Single concentration validation of uHTS antagonist hits from Gli-SUFU in a luminescent cytotoxicity assay
The Hh pathway plays a critical role in the patterning of certain embryonic tissues and contributes to their neoplastic transformation later in life. Hh signaling regulates cerebellar patterning by promoting the proliferation of neuronal precursor cells, and constitutive Hh target gene expression can lead to medulloblastoma, the most common pediatric brain tumor(1). ..more
BioActive Compounds: 786
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network(MLPCN)
Grant Number: 1R03MH094195-01
Assay Provider: James Chen, Ph.D.,Stanford University, Stanford California
The Hh pathway plays a critical role in the patterning of certain embryonic tissues and contributes to their neoplastic transformation later in life. Hh signaling regulates cerebellar patterning by promoting the proliferation of neuronal precursor cells, and constitutive Hh target gene expression can lead to medulloblastoma, the most common pediatric brain tumor(1).
Hh signaling is normally initiated by the binding of Hh ligands to the twelve-pass transmembrane protein Ptch1(2) inducing its exit from the cilium, leading to Smo accumulation and activation within the antenna-like organelle. Activated Smo then shifts the balance between repressor and activator forms of the Gli transcription factors (Gli1, Gli2, and Gli3) by promoting three events: their stabilization from proteolytic processing into N-terminal repressors (Gli2/3R), their conversion into transcriptional activators (Gli2/3A), and their accumulation at the distal tip of the cilium. The process by which Smo regulates Gli function appears to involve the nucleocytoplasmic protein Sufu, which can directly inhibit the Gli proteins and facilitate their proteolysis. The activator form of Gli2, and to a lesser extent that of Gli3, subsequently drives the expression of Hh target genes, including cell cycle regulators, oncogenes, Ptch1, and the constitutively active transcription factor Gli1. Oncogenic Hh pathway activation can be initiated at multiple points within the signaling mechanism described above.
As a GPCR-like signaling protein, Smo is perhaps the most "druggable" target within the Hh pathway, and Smo inhibitors have demonstrated efficacy in murine tumor models(3-9) and human clinical trials(10,11). The excitement generated by these findings is tempered by the emergence of GDC-0449-resistant tumors, which either contain point mutations in Smo that prevent drug binding or sustain Hh target gene expression in a Smo-independent manner(11). Tumors expressing GDC-0449-resistant Smo were also insensitive to cyclopamine, a structurally distinct Smo inhibitor(12), suggesting that the development of additional Smo antagonists will not be an adequate solution to this problem. Moreover, certain cancers such as Ewing's sarcoma and KRAS-induced pancreatic adenocarcinoma can proliferate in response to Gli activation through non canonical mechanisms(13-17). Chemical inhibitors that act downstream of Smo therefore constitute an important therapeutic strategy for the treatment of Hh pathway-dependent cancers. Due to the high susceptibility of Smo to small-molecule modulation, high-throughput screens using Hh ligand-stimulated cells are overwhelmingly dominated by Smo antagonists(18)
This lead discovery project employs a cell-based reporter that lacks Sufu and exhibits constitutive Hh target gene expression in response to endogenous Gli activators in order to find Smo-independent modulators of Hh pathway. The Sufu-KO-LIGHT cells used for this project rely on endogenous rather than overexpressed Gli for greater sensitivity toward the goal of identifying non-Smo Gli antagonists.
In this cell-based luminescent reporter assay, Smo-independent Hh gene activity is monitored in Sufu-KO-LIGHT cells through a co-transfection of eight tandem Gli-binding sites with a firefly luciferase reporter construct driven by Gli.
The purpose of this assay was to detect cytotoxic compounds among the confirmed antagonist hits from the primary Gli-SUFU luminescent reporter assay (AID: 602428).
1. Wechsler-Reya, R., and Scott, M. P. (2001). The developmental biology of brain tumors. Annu Rev Neurosci 24: 385-428.
2. Jiang, J., and Hui, C.-c. (2008). Hedgehog signaling in development and cancer. Dev Cell 15:801-812.
3. Berman, D., Karhadkar, S., Hallahan, A., Pritchard, J., Eberhart, C., Watkins, D., Chen, J., Cooper, M., Taipale, J., Olson, J., and Beachy, P. (2002). Medulloblastoma growth inhibition by hedgehog pathway blockade. Science 297: 1559-1561.
4. Lucas, B. S., Aaron, W., An, S., Austin, R. J., Brown, M., Chan, H., Chong, A., Hungate, R., Huang, T., Jiang, B., Johnson, M. G., Kaizerman, J. A., Lee, G., McMinn, D. L., Orf, J., Powers, J. P., Rong, M., Toteva, M. M., Uyeda, C., Wickramasinghe, D., Xu, G., Ye, Q., and Zhong, W. (2010). Design of 1- piperazinyl-4-arylphthalazines as potent Smoothened antagonists. Bioorg Med Chem Lett 20: 3618-3622.
5. Miller-Moslin, K., Peukert, S., Jain, R., McEwan, M., Karki, R., Llamas, L., Yusuff, N., He, F., Li, Y., Sun, Y., Dai, M., Perez, L., Michael, W., Sheng, T., Lei, H., Zhang, R., Williams, J., Bourret, A., Ramamurthy, A., Yuan, J., Guo, R., Matsumoto, M., Vattay, A., Maniara, W., Amaral, A., Dorsch, M., and Kelleher, J. (2009). 1-amino-4-benzylphthalazines as orally bioavailable smoothened antagonists with antitumor activity. J Med Chem 52: 3954-3968.
6. Pan, S., Wu, X., Jiang, J., Gao, W., Wan, Y., and Cheng, D. (2010). Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist. ACS Medicinal Chemistry.
7. Robarge, K. D., Brunton, S. A., Castanedo, G. M., Cui, Y., Dina, M. S., Goldsmith, R., Gould, S. E., Guichert, O., Gunzner, J. L., Halladay, J., Jia, W., Khojasteh, C., Koehler, M. F., Kotkow, K., La, H., Lalonde, R. L., Lau, K., Lee, L., Marshall, D., Marsters, J. C., Jr., Murray, L. J., Qian, C., Rubin, L. L., Salphati, L., Stanley, M. S., Stibbard, J. H., Sutherlin, D. P., Ubhayaker, S., Wang, S., Wong, S., and Xie, M. (2009). GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorg Med Chem Lett 19: 5576-5581.
8. Romer, J. T., Kimura, H., Magdaleno, S., Sasai, K., Fuller, C., Baines, H., Connelly, M., Stewart, C. F., Gould, S., Rubin, L. L., and Curran, T. (2004). Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice. Cancer Cell 6: 229-240.
9. Tremblay, M., Lescarbeau, A., Grogan, M., Tan, E., Lin, G., Austad, B., Yu, L., Behnke, M., Nair, S., Hagel, M., White, K., Conley, J., Manna, J., Alvarez-Diez, T., Hoyt, J., Woodward, C., Sydor, J., Pink, M., MacDougall, J., Campbell, M., Cushing, J., Ferguson, J., Curtis, M., McGovern, K., Read, M., Palombella, V., Adams, J., and Castro, A. (2009). Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926). J Med Chem 52: 4400-4418.
10. Von Hoff, D. D., LoRusso, P. M., Rudin, C. M., Reddy, J. C., Yauch, R. L., Tibes, R., Weiss, G. J., Borad, M. J., Hann, C. L., Brahmer, J. R., Mackey, H. M., Lum, B. L., Darbonne, W. C., Marsters, J. C., de Sauvage, F. J., and Low, J. A. (2009). Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 361: 1164-1172.
11. Yauch, R. L., Dijkgraaf, G. J. P., Alicke, B., Januario, T., Ahn, C. P., Holcomb, T., Pujara, K., Stinson, J., Callahan, C. A., Tang, T., Bazan, J. F., Kan, Z., Seshagiri, S., Hann, C. L., Gould, S. E., Low, J. A., Rudin, C. M., and de Sauvage, F. J. (2009). Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326: 572-574.
12. Chen, J., Taipale, J., Cooper, M., and Beachy, P. (2002). Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16: 2743-2748.
13. Joo, J., Christensen, L., Warner, K., States, L., Kang, H.-G., Vo, K., Lawlor, E. R., and May, W. A. (2009). GLI1 is a central mediator of EWS/FLI1 signaling in Ewing tumors. PLoS One 4:e7608.
14. Ji, Z., Mei, F. C., Xie, J., and Cheng, X. (2007). Oncogenic KRAS activates hedgehog signaling pathway in pancreatic cancer cells. J Biol Chem 282: 14048-14055.
15. Nolan-Stevaux, O., Lau, J., Truitt, M. L., Chu, G. C., Hebrok, M., Fernandez-Zapico, M. E., and Hanahan, D. (2009). GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation. Genes Dev 23: 24-36.
16. Dennler, S., Andre, J., Verrecchia, F., and Mauviel, A. (2009). Cloning of the human GLI2 Promoter: transcriptional activation by transforming growth factor-beta via SMAD3/beta-catenin cooperation. J Biol Chem 284: 31523-31531.
17. Beauchamp, E., Bulut, G., Abaan, O., Chen, K., Merchant, A., Matsui, W., Endo, Y., Rubin, J. S., Toretsky, J., and Uren, A. (2009). GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein. J Biol Chem 284: 9074-9082.
18. Chen, J., Taipale, J., Young, K., Maiti, T., and Beachy, P. (2002). Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A 99: 14071-14076.
Sufu-KO-LIGHT cell line GROWTH MEDIUM
DMEM Phenol Red containing (Hyclone #SH30243.02)
HI-FBS characterized (Hyclone SH30396.03 HI or equivalent) 10% Final
L-glutamine (Cellgro # 25-005-CI) 200 mM 10 mL-2 mM (1:100) Final
Penn/Strep (Cellgro #30-0020CI) 5000 IU/mL 100 mL-50 IU/mL (1:100) Final
Zeocin (Invitrogen R25005 or Sigma 46-0072) 5 g in 50 mL-(100 mg/mL) 0.15 mg/mL Final(1:666.67)
Sufu-KO-LIGHT cell line ASSAY MEDIUM
DMEM phenol red free (Hyclone SH30585.02)
HI-FBS characterized (HYCLONE SH30396.03 HI or equivalent) 10% Final
L-glutamine (Cellgro # 25-005-CI) 200 mM 100 mL-2 mM (1:100) Final
Na-pyruvate (Sigma S8636-100ML) 100 mM-1 mM (1:100) Final
Penn/Strep (Cellgro #30-0020CI) 5000 IU/mL 100 mL-50 IU/mL(1:100) Final
HEPES (Omega Scientific # HB-20) 100 mL 1M 25 mM (1:40) Final
Zeocin (Invitrogen R25005 or Sigma 46-0072) 5g in 50 mL-(100 mg/mL) 0.15 mg/mL Final(1:666.67)
Sufu-KO-LIGHT cell line (Assay Provider)
PBS (Phosphate Buffered Saline)
TrypLE Express cell dissociation reagent (Life Technologies)
T225 tissue culture flasks (Corning)
1536 well tissue culture plates Aurora & Corning
1536 well Echo compatible Cyclic Olefin Copolymer (COC) compound storage plates (Corning or Labcyte)
384 well low volume Echo compatible COC compound storage plates (labcyte)
ATP-Light luminescence detection reagent (Promega)
Automation & Instrumentation:
HighRes Biosolutions (HRB) MicroStar robotics platform with Cellario scheduling software integrating the following instruments:
Viewlux microplate imager (PerkinElmer)
VSpin microplate centrifuge (Velocity11/Agilent)
Multidrop Combi liquid handler/dispenser (Thermo)
Liconic tissue culture incubator (Liconic)
Echo 550 acoustic liquid handler (Labcyte)
Biotek Microflo Select peristaltic liquid handler/dispenser
Thermo Centra CL2 Clinical Centrifuge
Nexcelom Bioscience Cellometer Auto T4 cell counter
Eppendorf 5810 centrifuge
Procedure for single concentration cytotoxicity assay:
1) Cells harvested from 2 hyperflasks at 80-90% confluency per screening day.
2) Cells suspended in Sufu-KO-LIGHT assay medium to a density of 1.0*10^6 cells/mL
3) 5 uL/well cell suspension was dispensed to columns 3-48. 5 uL/well assay medium alone (without cells) was dispensed to columns 1-2 (positive control) in Corning white polystyrene tissue treated 1536 well assay plates (#3727) or Aurora white polystyrene tissue treated 1536 well Low Base square well assay plates (#00029846) using Biotek Microflo Select (c) peristaltic liquid handler/dispenser.
4) Plates were centrifuged 1 min at 1000 rpm (200xG) on an Eppendorf 5810 centrifuge
5) Plates were covered with Kalypsys brand stainless steel assay plate lids and placed over night (16-18 hrs) in humidified Liconic brand automated tissue culture incubator. Plates were stacked vertically in towers which rotate intermittently within the incubator. 37 oC, 5% CO2
1) Kalypsys stainless steel lids were removed and 2.5 nL MLSMR test agents at 10 mM in DMSO stored in Labcyte Echo (c) compatible Corning 1536 well Cyclic Olefin Copolymer (COC) plates were applied to assay wells (columns 5-48) using a Labcyte Echo 550 acoustic liquid handling system. Final assay concentration of test agents in the assay was 5 uM. 2.5 nL DMSO controls were also added to control wells (columns 1-4). Final DMSO concentration in the assay was 0.05%.
2) Plates were centrifuged 1 min at 1000 rpm (200xG) on an Eppendorf 5810 centrifuge
3) Plates were covered with Kalypsys brand stainless steel assay plate lids and place over night (16-18 hrs) in humidity controlled Liconic Automated tissue culture incubator. Incubator stacks plates vertically in towers. 37 oC, 5% CO2
1) Kalypsys stainless steel lids were removed and replaced with plastic assay plate lids from Corning 1536 well assay plates (#3727) and plates returned to incubator.
2) 3 uL/well ATP-Light luminescence detection reagent (Promega) was added to assay wells using Multidrop Combi liquid handler (Thermo) and immediately centrifuged for 30 seconds on a VSpin integrated microplate centrifuge at 1500 rpm (Velocity11/Agilent Technologies) and incubated for 10 minutes uncovered at room temperature. Plastic assay lids were removed by the HRB robotic system before addition of ATP-Light and NOT replaced. Luminescence was read on a Viewlux microplate imager (PerkinElmer). Read time: 2 sec, Binning: 2X.
Compounds that demonstrated a final average result of >= 25 % at 5 uM are considered re-confirmed and are prioritized for further tests and development.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)