnicto fRpmcna nFB1-idn Ligand. FKBP12-Binding an as Rapamycin of Identiﬁcation 01 02 033 30 25 20 15 10 5 1 1101520253033 5 1 2 (Gene no. ofGLORIA) (Gene no. ofGLORIA) Rapamycin 1 B and (B) 2 (C). 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 HPSE Q9Y251 26 MMP3 P08254 51 ERP27 Q96DN0 76 PARK7 Q99497 2 FKBP1A P62942 27 ARF6 P62330 52 TXNDC4 Q9BS26 77 CDA P32320 3 PCBD1 P61457 28 MOCS2 O96033 53 PER1 Q6IN51 78 ASF1A Q9Y294 4 TIMP1 P01033 29 CDC42EP3 Q9UKI2 54 PSMG2 Q6IAH4 79 ARF1 P84077 5 AGR2 O95994 30 FDPS P14324 55 NDUFC2 O95298 80 DSTN P60981 6 RNF34 Q969K3 31 MMP13 P45452 56 TUBA1A Q71U36 81 MDM2 Q00987 7 PPP6C O00743 32 TIMP2 P16035 57 TUBG1 P23258 82 DNAJC10 Q8IXB1 8 BCL2L1 Q07817 33 GGPS1 O95749 58 PPME1 Q9Y570 83 PTGS1 P23219 9 MAD2L2 Q9UI95 34 DHFR P00374 59 ERP29 P30040 84 MED31 Q9Y3C7 10 SFN P31947 35 BCL2L1 Q07817-2 60 BLMH Q13867 85 MYCBP Q99417 11 S100A4 P26447 36 HIG2 Q9Y5L2 61 TGFA P01135 86 SUB1 P53999 12 S100A10 P60903 37 MAPK1 P28482 62 ATF1 P18846 87 COX7A2L O14548 13 CDC37 Q16543 38 PCOTH Q58A44 63 TBCA Q6FGD7 88 E2F6 O75461 14 AURKB Q96GD4 39 PPP2CA P67775 64 PCBD2 Q9H0N5 89 IL1A P01583 15 CA2 P00918 40 BIRC5 O15392 65 HBXIP O43504 90 GARS P41250 16 NME2 P22392 41 PDPN Q86YL7 66 MMP8 P22894 91 SKP1 P63208 17 IGF2 P01344 42 NME1 P15531 67 MMP10 P09238 92 RBX1 P62877 18 EIF4EBP1 Q13541 43 MIRH1 Q75NE6 68 PLAU P00749 93 AES Q08117 19 GRB2 P62993 44 YARS P54577 69 MAP2K1IP1 Q9UHA4 94 REG4 Q9BYZ8 20 PDGFB P01127 45 WARS P23381 70 TP53AP1 Q9Y2A0-4 95 NUDT1 P36639 21 HSPB1 P04792 46 IARS P41252 71 C2orf12 Q8TDM3 96 NEU1 Q99519 22 P4HB P07237 47 SARS P49591 72 MAX P61244 97 TUBB P07437 23 MAP2K1 Q02750 48 IGF1 Q9NP10 73 DEFA1 P59665 98 MMP9 P14780 24 CENPA P49450 49 PDIA3 P30101 74 KARS Q15046 99 PPP1CA P62136 25 PDIA4 P13667 50 TXNDC12 O95881 75 RAN P62826 100 JUN P05412

Gene names and UniProt nos. are based on the UniProt website (http://beta.uniprot.org/). , splicing variant.

Depository) chemical library (Fig. 1). To use GLORIA array method can be used in the detection of known in connection with the robust chemical array system, we interactions, we used 2,011 compounds from NPDepo. cloned 100 genes and designed them to be able to express Cell lysates that expressed RFP-fused FKBP12 protein RFP-fused proteins of interest. HEK293T cells were and RFP were prepared. After incubation of glass slides transfected with vectors that encoded RFP-fused proteins with the cell lysates, the images treated with cell lysates of interest or RFP, and cell lysates were prepared. There that expressed RFP-fused FKBP12 or RFP were colored are several reasons to use cell lysates instead of purified red and green respectively, and the two images were proteins for high-throughput screening: (i) cell lysates merged onto a map. We detected rapamycin specifically are very easy and fast to prepare, and (ii) target proteins as an FKBP12-binding ligand from glass slides that must be folded in their natural conformation and were spotted in duplicate with the 2,011 compounds modified properly. Moreover, this chemical array screen- (Fig. 2A). To determine whether the signal:noise ration ing method contains another property called merged was changed by tetrameric or monomeric RFP, the signal display analysis, which excludes false positive signals. intensities of the slides treated with FKBP12-RFP and Merged display analysis is done by the following steps: FKBP12-mRFP were compared. Although binding sig- Positive signals on glass slides after incubation with cell nals due to cell lysates expressing FKBP12-RFP and lysates that express RFP-conjugated proteins of interest -mRFP were clearly observed, the use of FKBP12-RFP or RFP are colored red and green respectively on a yielded stronger signals. The S/N ratio of a spot treated computer. Then the colored figures are merged onto one by FKBP12-RFP was 2–3 fold higher than that of a spot map. It can highlight false positive signals as yellow, treated by FKBP12-mRFP (Fig. 2B). Hence we decided caused by binding of ligands to RFP or by autofluor- to use RFP-fused proteins of interest with our screening escent signal of the ligand itself. Hence it is suggested system. that the results due to this method give only real-hit compounds, without false positive signals. Development of GLORIA to be applied to chemical array screening with cell lysates Identification of rapamycin as an FKBP12-binding Next we sought to construct a human gene library, protein by this method GLORIA, for our platform. We selected many human NPDepo was established recently by our laboratory.24) disease-associated genes, including cancer and glauco- The compounds from NPDepo were spotted in duplicate ma. We cloned 100 genes and inserted them into onto PALC glass slides6) so that binding signals and pDsRed Express-N1 vector, because the interaction of noise could be easily distinguished by comparing small molecules with fluorescent protein-fused proteins duplicate slides. To confirm whether the unique chemical is very easy to detect (Table 1). The proteins encoded in Identification of Novel Inhibitors of Carbonic Anhydrase II 2745 A

Compound Structure IC50 (nM) F H H 1 N N 24 2.9 O S SO2NH2 O H H 2 O N N 50 12 O S SO2NH2 O H H O N N 3 O N 5400 67 O S S N N O H

N N acetazolamide 34 3.0 SO2NH2 H3COCHN S

1 2

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)of1 (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 forms, such as membrane proteins (e.g., Bcl-xL and activity (7%), hydrolase activity, (15%), lyase activity Aggrus). Furthermore, many kinds of post-translational (2%), and non-enzyme proteins (60%). For example, modiﬁcations occur in the proteins encoded in GLORIA, transferases contain protein kinases, which include some but if these proteins were expressed by E. coli, they clinical therapeutic targets (e.g., aurora kinase B and would not be modiﬁed. In sum, it appears that the MEK1). Although many proteins are expressed in full- proteins encoded in GLORIA might be useful for our length, some were designed to be expressed in truncated chemical array method. 2746 I. MIYAZAKI et al. A

H N R1

IC IC CompoundR R 50 Compound R R 50 1 2 (nM) 1 2 (nM)

O H H 4 N (p-)-SO2NH2 31 2.3 12 O N (p-)-SO2NH2 43 6.0 O S O S

O H H N 13 N 5 O (m-)-SO2NH2280 29 O (p-)-SO2NH2 36 2.1 O S O S

H H 6 N (p-)-SO NH 14 N (p-)-SO2NH2 F3C 2 2 60 2.9 54 11 O S O O S

H O N H (p-) 7 (p-)-SO NH 15 N O 2 2 30 4.2 N >10000 O S S N N O S O H

Cl O H H 8 Cl N 16 (p-)-CONH2 (p-)-SO2NH2 44 12 O N >10000 O S O S

O O O 9 H (p-)-SO NH 17 (p-)-SO NH O N 2 2 52 22 O 2 2 6.8 0.79 O S O

H O N 10 18 (p-)-SO2NH2 (p-)-SO2NH2 47 4.7 320 98 O S O O

O H 11 O N (p-)-SO2NH2 39 2.5 19 H (p-)-SO2NH2 1700 610 O S

Fig. 5. Continued.

Identification of novel ligands for carbonic anhydrase To evaluate the inhibitory activities and binding II by the chemical array platform abilities of 1 and 2 to CAII, we performed an enzyme To obtain novel ligands for CAII, we spotted about assay and ITC measurement. The enzyme assay was 10,000 compounds from NPDepo, and then carried out done in buffer with CAII and 4-nitrophenylacetate as screening with cell lysates from HEK293T-expressed substrates.21,22) 1 and 2 showed strong inhibitory CAII-RFP. After merged display analysis, we identified activity, the same as acetazolamide, a well-known CAII two compounds as CAII-binding compounds, 1 and 2, inhibitor (Fig. 4A). Moreover, ITC measurements dem- out of 10,000 compounds (Fig. 3A). To investigate the onstrated that the Kd values of 1 and 2 for CA were specificity of 1 and 2 to CAII, we prepared cell lysates 159 nM and 508 nM, respectively (Fig. 4B and C). On of HEK293T cells that expressed 33 different RFP- the other hand, compound 3, which lacks the sulfona- fused proteins (gene numbers 1–33 of GLORIA, mide moiety changed to 4,6-dimethylpyrimidine, was Table 1), including CAII (no. 15 of GLORIA). Among not detected by the array analysis. 3 shows very weak the 33 proteins tested, 1 (Fig. 3B) and 2 (Fig. 3C) were inhibitory activity (Fig. 4A) and has undetectable Kd bound specifically to cell lysates expressed CAII-RFP. value (data not shown). It has been found that the Identification of Novel Inhibitors of Carbonic Anhydrase II 2747 C

O H N O O 17 SO2NH2

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 Discussion interacting with CAII. Taken together with previous CA inhibitor reports,16,25) these data strongly suggest that 1 Although chemical arrays represent one of the most and 2 bound to active site of CAII, and chelated zinc promising and high-throughput approaches to searching ion in the active site with the sulfonamide moiety. This ligands against proteins of interest, the discovery of binding might have resulted in the enzymatic inhibitory novel interactions between proteins and small molecules effect. using chemical arrays has been limited.7,8,26,27) It has been reported that the ratio is one hit per 120,000 Structure-activity relationship study compounds for enzyme inhibitors.28) Hence efficient To obtain more potent inhibitors of CAII, we screening of a tremendous number of combinations of synthesized compounds 5–6 and 8–17, and purchased proteins and small molecules is necessary. 4, 7, 18, and 19 (Fig. 5A and B). The meta-position of Matsuyama et al. have reported a large-scale study sulfonamide moieties on the phenyl ring (5) resulted in a of the fission yeast Schizosaccharomyces pombe based loss of potency against CAII as compared with the para- on cloning of the ORFeome.29) Their system has made position (2). Compounds 15 and 16 lacked sulfonamide it possible to screen for ligands to yeast proteins in a moieties on R2, which are thought to be an important large-scale manner, and it can be applied to the group for interaction with the zinc ions of enzymes.25) chemical array screening method. However, some As expected, the inhibitory activities of 15 and 16 for proteins associated with human diseases have potential CAII dramatically decreased. Although modification of as drug targets, and they are expressed only in R1 appeared to be tolerant of inhibitory activity, 18 and mammalian cells (cf. growth factors). In this study, 19 showed weak inhibitory activities. When exploring we established that GLORIA contained 100 genes variations at the R1 position, we found that 17 showed many of which play important roles in human diseases. the strongest inhibitory activity among the derivatives We also recognized that GLORIA and NPDepo are not tested and had strong binding affinity, with a Kd value of definitive. For example, the current number of com- 66 nM (Fig. 5C). pounds and proteins in the libraries described here is 2748 I. MIYAZAKI et al. not sufficient to cover the vast interactions between Davenport, L., Desilets, R., Dietz, S., Dodson, K., Doup, proteins of interest and small molecules. Cloning of L., Ferriera, S., Garg, N., Gluecksmann, A., Hart, B., 100 genes is nothing extraordinary in modern molecu- Haynes, J., Haynes, C., Heiner, C., Hladun, S., Hostin, lar biology, and hence we are trying to increase the D., Houck, J., Howland, T., Ibegwam, C., Johnson, J., number of genes and proteins in GLORIA and Kalush, F., Kline, L., Koduru, S., Love, A., Mann, F., May, D., McCawley, S., McIntosh, T., McMullen, I., NPDepo. Moy, M., Moy, L., Murphy, B., Nelson, K., Pfannkoch, In summary, our screening method can perform C., Pratts, E., Puri, V., Qureshi, H., Reardon, M., large-scale chemical array screening in combination Rodriguez, R., Rogers, Y. H., Romblad, D., Ruhfel, B., with a gene library, GLORIA, and a chemical library, Scott, R., Sitter, C., Smallwood, M., Stewart, E., Strong, NPDepo. The development of CAII inhibitors indicates R., Suh, E., Thomas, R., Tint, N. N., Tse, S., Vech, C., the utility of our screening method in the identification Wang, G., Wetter, J., Williams, S., Williams, M., of useful ligands to human protein. To discover of Windsor, S., Winn-Deen, E., Wolfe, K., Zaveri, J., these ligands for human proteins, the numbers of genes Zaveri, K., Abril, J. F., Guigo´, R., Campbell, M. J., in GLORIA and of compounds in NPDepo must be Sjolander, K. V., Karlak, B., Kejariwal, A., Mi, H., increased. This should help to remove one of the Lazareva, B., Hatton, T., Narechania, A., Diemer, K., bottlenecks to in the discovery of useful tools for Muruganujan, A., Guo, N., Sato, S., Bafna, V., Istrail, S., Lippert, R., Schwartz, R., Walenz, B., Yooseph, S., modern chemical biology studies and drug discovery Allen, D., Basu, A., Baxendale, J., Blick, L., Caminha, research. M., Carnes-Stine, J., Caulk, P., Chiang, Y. 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