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Maxim Pharmaceuticals, Inc., San Diego, California

Requests for reprints: Shailaja Kasibhatla, Maxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, CA 92121. Phone: 858-202-4042; Fax: 858-202-4000. E-mail: skasibhatla{at}maxim.com

	Abstract

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A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics–based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-ß1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.

Key Words: apoptosis • cyclin D1 • high throughput screening • chemical genetics • Caspases • New targets • Combination chemotherapy

	Introduction

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Many chemotherapeutic drugs are known to induce apoptosis. Abnormal proliferation and failure to activate apoptosis are both the major contributors leading to malignant cellular transformation. Many of the current chemotherapeutics, designed to inhibit proliferation or induce apoptosis, are often less selective and may result in damage to normal cells limiting their clinical potential. However, selectively targeting apoptotic abnormalities in tumor cells could generate a potent, and possibly selective, proapoptotic stimulus leading to tumor cell destruction.

A family of cysteine proteases called caspases are key mediators of apoptosis (1, 2). Although the major pathways of apoptosis are well characterized, the numerous molecular mechanisms by which many cytotoxic agents induce apoptosis are not easily elucidated by genomic methods. The molecular elucidation of therapeutic targets and pathways involved in the induction of apoptosis by pharmacologic agents would be valuable for the discovery of new targets as well as in defining new functions for proteins not previously recognized to be coordinated with apoptosis.

Chemical genetics is defined as the use of chemical compounds to perturb systematically and determine the functions of cellular proteins similar to mutational genetics (3, 4). We combined our cell-based screening with chemical genetics and identified small molecules that induce an apoptotic phenotype in cells and subsequently identify the cellular target involved in perturbing the biological pathway.

In this report, we report the discovery of 3,5-diaryl-[1,2,4]-oxadiazoles, as apoptosis-inducers with potential tumor-specific activity. Through structure-activity relationship studies, MX-126374 is identified as a lead compound with in vivo activity. To establish the mechanism of action, we used Affymetrix (Santa Clara, CA) gene arrays (U133) and profiled the expression with probe sets using two of the potent analogues, MX-74420 and MX-126374. The genes that were differentially expressed were verified by real-time quantitative PCR. Some of the key genes that were identified during pathway analysis [transforming growth factor (TGF)-ß1, cyclin D1, p21, and insulin-like growth factor (IGF)-BP3] were further investigated. Based on structure-activity relationship studies, an azido- and tritium-labeled photoaffinity agent was prepared and used for the identification of tail-interacting protein 47 (TIP47), a 47 kDa protein that binds selectively to the cytoplasmic domains of cation-independent and cation-dependent IGF-II/M-6-P receptor, as the molecular target for these compounds (5). Understanding the mechanism of apoptosis in the above context helped to validate TIP47 as a potential therapeutic target. The current work reports on a novel screening platform that allows the identification of compounds that target known and novel pathways to induce apoptosis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells Lines and Reagents
T47D, SKBr-3, and SW620 cells were purchased from American Type Tissue Culture (Manassas, VA) and were cultured in RPMI 1640 with 25 mmol/L Hepes, with L-glutamine, plus 10% fetal bovine serum and penicillin/streptomycin. Human umbilical vascular endothelial cells (HUVEC) and human mammary epithelial cells (HMEC) were purchased from Cambrex Corporation (East Rutherford, NJ) and were grown in endothelial growth medium and mammary epithelial growth medium, respectively. The 3,5-diaryl-oxadiazoles used include ³H-MX-126911 (American Radiolabeled Chemicals, St. Louis, MO), MX-126911, MX-126374, and MX-74420.

Chemical Synthesis
3-(4-Chloro-phenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole (MX-74420): a solution of 3-chloro-thiophene-2-carbonyl chloride (1.45 g, 8 mmol) and 4-chlorobenzamidoxime (1.37 g, 8 mmol) in dioxane/pyridine (10:1, 110 mL) was refluxed for 12 hours and cooled to room temperature. To produce precipitates, 200 mL of water was added to the stirred solution. The solid was collected by filtration and washed with water (4 x 20 mL), dried to yield 2.36 g colorless sample, which was further purified by column chromatography (silica gel, ethyl acetate/hexane, 1:10) to yield 2.01 g (85%) of the title compound. [¹H]-Nuclear magnetic resonance (CDCl₃): 8.10 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 5.1 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 5.1 Hz, 1H).

3-(5-Chloro-pyridin-2-yl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole (MX-126374) was prepared similar to MX-74420 from reaction of 5-chloro-pyridine-2-amidoxime and 3-chloro-thiophene-2-carbonyl chloride. [¹H]-Nuclear magnetic resonance (CDCl₃): 8.78 (dd, J = 2.47, 0.83 Hz, 1H), 8.18 (dd, J = 8.38, 0.69 Hz, 1H), 7.86 (dd, J = 8.51, 2.47 Hz, 1H), 7.64 (d, J = 5.22 Hz, 1H), 7.14 (d, J = 5.22 Hz, 1H).

5-(3-Chlorothiophen-2-yl)-3-(pyridin-2-yl)-[1,2,4]-oxadiazole (MX-116839) was prepared similar to MX-74420 from reaction of pyridine-2-amidoxime and 3-chloro-thiophene-2-carbonyl chloride. [¹H]-Nuclear magnetic resonance (CDCl₃): 8.85 (d, J = 4.67 Hz, 1H), 8.22 (d, J = 7.97 Hz, 1H), 7.88 (td, J = 7.76, 1.74 Hz, 1H), 7.63 (d, J = 5.49 Hz, 1H), 7.46 (dd, J = 7.69, 4.67 Hz, 1H), 7.14 (d, J = 5.22 Hz, 1H).

3-(4-azidophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole (MX-126911): sodium nitrite (3.8 mg, 0.055 mmol) in water (0.5 mL) was added to a mixture of 3-(4-aminophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole (15.5 mg, 0.05 mmol) in acetic acid (2 mL) and sulfuric acid (0.3 mL). The mixture was stirred vigorously at 0 to 5°C for 20 minutes, and then sodium azide (3.6 mg, 0.055 mmol) in water (0.5 mL) was added. It was stirred at 0 to 5°C for 3 hours and then poured into ice water (30 mL). The resultant mixture was extracted with ethyl acetate (3 x 10 mL). The organic layer was washed with water, dried over anhydrous sodium sulfate, and evaporated. The crude residue was purified by flash chromatography to yield 16 mg (100%) of the title compound. [¹H]-Nuclear magnetic resonance (CDCl₃): 8.18 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 5.4 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 5.4 Hz, 2H).

3-(3,5-Ditritium-4-azidophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole (³H-MX-126911): the tritiated-azido compound was prepared by a procedure similar as the nonlabeled compound by using 3-(3,5-ditritium-4-aminophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole as the starting materials. 3-(3,5-Ditritium-4-aminophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole was prepared by reaction of 3-(4-amino-3,5-diiodophenyl)-5-(3-chloro-thiophen-2-yl)-[1,2,4]-oxadiazole with T₂ in the presence of a metal catalyst. The tritiated azido compound was purified by HPLC, with chemical and radiochemical purity of >98%, and specific activity of 40 to 50 Ci/mmol.

Caspase Induction Assay
Activity of caspase 3 was measured using the methods described previously (6). Briefly, cells were incubated with the test compounds in 384-well plates for 24 hours and the samples were treated with the profluorogenic substrate, N-(Ac-DEVD)-N'-ethoxycarbonyl-R110 (7), and incubated for 3 hours in caspase buffer containing 10 mmol/L DTT. The fluorescent signal was measured using a fluorescent plate reader (Tecan Model Spectraflour Plus). The EC₅₀ was determined by a sigmoidal dose-response calculation (XLFit3, IDBS) and represents the concentration of compound that produces the 50% maximum response.

Cell Proliferation Assay
The CellTiter 96 AQ_ueous assay by Promega (Madison, WI) was used to determine the 50% growth inhibition (GI₅₀) values for the compounds according to the manufacturers instructions. Briefly, cells were plated at 2 x 10³ cells/96-well plates. Compounds were serially diluted and then added to the plates containing the cells. The plates were then incubated at 37°C for 48 hours, followed by the addition of 20 µL of soluble tetrazolium salt (MTS) and read at 490 nm after 4 hours.

RNA Analysis, cDNA Synthesis, Quantitative Reverse Transcriptase-PCR, and Small Interfering RNA Analysis
T47D cells were plated at 1 x 10⁶ cells/100 mm plate and were allowed to attach overnight. Cells were then treated with DMSO or MX-77356 analogues at 2 x EC₅₀ (5–7 µmol/L) for 18 hours. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total RNA was quantitated, denatured, and electrophoresed in an agarose-formaldehyde gel to determine integrity of total RNA.

cDNA synthesis and quantitative PCR were done by standard methods. Briefly, 2 µg of total RNA was then used to make cDNA by reverse transcription using the Retroscript cDNA synthesis kit (Ambion, Austin, TX) according to the manufacturer's instructions. Quantitative PCR was done by Sybrgreen incorporation using the Quantitect kit (Qiagen, Valencia, CA) as measured on a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) using standard conditions. Data was normalized against the housekeeping gene, cyclophylin. Results for cells transfected with cyclophilin as a control were normalized against glyceraldehyde-3-phosphate dehydrogenase.

TIP47 small interfering RNA (siRNA) oligos were chemically synthesized by Ambion. The target sequence for TIP47 siRNA was 5' AAC AGA GCT ACT TCG TAC GTC' (obtained from Genbank AF057140), the control siRNA oligo, human cyclophilin was also from Ambion (8). T47D cells were preplated overnight and grown to 50% confluency. siRNAs were transfected into the cells using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, siRNA (at a final concentration of 100 nmol/L) and lipid were individually diluted in low serum media, Opti-MEM (Invitrogen), and allowed to incubate for 10 to 30 minutes after which they were combined and allowed to form lipid complexes for 20 minutes. The lipid complexes were added onto the cells and allowed to incubate for 48 hours. Cells were then harvested for RNA and protein as described above and below.

DNA Microarray
RNA was isolated from T47D cells for microarray analysis using a single-step extraction with TRIzol reagent (Invitrogen, Carlsbad, CA). RNA quality and integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and absorbance at A260/A280. Only high-quality RNA, having a 28S/18S rRNA ratio of 1.5:2 and an A260/280 ratio of 1.8:2 was used for further experimentation.

Double-stranded cDNA synthesis, biotin-labeled cRNA synthesis, and cRNA fragmentation were all conducted as described in the Affymetrix GeneChip expression analysis protocol. According to this protocol, first strand synthesis of cDNA was done using total RNA, a specialized T7- (dT) 24 primer (Genset, San Diego, CA), and Superscript (SSII) reverse transcriptase (Invitrogen). Second strand synthesis was completed with T4 DNA Polymerase, RNase H, and DNA Ligase. Double-stranded cDNA was then cleaned by phenol-chloroform extraction with Phase Lock Gels (Eppendorf, Westbury, NY), ethanol-precipitated, and resuspended in RNase-free water. Purified cDNA was then used as a template to develop biotin-labeled cRNA by in vitro transcription labeling with a HighYield BioArray RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Labeled cRNA was cleaned with RNeasy columns (Qiagen) and fragmented to a length of 35 to 200 bases. The quality of in vitro transcription and fragmentation products was assessed using the Agilent 2100 Bioanalyzer.

Array Hybridization
Affymetrix HG-U133A oligonucleotide arrays, representing approximately 22,500 transcripts, were hybridized at 45°C overnight with 15 µg of fragmented, biotin-labeled cRNA as defined in the Affymetrix expression analysis protocol. The hybridization buffer contained 100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L EDTA, 0.01% Tween 20, four eukaryotic hybridization controls (1.5 pmol/L BioB, 5 pmol/L BioC, 25 pmol/L BioD, and 100 pmol/L cre), 0.1 mg/mL herring sperm DNA (Promega), and 0.5 mg/mL of acetylated bovine serum albumin. After hybridization, the arrays were washed and stained with an Affymetrix fluidics station 450 following the antibody amplification washing and staining protocol (Affymetrix).

The arrays were scanned with the GeneChip Scanner 3000 (Affymetrix). GeneChip operating software was used for data output and the average intensity for each array was normalized by scaling to a target intensity value of 125.

Data Analysis
The cel files were exported and analyzed by ArrayAssist (Iobion, Inc., La Jolla, CA). ArrayAssist calculates the probe intensities by using robust multiarray (9), which adjusts background, normalizes, and log-transforms the PM values. Volcano plots were used to identify differentially expressed genes, between the control and drug treatments, using a fold change of 2.0 and P < 0.05 with a variance-stabilized unpaired t test. In order to understand the relationship between the samples and genes, hierarchical clustering as well as Venn diagram was used.

Identification of Drug Target
A photoaffinity labeling agent ³H-MX-126911 was designed and synthesized for target identification studies. Confluent T47D cells were scraped off dishes, washed with PBS and then lysed in cell lysis buffer (10 mmol/L HEPES, 10 mmol/L NaCl, 1 mmol/L KH₂PO₄, 5 mmol/L NaHCO₃, 1 mmol/L CaCl₂, 0.5 mmol/L MgCl₂, 5 mmol/L EDTA) plus 0.1% protease inhibitor cocktail (Sigma, St. Louis, MO). Cells were allowed to swell and then were homogenized with a Dounce homogenizer. Lysate was spun at 2,200 x g for 5 minutes and at 4°C. The supernatant was spun 100,000 x g, 40 minutes at 4°C. This resulting supernatant was called T47D cytosol. 300 µg T47D cytosol in 100 µL cell lysis buffer was pretreated with DMSO or 100 µmol/L cold analogue, MX-74420, rocking for 30 minutes at room temperature. ³H-MX-126911 (200 nmol/L; stock was 20 µmol/L, 1 mCi/mL, 50 Ci/mmol) was added to the well and allowed to rock for 30 minutes at room temperature. The plate was then exposed to a short wave UV source (254 nm) for 10 minutes at a distance of 3.5 cm.

For one-dimensional analysis, 5x sample buffer [150 mmol/L Tris (pH 6.8), 50% glycerol, 1% SDS, 62 mg/mL bromophenol blue] plus 40 mmol/L DTT was added to the labeled lysate. Samples were subjected to SDS-PAGE and the gel stained with Coomassie, destained, and incubated in Amplify (Amersham, Piscataway, NJ) then dried down on a gel dryer. The dried gel was put on Hyperfilm (Amersham) and placed at –80°C. Film was developed 5 to 7 days later.

For two-dimensional analysis, lysates were concentrated after UV exposure in a YM-30 Microcon concentrator (Millipore, Billerica, MA) according to the manufacturer's instructions. Ten microliters (300 µg) of protein sample was added to pH 4 to 7/6 to 9 rehydration buffer (Invitrogen) with 20 mmol/L DTT to a final volume of 155 µL. Sample/rehydration buffer (155 µL) was loaded into the sample loading well of the IPGRunner (Invitrogen) cassette. A nonlinear Zoom strip (pH 3–10) was then inserted into the sample well of the cassette. The cassette was placed in the IPG Runner and isoelectric focusing (first dimension) was done according to the manufacturer's instructions. SDS-PAGE (second dimension) was done by inserting the strip into a two-dimensional well of a 10% Tris-glycine gel (Invitrogen). Gels were Coomassie-stained/destained, treated with Amplify, dried and put on film as above. A duplicate two-dimensional gel of lysates that were not treated with ³H-MX-126911 was left in destain solution until autoradiography films were developed. The film (which revealed the location of a single spot) was compared with the duplicate gels to identify the location of the labeled protein (50 kDa and pI 5.3). The protein spot was excised out of the gel, analyzed by liquid chromatography and tandem mass spectroscopy sequencing at the Centre Proteomique de l'Est du Quebec (Quebec, Canada).

Proteins were identified by mass spectrometry. An ion trap mass spectrometer, liquid chromatography and tandem mass spectroscopy LCQ Deca XP (ThermoFinnigan) enabled the identification of proteins from their amino acid sequence (tandem mass spectroscopy). The protein was identified using the individual ions score threshold of 30. The 12 actual peptides matching TIP47 are as follows: DTVATQLSEAVDATR (141–155); GLDKLEENLPILQQPTEK (99–116); IATSLDGFDVASVQQQR (214–230); LEPQIASASEYAHR (85–98); LGQMVLSGVDTVLGK (181–195); QEQSYFVR (231–238); QLQGPEKEPPKPEQVESR (308–325); SEEWADNHLPLTDAELAR (196–213); SVVTGGVQSVMGSR (167–180); TLTAAAVSGAQPILSK (69–84); VASMPLISSTCDMVSAAYASTK (29–50); VSGAQEMVSSAK (129–140).

Glutathione-S-transferase-TIP47 Construction and Polyclonal TIP47 Antibody Production
Full-length TIP47 cDNA was cloned into the pGEX-4T-1, a glutathione S-transferase gene fusion system (Amersham) using standard methods. Briefly, PCR primers to the 5' and 3' region of the gene were designed to contain restriction sites that allowed for the in-frame cloning of TIP47 into the pGEX-4T-1 vector. Subsequent to sequence verification, the pGEX-TIP47 construct was transformed into the E. coli BL-21 strain. TIP47 was then expressed by growing the E. coli cells containing the pGEX-TIP47 for 6 hours after isopropyl-L-thio-ß-D-galactopyranoside induction and purified according to the manufacturer's suggested protocol. Briefly, the induced E. coli cells containing the pGEX-TIP47 were sonicated and the eluate was purified using glutathione S-transferase-Sepharose followed by Precision Protease digestion (Amersham) as described in detail in the manufacturer's instruction manual.

TIP47 antigen was provided to Genway (Genway, San Diego, CA) in the form of purified glutathione S-transferase-TIP47, which was then used to generate an anti-TIP47 IgY antibody in chicken.

Immunoprecipitation of ³H-MX-126911 Labeled TIP47
T47D cells were scraped off dishes, washed in PBS and then resuspended in cell lysis buffer as above. Cell lysis buffer cytosol was prepared as above. For competition experiments, cytosolic lysates (1 mg T47D cytosol in 1 mL cell lysis buffer) were pretreated with either DMSO or 2 µmol/L cold MX-126911 for 30 minutes at room temperature. ³H-MX-126911 (200 nmol/L) was added to the T47D cytosol and allowed to incubate, rocking, for 30 minutes at room temperature. Lysates were then exposed to a short wave UV source (254 nm) for 10 minutes.

Labeled lysates were precleared with 50 µL solution of protein A Sepharose (Zymed, South San Francisco, CA) for 2 hours at 4°C. Ten micrograms of either chicken IgY (Santa Cruz Biotechnology, Santa Cruz, CA) or chicken anti-TIP47 IgY (Genway) were incubated with the lysates for 2 hours at 4°C. Twenty-five micrograms of rabbit anti-chicken IgG was then added to the lysates and incubated for 2 hours at 4°C. To bring down the complex, 50 µL protein A Sepharose was incubated with the lysate and rocked over night at 4°C. The Sepharose was washed six times in cell lysis buffer and resuspended in 2x sample buffer (Invitrogen) plus 40 mmol/L DTT. Samples were subject to SDS-PAGE and processed for autoradiography as above.

Protein Extraction and Western Blot Analysis
Transfected cells were washed in PBS and lysed in radioimmunoprecipitation assay buffer (Upstate Biotechnologies, Lake Placid, NY). Thirty-five micrograms of protein were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Invitrogen) for Western analysis. Primary antibodies used include goat anti-actin (Santa Cruz Biotechnology), mouse anti-p21 and mouse anti-cyclin D1 (BD Biosciences PharMingen, San Diego, CA), and chicken anti-TIP47 (Genway), all used at 1 µg/mL. Secondary antibodies used include horseradish peroxidase-conjugated bovine anti-goat (Santa Cruz Biotechnology), goat anti-mouse (Bio-Rad, Hercules, CA), and goat anti-chicken (Genway). Proteins were visualized with super signal West-Pico luminol enhancer solution (Pierce, Rockford, IL).

MX-1 In vivo Tumor Model
Inbred female athymic NCr mice weighing 20 to 30 g (for the MX-1 model) were obtained from Charles River Laboratories (Wilmington, MA). Female NCr nu/nu mice were implanted s.c. by trocar in the right auxillary area with MX-1 tumor fragments. Animals were divided into control groups that received i.p. either vehicle, MX-126374, paclitaxel or in combination at the indicated dosing regimen. The compound was formulated in vehicle containing cremophor, tartaric acid, and saline. All treatments started when tumors reached an average size of approximately 100 mm³.

The tumor mass was measured thrice a week in two dimensions by calipers, and tumor volume was calculated according to the equation (l x w²)/2; where l, length; w, width (10). We did two-tailed Student's t test statistics (Prism software). To monitor the drug-associated toxicity, mice were weighed at least twice a week and inspected daily for any signs of abnormalities. The maximum tolerated dose was defined as the dose at which we observed <15% body weight loss and reversible clinical signs.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemical Genetics-Based Screening Process
A large collection of compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. We have developed a chemical genetics approach for the discovery of novel anticancer agents and molecular targets that cause apoptosis in tumor cells (Fig. 1). Starting with our novel cell-based high-throughput screening assays, chemical compounds (hits) that perturb the apoptosis pathways and induce apoptosis are identified (6). Through structure-activity relationship studies, compounds (leads) with improved chemical, pharmacologic and in vivo tumor growth inhibitory properties are selected. The mechanism of action of the leads is established through gene profiling and pathway analysis. Applying the knowledge obtained from the structure-activity relationship studies, reagents are designed, synthesized, and used for the identification of the molecular targets.

View larger version (20K): [in this window] [in a new window]   Figure 1. A schematic of the screening approach using chemical genetics.

View larger version (21K): [in this window] [in a new window]   Figure 2. A, structure of MX-74420 R2, R4 = carbon, R1, R3 = chloride, A and B = carbon. B, structure of MX-126374 R2, R4 = carbon, R1, R3 = chloride, A = nitrogen and B = carbon. C, structure of MX-116839. D, structure of MX-126911.

In the MX-1 breast cancer xenograft model, MX-126374 was tested both as a single agent and in combination with paclitaxel. Drug treatment was started after tumors, derived from cell aggregates, were established and reached an average size of approximately 100 mm³. Paclitaxel was used at its half maximum dose (10 mg/kg) to understand the potential benefit of the compound in combination. Tumor measurements were evaluated thrice a week. Tumor growth inhibition was seen with a single agent; however, the greatest inhibition (83% inhibition at day 15) was seen in combination with paclitaxel (Fig. 3). The drug was well tolerated by the animals in all arms of the study. In a preliminary safety evaluation study, MX-126374 did not show any adverse effects on the major organs including the lack of any bone marrow toxicity (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 3. MX-126374 inhibits tumor growth in vivo. Female NCr nu/nu mice were implanted s.c. with MX-1 tumor fragments. Animals were divided into control groups that received i.p. either vehicle, MX-126374, paclitaxel, or in combination at the indicated dosing regimen. A tumor growth inhibition of 83% in the combination arm at day 15 was observed. *, P < 0.0002.

The hierarchical clustering of the two analogues of 3,5-diaryl-oxadiazoles, MX-74420 and MX-126374 and other small molecules identified as apoptosis inducers in T47D breast cancer cells are shown in Fig. 4 along with the corresponding gene list (Supplemental Information).² The clustering analysis on the 555 genes was done with MX-126374 as the lead compound using a +2/–2 threshold. MX-126374 and MX-74420 cluster together, indicating a similarity in their structures and their mode of action. Compounds 3, 4, and 5 cluster closer to MX-74420 and MX-126374 in their hierarchy. In an independent experiment, compounds 3, 4, MX-74420, and MX-126374 caused an arrest in G₁ phase of the cell cycle as indicated by DNA content and hypophosphorylation of the retinoblastoma tumor suppressor protein (data not shown), whereas compound 5 caused a G₂-M arrest. The other G₂-M compounds acting through tubulin inhibition to induce apoptosis, cluster together indicated as compounds 1 and 2. Thus, using expression analysis, we were able to categorize the compound hits by their potential biochemical mechanisms and not necessarily by their effects on the cell cycle.

View larger version (31K): [in this window] [in a new window]   Figure 4. 3,5-diaryl-oxadiazole analogues cluster together as a subgroup of breast-specific compounds. Hierarchical clustering of 555 genes with differential expression between MX-126374 and untreated T47D cells using a 2-fold threshold. Untreated controls and seven compounds were used in this hierarchical clustering representing different modes of action. Two tubulin inhibitors (compounds 1 and 2) cluster together, whereas the breast-specific compounds cluster together (compounds 3, 4, 5, MX-74420, and MX-126374). MX-74420 and MX-126374 of this series cluster with one another.

View larger version (23K): [in this window] [in a new window]   Figure 5. Selected gene expression profile of T47D cells in the presence of MX-126374. T47D cells were treated for 18 h with MX-126374 (5 µmol/L), corresponding cDNAs were used for the validation of selected genes using quantitative real-time PCR.

View larger version (28K): [in this window] [in a new window]   Figure 6. MX-126374 alters the expression of selected genes in a cell-specific and time-dependent manner. Two breast-specific cell lines, T47D and SK-Br3; two normal cell lines, HMEC and HUVEC; and an insensitive lung carcinoma line, SW620, were treated with MX-126374 (5 µmol/L) for 2, 4, 6, 16, 24 h. The expression of cyclin D1 (A), p21 (B), TGF-ß1 (C), and IGF-BP3 (D) genes were quantitated using real-time PCR. mRNA levels were normalized against cyclophilin, a housekeeping gene.

View larger version (23K): [in this window] [in a new window]   Figure 7. The tritiated photoaffinity-labeled compound 3H-MX-126911 binds to 50 kDa cytosolic protein. Cytosolic lysates of T47D cells were treated with DMSO, cold active, or inactive (100 µmol/L) analogues before the addition of 200 nmol/L 3H-MX-126911 and subsequently subjected to UV waves. Samples were subjected to one-dimensional (A) or two-dimensional (B) analysis and exposed to film. A, one-dimensional analysis of the samples. Arrow, band that is competed with the active competitor and is present with the inactive competitor. B, two-dimensional analysis of the sample represented in one-dimensional analysis (lane 1). Arrow, a single labeled spot corresponding to an approximately 50 kDa protein size with a pI of 5.3. C, T47D cytosolic lysates were pretreated with DMSO or unlabeled MX-126911 prior to the addition of 3H-MX-126911. Immunoprecipitations were done using the TIP47 IgY, chicken IgY, and beads alone (used as negative controls). Samples were subjected to SDS-PAGE. Dried gels were placed against film. Lane 1, beads alone; lane 2, chicken IgY immunoprecipitation; lane 3, TIP47 IgY immunoprecipitation; and lane 4, TIP47 IgY immunoprecipitation from lysates competed with unlabeled MX-126911. Arrow, TIP47 band, which corresponds to the tritiated band.

TIP47 Knockdown and Pathway Verification
To further validate that TIP47 is the target for 3,5-diaryl-[1,2,4]-oxadiazoles, we used RNA interference assays with siRNA duplexes to inhibit the expression of TIP47 in T47D cells (18, 19). The concentration of TIP47 siRNA (100 nmol/L) was optimized to result in specific RNA interference and avoid any nonspecific effects of the chemically synthesized RNA duplexes (data not shown). We then did studies to evaluate the sensitivity of the transfected cells toward MX-126374. Inhibition of endogenous TIP47 mRNA expression after transfection with the siRNA oligonucleotide was observed (Fig. 8A) relative to T47D cells transfected with the control siRNA oligonucleotide, cyclophillin, or lipid alone. TIP47-specific siRNA transfections showed an approximately 90% inhibition of TIP47 expression after 48 hours. With the down-regulation of TIP47, we were able to show by quantitative PCR analysis, significant changes in the gene expression levels of cyclin D1 and p21, resulting in a 2-fold decrease and a 4-fold increase, respectively (Fig. 8B). In addition, inhibition of endogenous TIP47 in T47D cells displayed a similar pathway profile when focusing on the genes of interest, namely cyclin D1 and p21 (Fig. 8C). Treatment of cells for 18 hours with 2x EC₅₀ of the compound leads to down-regulation of cyclin D1 and up-regulation of p21. However, in TIP47 down-regulated cells, we observed that a lesser amount of the drug was needed to obliterate the rest of TIP47 as well as to enhance the effects observed on these genes. T47D cells were treated with only 0.5x EC₅₀ of MX-126374 for 6 hours, after which protein lysates were prepared. Cyclin D1 at this concentration was only slightly decreased in the treated lanes of control transfected cells compared with TIP47 siRNA transfected cells (Fig. 8C). The down-regulation of TIP47 alone resulted in a significant decrease in cyclin D1 level compared with lipid and cyclophillin controls. In the case of p21 protein levels, the presence of MX-126374 increased the p21 protein level in both lipid alone and cyclophillin siRNA-transfected cells. However, TIP47 down-regulation alone resulted in significant increase in p21 levels and displayed further up-regulation in the presence of MX-126374. In summary, validation studies in TIP47 down-regulated cells indicated that down-regulation of TIP47 in cancer cells by siRNA shows a similar pathway profile as compound treatment.

View larger version (29K): [in this window] [in a new window]   Figure 8. Down-regulation of TIP47 leads to altered expression of specific genes and sensitization to MX-126374. A, real-time PCR showing the down-regulation of the TIP47 at the mRNA level. T47D cells were transfected for 48 h as lipid alone, cyclophillin (100 nmol/L), and TIP47 siRNA (100 nmol/L). TIP47 mRNA levels were normalized to cyclophillin, a housekeeping gene. Cyclophillin down-regulation was normalized to glyceraldehyde-3-phosphate dehydrogenase. Confirmed in three independent experiments. B, real-time PCR was used to look at the expression of cyclin D1 and p21 post TIP47 siRNA transfection. Data was normalized against cyclophillin or glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. C, down-regulation of TIP47 mRNA leads to a sensitization to MX-126374: T47D cells were transfected for 48 h as described in (A) after which they were treated with DMSO or MX-126374 (1.25 µmol/L) for 6 h. Protein extracts were prepared for Western blot analysis. TIP47, cyclin D1, and p21 antibodies were used to detect the levels of these proteins. Equal loading was confirmed by actin levels (bottom).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

TIP47 is a 47 kDa protein that binds selectively to the cytoplasmic domains of cation-independent and cation-dependent IGF-II/M-6-P receptor (IGF-IIR). IGF-IIR is important for modulating IGF-II and also for the activation of tumor growth factor ß1, a potent growth inhibitor for most cell types. TIP47 also binds and transports newly synthesized lysosomal hydrolases including cathepsins (5). Binding of IGF-II to the IGF-IIR results in the internalization and eventual degradation of the ligand in the lysosomes (22), thus suppressing mitogenesis by reducing IGF-II availability for binding to the IGF-I receptor. Additionally, IGF-IIR has been identified as a potential tumor suppressor gene. A loss of heterozygosity at the IGF-IIR locus (chromosome 6) coupled with mutations in the remaining allele are thought to be early events in human hepatocarcinoma (23). This loss of heterozygosity at chromosome 6 has been identified in hepatocellular carcinomas, breast cancer, ovarian cancer, renal cell carcinoma, and squamous cell carcinoma (23–25), non–Hodgkins lymphoma (26), malignant melanoma (27). A deficiency in functional IGF-IIR, may also contribute to tumor growth and metastasis of some cancer types (28).

The presence of TIP47 in the cytosol and on endosomes is a requirement for IGF-IIR transport from endosomes to the trans-Golgi network (17). Differential expression levels of IGF-IIR in breast cancer cell lines of different invasive potential indicates its key role in tumor progression and metastasis (28). TIP47 is also known to be overexpressed in cervical dysplasias and carcinomas (29). It is not known if TIP47 has any role in tumors with defective IGF-IIR and if manipulating TIP47 levels in tumors would have therapeutic advantages.

The identification of TIP47 in our study may not necessarily imply it as the only target for this series of compounds but certainly highlights TIP47 as a key player in 3,5-diaryl-oxadiazoles-mediated apoptotic signaling pathway. Some of the validation studies done here may suggest a regulatory mechanism connecting TIP47 and IGF-IIR to apoptosis that involves the key genes identified in this study. A further analysis of this pathway may also identify other approaches to regulate IGF-IIR in tumor cells that may be useful in enhancing its proapoptotic capacity. Further investigation may unravel the complexity of TIP47 and IGF-IIR regulation as it pertains to tumor growth and metastasis. Although TIP47 is a known protein, it is primarily considered as a chaperone or cargo protein. An analysis of the expression profile in TIP47 down-regulated cells in comparison to the compound-treated cells indicates an overlap of 92 genes that are common to both (+2/–2-fold differentially expressed genes). A further understanding of these genes and those that are selective to TIP47 down-regulation may indeed lead to interesting new pathways, and/or targets. Whether the selectivity observed here is truly based on the tissue type or if there are other common mechanisms underlying the general tumor development is intriguing and is being explored further.

Taken together, our data shows that by combining various drug discovery approaches that include cell-based screening programs, expression profiling, pathway analysis, and target identification, we can discover new or novel therapeutic targets. The pathway analysis as done here can also be useful in identifying certain treatment-dependent biomarkers. This approach not only allows the discovery of new targets or new functions for known targets but also leads to the identification of new signaling pathways that could be selectively exploited to achieve effective therapies.

	Acknowledgments

 
We thank Candace Crogan-Grundy for technical assistance, and Dr. Jeff Gregg and technical staff at UC Davis for their help and expertise in expression profiling.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org).

Received 12/10/04; revised 1/24/05; accepted 3/ 1/05.

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