uHTS for identification of Inhibitors of Mdm2/MdmX interaction in luminescent format.
A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent more ..
BioActive Compounds: 10022
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: R03 MH089489-01
Assay Provider: Dr. Geoffrey M. Wahl, Salk Institute for Biological Studies, La Jolla, CA
A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent transcription of genes required for tumor suppression. Efforts to reactivate p53 with small molecules have focused on inhibition of the mdm2/p53 interaction, which leads to increased p53 levels and activity. However, recent reports indicate that targeting the mdm2/p53 interaction alone is insufficient to induce apoptosis in some cancer cell lines, particularly in the presence of high levels of mdmx (3-5). The most potent mdm2 antagonists do not effectively block mdmx, due to subtle structural alterations of the mdmx hydrophobic pocket (6). Since mdmx is overexpressed in many human tumors with wild type p53, this presents a barrier to effective p53 activation - additional strategies to antagonize mdm2 and mdmx are warranted.
Mdm2 is a member of the E3 ubiquitin ligase family of proteins, which facilitate the transfer of ubiquitin (Ub) molecules from E2 Ub-conjugating enzymes to target substrates (7, 8). This in turn targets the substrate for proteasome-dependent degradation. The C-terminal RING domain of mdm2 is required for ubiquitination and degradation of p53 (7). Although mdmx also has a RING domain, it has no intrinsic ubiquitin ligase activity (9). However, mdm2 and mdmx can homo- or heterodimerize via their RING domains, with a heterodimer being the more stable complex (10, 11). The RING/RING heterodimerization is thought to create an optimal scaffold for positioning and/or processivity of the E2 Ub conjugating enzyme. Therefore, identification of small molecules that disrupt the mdm2/mdmx interaction will provide an additional strategy for p53 reactivation. Development of a small molecule inhibitor of the mdm2/mdmx interaction will permit reversible inactivation of endogenous complexes, thus making analysis of p53 pathway more tractable. The aims of this proposal will be to identify a novel class of p53 activating compounds.
This reporter cell-based assay is based on bimolecular luciferase-based complementation (12-14), which relies on the reconstitution of the luciferase activity from two fragments of the enzyme. Corresponding halves of the luciferase cDNA are fused to the RING domains of Mdm2 and MdmX proteins, resulting in luciferase activity only if the two RING domains interact. The constructs are expressed from a bidirectional doxycycline-inducible promoter, affording precise control over the timing and expression level of Luc-RING fusions.
1. Montes de Oca Luna, R., D.S. Wagner, and G. Lozano, Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature, 1995. 378(6553): p. 203-6.
2. Parant, J., A. Chavez-Reyes, N.A. Little, W. Yan, V. Reinke, A.G. Jochemsen, and G. Lozano, Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet, 2001. 29(1): p. 92-5.
3. Wade, M., E.T. Wong, M. Tang, J.M. Stommel, and G.M. Wahl, Hdmx modulates the outcome of p53 activation in human tumor cells. J Biol Chem, 2006. 281(44): p. 33036- 44.
4. Patton, J.T., L.D. Mayo, A.D. Singhi, A.V. Gudkov, G.R. Stark, and M.W. Jackson, Levels of HdmX expression dictate the sensitivity of normal and transformed cells to Nutlin-3. Cancer Res, 2006. 66(6): p. 3169-76.
5. Hu, B., D.M. Gilkes, B. Farooqi, S.M. Sebti, and J. Chen, MDMX overexpression prevents p53 activation by the MDM2 inhibitor Nutlin. J Biol Chem, 2006. 281(44): p. 33030-5.
6. Popowicz, G.M., A. Czarna, and T.A. Holak, Structure of the human Mdmx protein bound to the p53 tumor suppressor transactivation domain. Cell Cycle, 2008. 7(15): p. 2441-3.
7. Fang, S., J.P. Jensen, R.L. Ludwig, K.H. Vousden, and A.M. Weissman, Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem, 2000. 275(12): p. 8945-51.
8. Glickman, M.H. and A. Ciechanover, The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev, 2002. 82(2): p. 373-428.
9. Stad, R., Y.F. Ramos, N. Little, S. Grivell, J. Attema, A.J. van Der Eb, and A.G. Jochemsen, Hdmx stabilizes Mdm2 and p53. J Biol Chem, 2000. 275(36): p. 28039-44.
10. Kawai, H., V. Lopez-Pajares, M.M. Kim, D. Wiederschain, and Z.M. Yuan, RING domain mediated interaction is a requirement for MDM2's E3 ligase activity. Cancer Res, 2007. 67(13): p. 6026-30.
11. Linke, K., P.D. Mace, C.A. Smith, D.L. Vaux, J. Silke, and C.L. Day, Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans. Cell Death Differ, 2008. 15(5): p. 841-8.
. Ozawa, T., A. Kaihara, M. Sato, K. Tachihara, and Y. Umezawa, Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem, 2001. 73(11): p. 2516-21.
13. Luker, K.E., M.C. Smith, G.D. Luker, S.T. Gammon, H. Piwnica-Worms, and D. Piwnica-Worms, Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci U S A, 2004. 101(33): p. 12288-93.
14. Wong, E.T., J.L. Kolman, Y.C. Li, L.D. Mesner, W. Hillen, C. Berens, and G.M. Wahl, Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange. Nucleic Acids Res, 2005. 33(17): p. e147.
1) p53-null SaOS-2 osteosarcoma cell line, expressing fusion proteins of the Mdmx (427-490 aa) and Mdm2 (429-491 aa) RING domains with the N- and C- terminal fragments of firefly luciferase from a bidirectional doxycycline-inducible promoter, was obtained from the assay provider's laboratory.
2) Saos-2 cells were expanded by passaging at 2 to 4-fold dilutions with the confluency kept between 30% - 95% in DMEM/High Modified (1x) supplemented with 10% HI FBS, 400 ug/mL G418, 10 ug/mL Cipro, 1/100 NaPyruvate, 1/100 L-Glutamine, 1/100 Penicillin/streptomycin.
3) Assay Media: Filtered DMEM/High Modified (1x) supplemented with 2% HI FBS, 400 ug/mL G418, 10 ug/mL Cipro, 1/100 NaPyruvate, 1/100 L-Glutamine, 1/100 Penicillin/streptomycin.
For screening prepare Saos-2 cell suspensions at 1.67 x 105 cells/mL in fresh Assay Media
4) Steady-Glo Luciferase Assay System (Promega, Cat #E2550)
4) 1536 well white solid bottom TC treated plate (Aurora Biotechnology, Cat#00029846)
Day 1 Compound Plating and Cell Seeding
1) Using LabCyte Echo, transfer 20 nL of 2 mM test compounds into columns 5 48 of the 1536 well assay plate (final concentration on Day 2 of test compounds is 8 uM, 0.4% DMSO), transfer 20 nL of DMSO into columns 1-4 (control wells).
2) Centrifuge plates at 1000 rpm for 1 minute on Vspin centrifuge.
3) Prepare Saos-2 cell suspension at 1.67 x 105 cells/mL in fresh assay media
4) Add 3 uL from 1.67 x 105 cells/mL Saos-2 cells in assay media (500 cells/well) into columns 1 48 of assay plate (on top of compounds and DMSO) using straight tip dispense on a Kalypsys dispenser.
5) Centrifuge plates at 500 rpm for 1 minute on Vspin centrifuge.
6) Incubate assay plates covered with Kalypsys metal lids overnight in a tissue culture incubator at 37o, 95% relative humidity, 5% CO2.
Day 2 Doxycycline Addition
1) Using Kalypsis dispenser, add 2 uL 50 ug/mL Doxycycline in warmed assay media to columns 3 48, and 2 uL assay media to columns 1 2.
2) Centrifuge plates at 1000 rpm for 1 minute on Vspin centrifuge.
3) Incubate assay plates overnight covered with Kalypsys metal lids in a tissue culture incubator at 37o, 95% relative humidity, 5% CO2.
Day 3 Detection
1) Following overnight incubation in the presence of doxycycline, remove lids and incubate plates for 10 min in at room T.
3) Bring Steady-Glo to room temperature. Using a Kalypsis dispenser, add 3 uL of Steady-Glo to columns 1 48.
4) Centrifuge plates at 2000 rpm for 2 minutes on Vspin centrifuge.
5) Lid the plates and incubate for 10 min at room T.
6) Read assay plates on Envision using luminescence protocol.
7) Data analysis was performed using CBIS software (ChemInnovations, Inc).
Compounds that demonstrated % activity of >= 50% at 8 uM concentration are defined as actives of the primary screen in this assay.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. Its utilization for the assay is described below.
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 and single-concentration confirmation screening data.
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
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
** Test Concentration.
Data Table (Concise)