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RESEARCH ARTICLE
Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay
Ariel G. Herman1, Miki Hayano2, Masha V. Poyurovsky3, Kenichi Shimada3,4,Rachid Skouta3,4, Carol Prives3, and Brent R. Stockwell3,4
Cancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst July 28, 2011; DOI: 10.1158/2159-8290.CD-11-0104
SEPTEMBERâ2011 CANCER DISCOVERY | 313
Mdm2 also has auto-ubiquitination activity, whereby it can regulate itself via protein degradation ( 9 ). In addition, a tran-scriptional target of p53 is the Mdm2 gene, creating an auto-regulatory negative feedback loop between Mdm2 and p53 ( 10 ). Once a stress response is removed or resolved, p53 returns to basal levels due to Mdm2-mediated degradation and inhibition. Although there are other E3 ligases capable of targeting p53 ( 3 ), Mdm2 is of primary importance in p53 regulation. This relationship is illustrated by the rescue of embryonic lethality of Mdm2 -/- mice by the concomitant deletion of p53 ( 11 , 12 ).
The Mdm2 homolog MdmX (also known as Mdm4 and used to denote both the human and mouse forms of the protein) is a nonredundant and essential p53 regulator ( 13â15 ). Similarly to Mdm2, MdmX is overexpressed in human tumors generally distinct from those containing p53 mutations ( 3 ). Evidence sug-gests that Mdm2 and MdmX function together to inhibit p53 activity. MdmX can interact with p53 and inhibit its transactiva-tion ability ( 16 ). Although MdmX has no intrinsic E3 ligase ac-tivity on its own, Mdm2 and MdmX can form hetero-oligomers through their RING domains ( 17 ), whereby MdmX can increase Mdm2 E3 ligase activity ( 18 , 19 ). Mdm2 can also directly ubiqui-tinate and degrade MdmX upon DNA-damage stimuli ( 20â22 ).
Mdm2 may be a promising target for therapeutics. Small molecule inhibitors of the Mdm2-p53 protein-protein interac-tion, such as Nutlin-3, and inhibitors of Mdm2 E3 ligase activ-ity, such as the HLI series of compounds, have been identified ( 23 , 24 ). These compounds revealed that inhibiting Mdm2 has therapeutic potential by reactivating p53 in vitro and in cell-based assays. However, Nutlin-3 does not inhibit Mdm2 E3 ligase activity and so does not block p53-independent func-tions of Mdm2; the HLI compounds have nonspecific effects at higher concentrations. For these reasons, there is a need for more specific and novel inhibitors of Mdm2 E3 ligase activity.
Here, we used a novel high-throughput cell-based auto-ubiquitination assay to identify inhibitors of Mdm2 E3 ligase activity. This is the first cell-based screen used to identify inhibitors of the E3 ligase activity of Mdm2 and can be readily adapted in order to identify inhibitors of other E3
E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to
target, as it is the key negative regulator of the tumor suppressor p53, which controls the tran-scription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inac-tivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small-molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents.
SIGNIFICANCE:â We identified a class of small-molecule inhibitors of the Mdm2-MdmX hetero-complex E3 ligase activity through a high-throughput cell-based Mdm2 ubiquitination screen. This is a new target for small-molecule therapeutics and may be developed to treat specific cancers. Cancer Discovery; 1(4); 312â25. ©2011 AACR.
ABSTRACT
INTRODUCTION The murine double minute 2 protein (Mdm2, also used to
denote the human protein) is a RING-domain-containing E3 ubiquitin ligase of paramount importance in cancer
biology because of its ability to regulate the p53 tumor suppressor protein. The p53 protein is a transcription factor that controls the activation of multiple genes involved in apoptosis and growth arrest following a wide range of cellular stresses ( 1 ). The importance of
the Mdm2-p53 pathway in tumor progression is shown by the fact that approximately 50% of tumors have a mutation
in the TP53 gene ( 2 ), and many other tumors have a deregu-lated p53 pathway ( 3 ).
In unstressed cells, p53 levels are kept low and it is held in a latent state by its negative regulator Mdm2, via two primary mechanisms. First, Mdm2 causes rapid degradation of p53 through ubiquitination and proteasomal degradation ( 4â6 ); second, Mdm2 binds the N-terminal transactivation domain of p53, preventing transcriptional activation of p53 target genes ( 7 ). Upon stress signals, such as hypoxia, DNA damage, or expression of oncogenes, post-translational modifications on both p53 and Mdm2 inhibit their interaction, stabilizing p53 and activating p53-mediated transcription ( 8 ).
Authors' Affiliations: Departments of 1 Biochemistry and Molecular Biophysics, 2 Pharmacology, and 3 Biological Sciences; 4 Howard Hughes Medical Institute, Department of Biological Sciences and Department of Chemistry, Columbia University, New York, New YorkMasha Poyurovsky is currently at the Kadmon Research Institute, 450 East 29th Street, New York, NY 10016Note: Supplementary data for this article are available at Cancer Discovery Online (http://www.cancerdiscovery.aacrjournals.org).
doi: 10.1158/2159-8290.CD-11-0104©2011 American Association for Cancer Research.
Corresponding Author: Brent R. Stockwell, Howard Hughes Medical Institute, Department of Biological Sciences and Department of Chemistry, Columbia University, Northwest Corner Building 12th Floor, 550 West 120th Street, MC 4846, New York, NY 10027. Phone: 212-854-2948; Fax: 212-854-2951; E-mail: [email protected]
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Herman et al.rEsEArCh ArtICLE
aâmetal-bindingâsiteâinâtheâRINGâdomain,âtherebyâinhibitingâMdm2â E3â ligaseâ activityâ (â9â).â Smallâ moleculesâ thatâ increaseâtheâ luminescenceâofâ theâMdm2(wt)-luciferaseâ fusionâproteinâwithoutâ increasingâ theâ luminescenceâ ofâ theâ Mdm2(C464A)-luciferaseâfusionâproteinâlikelyâinhibitâMdm2âE3âligaseâactiv-ityâorâproteasomalâdegradationâofâMdm2.âAlternatively,âsmallâmoleculesâthatâincreaseâtheâluminescenceâofâbothâfusionâpro-teinsâ areâ likelyâ affectingâ transcription,â translation,â orâ haveâ aâgeneralâcellularâimpactâthatâisânotâdependentâonâtheâE3âligaseâactivityâofâMdm2âorâonâproteasomalâdegradation.â
âWeâ firstâ comparedâ theâ expressionâ levelsâ ofâ theâ twoâ fusionâproteinsâ inâ 293Tâ cells.â Consistentâ withâ auto-ubiquitination,âMdm2(wt)-luciferaseâproteinâ isâexpressedâatâ lowerâ levelsâthanâtheâmutantâMdm2(C464A)-luciferaseâproteinâ(âFig.â1Aâ).âToâcon-firmâthatâtheâdifferenceâinâluminescenceâlevelsâbetweenâtheâtwoâconstructsâwasâtheâresultâofâaâdifferenceâinâdegradationârates,âweâmadeâ293Tâcellâlinesâstablyâexpressingâbothâconstructsâandâtreatedâthemâwithâaâproteasomeâinhibitor,âMG132â(Z-Leu-Leu-Leu-aldehyde).âInâmultipleâclonalâcellâlines,âproteasomeâinhibi-tionâ increasedâ theâ levelsâ ofâ theâ Mdm2(wt)-luciferaseâ protein,âwhileâ Mdm2(C646A)-luciferaseâ levelsâ remainedâ unaffectedâ(âFig.â1Bâ;âandâSupplementaryâFig.âS1).âThisâ isâconsistentâwithâtheâ wild-typeâ proteinâ beingâ degradedâ throughâ aâ ubiquitin-dependentâ mechanism.â Treatmentâ withâ cycloheximideâ selec-tivelyâdecreasedâtheâluminescenceâofâtheâMdm2(wt)-luciferaseâ
ligases.âThisâscreenâyieldedâtwoârelatedâcompounds,âwhichâweânamedâ Mdm2â E3â Ligaseâ Inhibitorsâ 23â andâ 24â (MEL23â andâMEL24).â Treatmentâ ofâ multipleâ cellâ linesâ withâ MEL23â andâMEL24â inhibitedâ Mdm2â andâ p53â ubiquitinâ conjugatesâ andâincreasedâ theâ stabilityâ ofâ Mdm2â andâ p53.â MELâ activityâ wasâshownâtoâbeâdependentâuponâMdm2âMdmXâinââin vitroââandâinâcell-basedâstudies.âMELâcompoundsâdecreasedâcellâsurvivalâinâaâp53-dependentâmannerâandâ increasedâ sensitivityâ toâDNA-damagingâ agents,â andâ thereforeâ representâ aâ usefulâ classâ ofâMdm2-targetedâsmallâmoleculeâinhibitors.ââ
Results high-throughput screening Using a Cell-Based Mdm2 stability Assay
âToâidentifyâMdm2âE3âligaseâinhibitors,âweâdesignedâaâhigh-throughputâcellularâassayâtoâmeasureâchangesâinâMdm2âauto-ubiquitinationâ andâ degradation.â Weâ tookâ advantageâ ofâ theâfactâ thatâ Mdm2â canâ regulateâ itselfâ inâ cellsâ throughâ ubiqui-tinationâ andâ degradationâ (â9â).â Inâ thisâ assay,â weâ usedâ 2â lucif-eraseâ fusionâ proteins:â aâ wild-typeâ Mdm2-luciferaseâ fusionâprotein,âwhichâcanâauto-ubiquitinateâandâtherebyâtargetâitselfâforâ degradation,â andâ aâ mutantâ Mdm2(C464A)-luciferaseâ fu-sionâprotein.âBothâfusionâproteinsâwereâconstructedâwithâlu-ciferaseâN-terminalâtoâMdm2.âTheâC464Aâmutationâdisruptsâ
Figure 1. Validation of cell-based luminescence assay for Mdm2 E3 ligase activity. A , Mdm2(C464A)-luciferase expression levels are higher than Mdm2(wt)-luciferase expression levels. 293T cells were transfected with Mdm2(wt)-luciferase, Mdm2(C464A)-luciferase, or a mock transfection. Cells were lysed 48 hours later and Mdm2 levels were analyzed by Western blotting with anti-Mdm2 and anti-actin antibodies. B , Mdm2(wt)-luciferase protein is degraded by the proteasome. Stable cell lines were seeded in 384-well plates and 24 hours later treated with MG132 for 2 hours or cycloheximide for 30 minutes at the indicated concentrations. Cells were lysed with a luminescence buffer and analyzed on a Victor3 Plate Reader. Each graph represents the median of 10 replicates normalized to a no-treatment control; one standard deviation is shown. C , Mdm2(wt)-luciferase, but not Mdm2(C464A)-luciferase, is ubiquitinated. Stable cell lines were transfected with HA-ubiquitin (5 ÎŒg) or a mock transfection. Forty-eight hours following transfection, cells were treated with 10 ÎŒM MG132 for 3 hours. Cells were lysed and immunoprecipitated with an anti-HA affinity matrix. Total lysates and immunoprecipitated samples were analyzed by Western blotting using an anti-Mdm2 antibody. D , levels of p53 protein are lower in Mdm2(wt)-luciferase cells than in Mdm2(C464A)-luciferase cells. 293T and stable cell lines were lysed and equal amounts of cell extracts were analyzed by Western blotting with anti-p53 and anti-eIF4E antibodies. NT, no-treatment control.
A
D
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Mdm
2-lu
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2 (C
464A
)-lu
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464A
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464A
)-lu
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Mdm2-luciferase Actin
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+ M
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+ N
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464A
)-lu
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[MG132] uM
0.01 1 10
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Mdm2-Luc
Mdm2 (C464A)-Luc
[Cycloheximide] uM
0.001 10 1000
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464A
)-lu
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Mdm2-luciferase Actin
Mdm2-luciferase
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- + N
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+ M
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+ N
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Mdm
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Mdm
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464A
)-lu
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p53
eIF4E
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Mdm2-Luc
Mdm2 (C464A)-Luc
[MG132] uM
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)-lu
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7953_CD-11-0104_pp312-325.indd 314 8/30/11 12:23 PMCancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst July 28, 2011; DOI: 10.1158/2159-8290.CD-11-0104
SEPTEMBERâ2011â CANCER DISCOVERYâ |â 315
Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay rEsEArCh ArtICLE
[Molecularâ Librariesâ Screeningâ Centerâ Networkâ (MLSCN)âlibrary,âPubchemâBioAssayâ IDâ1442,â1230,â1444,âandâ1394].âAllâcompoundsâwereâincubatedâwithâtheâMdm2(wt)-luciferaseâcellâ lineâ forâ 2â hours,â afterâ whichâ relativeâ luminescenceâ wasâdetectedâbyâtheâadditionâofâaâluminescenceâbuffer.âTheâtimeâpointâofâ2âhoursâwasâchosenâbecauseâitâisâpossibleâtoâseeâin-creasesâinâtheâMdm2âproteinâlevelâdueâtoâitsâshortâhalfâlifeâofâapproximatelyâ20âminutesâ (â25â),âandâ itâminimizesâsecondaryâeffectsâ thatâ affectâ luminescenceâ levels.â Compoundsâ thatâ in-creasedâ luminescenceâ inâtheâprimaryâscreenâwereâtestedâ inâaâcounterâ screenâ inâ theâ mutantâ Mdm2(C464A)-luciferaseâ cellâlineâtoâeliminateâcompoundsâwithâeffectsânotâdependentâonâMdm2âE3âligaseâactivityâorâproteasomalâdegradation.â
Weâidentifiedââ57âselectiveâcompounds.âAâseriesâofâsecondaryâassaysâ wasâ performedâ toâ validateâ theseâ potentialâ hits.â First,âtheseâcompoundsâwereâtestedâinâaâdilutionâseriesâinâbothâlucif-eraseâcellâlines.âThen,âtheâeffectsâofâtheâcompoundsâonâendog-enousâ p53â andâ Mdm2â levels,â andâ onâ Mdm2â ubiquitinationâactivity,â wereâ testedâ inâ multipleâ cellâ lines.â Twoâ structurallyâsimilarâcompounds,âMEL23âandâMEL24,âwereâvalidatedâtoâbeâeffectiveâMdm2âinhibitorsâinâtheseâassaysâandâfurtherâstudied.â
âMEL23â andâ MEL24â increasedâ theâ luminescenceâ ofâMdm2(wt)-luciferaseâcellsâtoâaâsimilarâlevelâasâMG132âtreatment,âandâ didâ notâ significantlyâ affectâ theâ luminescenceâ ofâ theâ con-trolâMdm2(C464A)-luciferaseâcellâlineâ(âFig.â2AâandâBâ).âTheâECâ50
protein,âsuggestingâthatâtheâobservedâdecreaseâinâstabilityâwasâdueâtoâaâpost-translationalâmechanismâ(âFig.â1Bâ).ââ
âToâ examineâ theâ ubiquitinâ ligaseâ activityâ ofâ theâ Mdm2-luciferaseâconstructs,âwild-typeâandâmutantâMdm2-luciferaseâcellâ linesâ wereâ transfectedâ withâ HA-ubiquitinâ andâ cellâ ly-satesâ wereâ immunoprecipitatedâ withâ anâ anti-HAâ antibody.âTheâ Mdm2(wt)-luciferaseâ proteinâ wasâ immunoprecipitatedâwithâ theâ anti-HAâ affinityâ matrix,â indicatingâ thatâ itâ isâ ubiq-uitinatedâ inâ cells,â whereasâ theâ Mdm2(C464A)-luciferaseâproteinâ wasâ immunoprecipitatedâ toâ aâ significantlyâ lesserâextentâ (âFig.â 1Câ).â Furthermore,â theâ Mdm2(wt)-luciferaseâ cellâlineâ hadâ overallâ lowerâ endogenousâ p53â levels,â comparedâ toâtheâ Mdm2(C464A)-luciferaseâ cellâ line,â suggestingâ thatâ theâMdm2(wt)-luciferaseâfusionâproteinâwasâableâtoâregulateâen-dogenousâ p53â (âFig.â 1Dâ).â Together,â theseâ resultsâ confirmedâthatâ Mdm2(wt)-luciferaseâ isâ rapidlyâ degradedâ afterâ auto-ubiquitinationâ throughâ theâ proteasome,â andâ itsâ stabilityâ isâregulatedâbyâanâintactâRINGâdomain.ââ
screening and Identification of MEL23 and MEL24 âTheâaboveâexperimentsâverifiedâthatâtheâcellâlinesâweâengi-
neeredâcouldâfunctionâasâaâvalidâsystemâtoâscreenâforâMdm2âE3â ligaseâ inhibitors.â Weâ proceededâ toâ screenâ 270,080â com-pounds;â51,356âwereâscreenedâatâ5.33âÎŒg/mLâinâ384-wellâfor-matâandâ218,724âwereâscreenedâatâ5âÎŒMâinâ1536-wellâformatâ
Figure 2. Structure and activity of MEL23, MEL24, and MEL analogs. A and B, structures of MEL23 and MEL24 and their activities in the cell-based auto-ubiquitination assay. Cells were seeded in 384-well plates and treated with MEL23 or MEL24 for 2 hours at the indicated concentrations. Cells were lysed with a luminescence buffer and analyzed on a Victor3 Plate Reader. Each graph represents the median of 5 replicates normalized to a no-treatment control; one standard deviation is shown. The EC 50 for MEL23 and MEL24 are 2.7 ÎŒg/mL and 3.0 ÎŒg/mL (7.5 ÎŒM and 9.2 ÎŒM), respectively. The graphs and EC 50 values were generated using GraphPad Prism. C , structure activity relationship of MEL23 and MEL24. MEL analogs were tested in the cell-based luminescence assay for activity (ranked from ***, most active to â, no activity).
MEL23 MEL24
-
R1 R
2 R
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H H
H
H
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H
H H
H
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H
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H
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-CH3 -CH
3
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2)3
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2
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-Bz
-Cl
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-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
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[MEL24] uM
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MEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
6
7
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9
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12 11 13
No
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Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
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orm
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Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
BMEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
6
7
8
9
10
12 11 13
No
rm
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esce
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ra
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Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
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2.5
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No
rm
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Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
A
C
MEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
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10
12 11 13
Norm
alized lu
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escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
MEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
6
7
8
9
10
12 11 13
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
MEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
6
7
8
9
10
12 11 13
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
MEL23 MEL24
-
R1 R
2 R
3
H H
H
H
H
H
H H
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H H
H
-CH3 -CH
3
-CH3(CH
2)3
-t(bu)
-CH2CH=CH
2
-Ph -Ph
-Bz
-Cl
-p-PhOCH3
-OCH3
-CH2CH
2=CH
2
H
-CH3
-CH3
-CH3
-CH3
-CH2CH
2OCH
3
H H H -Sec-butyl
-Sec-butyl
Activity at 15 ”M
*
**
*
*
**
*
-
**
***
-
-
**
Compound
MEL23
MEL24
1
2
3
4
5
6
7
8
9
10
12 11 13
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL23] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
Norm
alized lu
min
escence ratio
0.5
Mdm2-Luc
Mdm2 (C464A)-Luc
[MEL24] uM
10 0.0
10 0.5
10 1.0
10 1.5
1.0
1.5
2.0
2.5
3.0
3.5
7953_CD-11-0104_pp312-325.indd 315 8/30/11 12:23 PMCancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst July 28, 2011; DOI: 10.1158/2159-8290.CD-11-0104
316â|â CANCER DISCOVERYâ SEPTEMBERâ2011â www.aacrjournals.org
Herman et al.rEsEArCh ArtICLE
areâ specificâ forâ theâ p53â substrateâ asâ theyâ didâ notâ affectâ theâlevelsâofâc-Mycâorâc-Jun,âtwoâshort-livedâproteinsâinvolvedâinâcellâsurvivalâ(SupplementaryâFig.âS6).ââ
âMdmXâ isâ stableâ inâ unstressedâ cells,â butâ uponâ DNA-damage,â MdmXâ isâ rapidlyâ degradedâ byâ Mdm2â E3â ligaseâ ac-tivityâ (â26â).â Accordingly,â whenâ MCF7â cellsâ wereâ treatedâ withâetoposideâ (30â ÎŒg/mL),â MdmXâ levelsâ decreasedâ substan-tially.â Thisâ degradationâ couldâ beâ preventedâ whenâ cellsâ wereâco-treatedâwithâ5âÎŒg/mLâMEL23âorâMEL24âforâ16âhoursâorâ50âÎŒMâALLNâ(N-Acetyl-Leu-Leu-Nle-CHO),âaâproteasomeâin-hibitorâ(âFig.â3Câ).âThus,âMELâcompoundsâinhibitâendogenousâMdmXâdegradation.â
âWeâwantedâtoâdetermineâifâtheâincreasesâinâp53âproteinâlev-elsâledâtoâaâcorrespondingâincreaseâinâtheâtranscriptionâofâp53âtargetâ genes.â Therefore,â severalâ p53â targetâ genesâ wereâ ana-lyzedâbyâquantitativeâreal-timeâPCRâ(qRT-PCR).âInâRKOâcellsâtreatedâ withâ 5â ÎŒg/mLâ MEL23,â mRNAâ levelsâ ofâ âMdm2, p21, Bax,ââandââpuma,ââallâestablishedâp53âtargetâgenesâ(â27â),âincreasedâwithinâ48âhoursâofâtreatmentâ(âFig.â3Dâ).âThisâincreaseâinâtran-scriptionâwasâlessâthanâobservedâwithâdoxorubicinâtreatment,âaâ DNA-damagingâ andâ p53-activatingâ agent,â butâ moreâ thanâwithâALLN,âaâproteasomeâinhibitorâ(SupplementaryâFig.âS7).âTherefore,âMELâcompoundsâactivateâtranscriptionâofâatâleastâaâsetâofâp53âtargetâgenesâbyâincreasingâp53âproteinâlevels.ââ
valuesâofâMEL23âandâMEL24âwereâdeterminedâtoâbeâ2.7âÎŒg/mLâandâ3.0âÎŒg/mLâ(7.5âÎŒMâandâ9.2âÎŒM),ârespectively.ââ
âTheâ MELâ compoundsâ areâ composedâ ofâ tetrahydro-b-carbolineâ andâ barbituricâ acidâ moietiesâ withâ slightlyâ differ-entâsubstituentsâ(âFig.â2AâandâBâ).âTestingâaâseriesâofâanalogsârevealedâ thatâ bothâ theâ tetrahydro-b-carbolineâ andâ barbitu-ricâ acidâ moietiesâ areâ necessaryâ forâ activity.â Structureâ activ-ityâ relationshipâ (SAR)â studiesâ identifiedâ substituentsâ thatâcouldâ increase,â decrease,â orâ eliminateâ theâ activityâ (âFig.â 2Câ;âSupplementaryâFig.âS2).ââ
MEL23 and MEL24 Increase Levels of Mdm2, p53, and MdmX and Activate p53
âAnâMdm2âE3âligaseâinhibitorâshouldâpreventâtheâdegrada-tionâofâMdm2âsubstrates,âincludingâMdm2,âp53,âandâMdmX.âAccordingly,â Mdm2â andâ p53â levelsâ increasedâ inâ 3â wild-typeâp53â cellâ linesâ (U2OS,â RKO,â HCT116),â followingâ incubationâwithâ5âÎŒg/mLâMEL23âorâMEL24âforâ6âhours,âtoâsimilarâlevelsâasâ withâ MG132â treatmentâ (âFig.â 3Aâ;â Supplementaryâ Figs.â S3âandâ S4).â Thisâ increaseâ inâ Mdm2â andâ p53â levelsâ occurredâ inâaâ concentration-dependentâ mannerâ (âFig.â 3Bâ).â Additionally,âweâwereâableâtoâshowâthatâp53âmRNAâlevelsâwereânotâaffectedâbyâ MELâ compoundâ treatmentâ (Supplementaryâ Fig.â S5).âFurthermore,âweâwereâableâtoâshowâthatâtheâMELâcompoundsâ
Figure 3. Effects of MEL23 and MEL24 on Mdm2, p53, and MdmX protein levels and on p53 target genes. A , Mdm2 and p53 protein levels are increased with MEL compound treatment. U2OS, RKO, and HCT116 cells (p53 wild-type) were incubated with 5 ÎŒg/mL (14 ÎŒM) MEL23, 5 ÎŒg/mL (15 ÎŒM) MEL24, or 10 ÎŒM MG132 for 6 hours. Cells were lysed and analyzed by Western blotting using anti-Mdm2, anti-p53, and anti-eIF4E antibodies. B , Mdm2 and p53 protein levels increase with MEL treatment in a concentration-dependent manner. RKO cells were incubated with decreasing concentrations of MEL23, starting from 10 ÎŒg/mL (28 ÎŒM) in a 2-fold, 5-point dilution series for 6 hours. Cells were lysed and analyzed by Western blotting using anti-Mdm2, anti-p53, and anti-eIF4E antibodies. C , MEL compounds inhibit etoposide-induced MdmX degradation. MCF7 cells (p53 wild-type) were incubated with 30 ÎŒg/mL (51 ÎŒM) etoposide alone or in combination with 50 ÎŒM ALLN, 5 ÎŒg/mL (14 ÎŒM) MEL23, or 5 ÎŒg/mL (15 ÎŒM) MEL24 for 16 hours. Cells were lysed and analyzed by Western blotting with anti-MdmX and anti-actin antibodies. D , MEL23 increases the transcription of p53 target genes, Mdm2, p21, puma, and bax . RKO cells were treated with 5 ÎŒg/mL (14 ÎŒM) MEL23 for 24 or 48 hours. qRT-PCR was used to analyze the mRNA levels of the Mdm2 , p21 , puma , and bax transcripts. All values are normalized to no-treatment, DMSO control. The figure is representative of three independent experiments and standard error bars represent intra-experimental error. NT, no-treatment control.
C D
U2OS RKO HCT116
Mdm2 p53
eIF4E
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
MdmX Actin
NT
Eto
posi
de
Eto
posi
de +
ALL
N
Eto
posi
de +
ME
L23
Eto
posi
de +
ME
L24
MCF7
NT
MEL23
Mdm2 p53
eIF4E
Fol
d in
crea
se o
ver
no tr
eate
men
t
mRNA levels of p53 target genes
9
8
7
6
5
4
3
2
1
0
Mdm2
NT
MEL23-24hrs
MEL23-48hrs
p21 puma bax
A
U2OS RKO HCT116
Mdm2 p53
eIF4E
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
MdmX Actin
NT
Eto
posi
de
Eto
posi
de +
ALL
N
Eto
posi
de +
ME
L23
Eto
posi
de +
ME
L24
MCF7
NT
MEL23
Mdm2 p53
eIF4E
Fol
d in
crea
se o
ver
no tr
eate
men
t
mRNA levels of p53 target genes
9
8
7
6
5
4
3
2
1
0
Mdm2
NT
MEL23-24hrs
MEL23-48hrs
p21 puma bax
U2OS RKO HCT116
Mdm2 p53
eIF4E
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
MdmX Actin
NT
Eto
posi
de
Eto
posi
de +
ALL
N
Eto
posi
de +
ME
L23
Eto
posi
de +
ME
L24
MCF7
NT
MEL23
Mdm2 p53
eIF4E
Fol
d in
crea
se o
ver
no tr
eate
men
t
mRNA levels of p53 target genes
9
8
7
6
5
4
3
2
1
0
Mdm2
NT
MEL23-24hrs
MEL23-48hrs
p21 puma bax
U2OS RKO HCT116
Mdm2 p53
eIF4E
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
NT
ME
L23
ME
L24
MG
132
MdmX Actin
NT
Eto
posi
de
Eto
posi
de +
ALL
N
Eto
posi
de +
ME
L23
Eto
posi
de +
ME
L24
MCF7
NT
MEL23
Mdm2 p53
eIF4E
Fol
d in
crea
se o
ver
no tr
eate
men
t
mRNA levels of p53 target genes
9
8
7
6
5
4
3
2
1
0
Mdm2
NT
MEL23-24hrs
MEL23-48hrs
p21 puma bax
B
7953_CD-11-0104_pp312-325.indd 316 8/30/11 12:23 PMCancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst July 28, 2011; DOI: 10.1158/2159-8290.CD-11-0104
SEPTEMBERâ2011â CANCER DISCOVERYâ |â 317
Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay rEsEArCh ArtICLE
aâ HECTâ domainâ E3â ligase,â andâ targetsâ p53â forâ degrada-tionâ(28).âAsâaâresult,âp53âlevelsâareâsignificantlyâreducedâinâRKO-E6âcellsâcomparedâtoâRKOâcellsâ(Fig.â4D,âNTâlanes).âAâcompoundâthatâspecificallyâ inhibitsâMdm2âE3â ligaseâactiv-ityâshouldâincreaseâp53âlevelsâinâRKOâcells,âbutânotâRKO-E6âcells.â Consistently,â treatmentâ withâ 5â ÎŒg/mLâ MEL23â orâMEL24âforâ6âhoursâincreasedâtheâlevelsâofâp53âinâRKOâcells,âbutâ notâ RKO-E6â cells,â whereasâ treatmentsâ withâ MG132,âdoxorubicin,âetoposide,âandâcamptothecinâincreasedâp53âinâbothâ cellâ linesâ throughâ proteasomeâ inhibitionâ orâ throughâtheâactivationâofâaâDNA-damageâresponseâ (Fig.â4D).âTheseâdataâ demonstrateâ thatâ MEL23â andâ MEL24â doâ notâ inhibitâtheâ proteasome,â doâ notâ inhibitâ E6APâ activity,â andâ doâ notâinduceâ aâ DNA-damageâ response,â suggestingâ thatâ MEL23âandâMEL24âstabilizeâMdm2âviaâaâmechanismâindependentâofâp53âtranscription.
Someâcompoundsâcanâreactânonspecificallyâwithâproteins,âDNAâ andâ glutathione,â leadingâ toâ off-targetâ affectsâ (29).â Inâorderâ toâ determineâ ifâ MELâ compoundsâ generateâ aâ Michaelâacceptorâ(anâelectophile)â in situâandâreactânonspecifically,âweâconductedâ experimentsâ toâ determineâ ifâ aâ MEL-glutathioneâadductâ wouldâ form.â MEL24â didâ notâ formâ anâ adductâ withâglutathioneâ asâ determinedâ byâ massâ spectrometry,â whereasâaâ knownâ Michaelâ acceptor,â phenylisothiocyanate,â formedâaâ glutathioneâ adductâ underâ theâ sameâ reactionâ conditionsâ(SupplementaryâFig.âS9).
Activity of MEL23 and MEL24 Is Mdm2-specific and Independent of p53 transcription
ManyâtypesâofâcompoundsâleadâtoâincreasesâinâbothâMdm2âandâp53âproteinâlevelsâbyâinducingâaâDNA-damageâresponse.âAâ seriesâ ofâ experimentsâ wasâ performedâ toâ excludeâ theâ pos-sibilityâ thatâ theâ MELâ compoundsâ wereâ inducingâ p53-medi-atedâstress,â therebyâ leadingâtoâtheâaccumulationâofâp53âandâMdm2.âFirst,âp53-nullâH1299âcellsâwereâtreatedâwithâ5âÎŒg/mLâMEL23âorâMEL24âforâ6âhours.âTheâlevelsâofâbothâendogenousâMdm2â proteinâ andâ ectopicallyâ expressedâ Mdm2â increasedâafterâ treatment,â whereasâ noâ effectâ onâ Mdm2â levelsâ wasâ ob-servedâ followingâ doxorubicinâ treatmentâ (Fig.â 4Aâ andâ 4B).âThisâ indicatesâ thatâ theâMELâcompoundsâstabilizeâMdm2âinâaâp53-independentâmanner.âFurtherâverificationâthatâMEL23âandâMEL24âareânotâgenotoxicâandâareânotâinducingâaâp53-sta-bilizingâstressâresponseâwasârevealedâbyâtheâlackâofâdetectableâphosphorylationâ ofâ p53â atâ serine-15â (Fig.â 4C)â orâ phosphor-ylationâ ofâ histoneâ H2A.Xâ atâ serine-139â (SupplementaryâFig.âS8)âwithâMEL23âorâMEL24â(5âÎŒg/mL);âtheseâphosphory-lationâeventsâwereâstronglyâinducedâbyâdoxorubicin-mediatedâDNAâdamage.
Next,âweâtestedâtheâspecificityâofâMEL23âandâMEL24âforâMdm2â inâ twoâ isogenicâ cellâ lines,â RKOâ andâ RKO-E6.â RKOâandâ RKO-E6â cellsâ haveâ wild-typeâ p53â andâ Mdm2,â butâRKO-E6âcellsâexpressâtheâHPV-E6âprotein.âTheâHPV-E6âpro-teinâ formsâ anâ activeâ E3â ligaseâ inâ associationâ withâ E6AP,â
Figure 4. MEL23 and MEL24 are specific toward Mdm2 and cause Mdm2 accumulation independent of p53 transcription. A and B, MEL23 and MEL24 stabilize Mdm2 protein in p53-null cells. H1299 cells were transfected with Flag-Mdm2 (5 ÎŒg) or left untransfected. Cells were treated with 5 ÎŒg/mL (14 ÎŒM) MEL23, 5 ÎŒg/mL (15 ÎŒM) MEL24, 10 ÎŒM MG132, or 1 ÎŒg/mL (1.7 ÎŒM) doxorubicin for 6 hours. Cells were lysed and analyzed by Western blotting using anti-Mdm2 and anti-actin antibodies. The upper band indicated with an arrow is the Mdm2 protein. C, MEL23 and MEL24 do not induce p53-ser15 phosphorylation. RKO cells were treated with 5 ÎŒg/mL (14 ÎŒM) MEL23, 5 ÎŒg/mL (15 ÎŒM) MEL24, 10 ÎŒM MG132, or 0.5 ÎŒg/mL (0.9 ÎŒM) doxorubicin for 6 hours. Cells were lysed and analyzed by Western blotting using anti-p53 and anti-phospho-ser15 p53 antibodies. D, MEL23 and MEL24 increase p53 levels in RKO cells, but not in RKO-E6 cells. RKO and RKO-E6 cells were treated with 5 ÎŒg/mL (14 ÎŒM) MEL23, 5 ÎŒg/mL (15 ÎŒM) MEL24, 10 ÎŒM MG132, 0.5 ÎŒg/mL (0.9 ÎŒM) doxorubicin, 0.5 ÎŒg/mL (1.4 ÎŒM) camptothecin, or 25 ÎŒg/mL (42 ÎŒM) etoposide for 6 hours. Cells were lysed and analyzed by Western blotting using anti-Mdm2, anti-p53, and anti-actin antibodies. The upper band indicated with an arrow is the Mdm2 protein. NT, no-treatment control.
Eto
posi
de
Transfected FLAG-Mdm2 Endogenous
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
Moc
k
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53 Actin
NT
ME
L23
ME
L24
Eto
posi
de C
ampt
otht
ecin
MG
132
Dox
orub
icin
Dox
orub
icin
RKO RKO E6 N
T
ME
L23
ME
L24
Cam
ptot
htec
in
MG
132
Mdm2
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53
pSer15
Eto
posi
de
Transfected FLAG-Mdm2 Endogenous
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
Moc
k
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53 Actin
NT
ME
L23
ME
L24
Eto
posi
de C
ampt
otht
ecin
MG
132
Dox
orub
icin
Dox
orub
icin
RKO RKO E6
NT
ME
L23
ME
L24
Cam
ptot
htec
in
MG
132
Mdm2
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53
pSer15
A B C
Eto
posi
de
Transfected FLAG-Mdm2 Endogenous
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
Moc
k
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53 Actin
NT
ME
L23
ME
L24
Eto
posi
de C
ampt
otht
ecin
MG
132
Dox
orub
icin
Dox
orub
icin
RKO RKO E6
NT
ME
L23
ME
L24
Cam
ptot
htec
in
MG
132
Mdm2
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53
pSer15
Eto
posi
de
Transfected FLAG-Mdm2 Endogenous
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
Moc
k
Mdm2
Actin
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53 Actin
NT
ME
L23
ME
L24
Eto
posi
de C
ampt
otht
ecin
MG
132
Dox
orub
icin
Dox
orub
icin
RKO RKO E6
NT
ME
L23
ME
L24
Cam
ptot
htec
in
MG
132
Mdm2
NT
ME
L23
ME
L24
MG
132
Dox
orub
icin
p53
pSer15
D
7953_CD-11-0104_pp312-325.indd 317 8/30/11 12:23 PMCancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst July 28, 2011; DOI: 10.1158/2159-8290.CD-11-0104
318â|â CANCER DISCOVERYâ SEPTEMBERâ2011â www.aacrjournals.org
Herman et al.rEsEArCh ArtICLE
theâ compoundsâ stabilizedâ p53â withoutâ ubiquitination.â Inâfact,â theâ half-lifeâ ofâ p53â increasedâ fromâ 1â hourâ inâ thisâ cellâlineâtoâgreaterâthanâ6âhoursâwithâ5âÎŒg/mLâMEL23âtreatmentâ(âFig.â5Bâ).âThus,âtreatmentâwithâMEL23âandâMEL24âinhibitsâubiquitinâ conjugationâ toâ Mdm2â andâ p53â thusâ stabilizingâtheseâproteins.â
âWeâ thenâ testedâ theâ specificityâ ofâ theâ MELâ compoundsâinâ âin vitroââ ubiquitinationâ reactions,â whichâ includedâ puri-fiedâ E1,â UbcH5C,â â32âP-labeledâ ubiquitin,â andâ ATPâ andâ de-terminedâtheâextentâofâubiquitinationâbyâautoradiography.âTheâ additionâ ofâ increasingâ amountsâ ofâ MEL23â toâ ubiq-uitinationâ reactionâ mixturesâ containingâ full-lengthâ flag-Mdm2â hadâ onlyâ modestâ inhibitoryâ effectâ onâ theâ Mdm2âligaseâ activityâ (âFig.â 6Aâ).â Becauseâ Mdm2â isâ ableâ toâ makeâ aâfunctionalâ complexâ withâ MdmX,â andâ thisâ complexâ isâ re-portedâtoâhaveâdifferentâactivityâandâspecificityâinâcells,âweâtestedâ theâ effectâ ofâ MEL23â onâ theâ purifiedâ Flag-Mdm2-HA-MdmXâ hetero-complexâ (purificationâ methodâ outlinedâinâ Supplementaryâ Fig.â S10).â Indeed,â MEL23â markedlyâ de-creasedâ theâ ligaseâ activityâ ofâ theâ Mdm2-MdmXâ complexâ(âFig.â 6Aâ).â Ofâ note,â Mdm2â isâ lessâ activeâ thanâ theâ Mdm2-MdmXâcomplexâasâanâE3â ligase,âbutâ theâ inhibitoryâeffectsâofâ MEL23â areâ stillâ notâ seenâ withâ Mdm2â whenâ equallyâ ac-tiveâamountsâofâproteinâareâusedâ(SupplementaryâFig.âS11,â50ângâMdm2/Xâvs.â100ângâMdm2).ââ
âBecauseâMdm2âandâMdmXâinteractâthroughâtheirâRINGâdomainsâ (â17â),âweâhypothesizedâ thatâ theâ targetâofâ theâMELâ
MEL23 and MEL24 Inhibit Mdm2-MdmX E3 Ligase Activity
âToâ determineâ whetherâ theâ MELâ compoundsâ inhibitâMdm2âandâp53âubiquitinationâinâcells,âweâanalyzedâMdm2âandâ p53â ubiquitinâ conjugatesâ inâ cell-basedâ ubiquitinationâassays.â Theâ Mdm2(wt)-luciferaseâ cellâ lineâ wasâ transfectedâwithâHA-ubiquitinâandâpretreatedâwithâ10âÎŒg/mLâMEL23âorâMEL24,â followedâ byâ 10â ÎŒMâ MG132.â Anâ increasedâ concen-trationâofâtheâcompounds,â10âÎŒg/mL,âwasâusedâtoâfacilitateâcompleteâinhibitionâofâubiquitination.âCellâlysatesâwereâim-munoprecipitatedâ withâ anâ anti-HAâ antibodyâ andâ analyzedâbyâWesternâblotting.âInâcellsâtreatedâwithâMEL23âorâMEL24,âsignificantlyâfewerâMdm2-ubiquitinâconjugatesâwereâimmu-noprecipitated,âindicatingâthatâtheâMELâcompoundsâinhibitâubiquitinationâofâMdm2â(âFig.â5Aâ).âTheâinhibitionâofâMdm2âubiquitinationâledâtoâaâstabilizationâofâMdm2âprotein,âasâin-dicatedâbyâitsâincreasedâhalf-lifeâfromâ1âhourâtoâ2âhoursâwithâ5âÎŒg/mLâMEL23âtreatmentâ(âFig.â5Bâ).ââ
âToâ testâ theâ effectsâ ofâ theâ MELâ compoundsâ onâ p53â ubiq-uitination,â H1299â cellsâ wereâ transfectedâ withâ plasmidsâ ex-pressingâ p53,â Mdm2,â andâ ubiquitinâ andâ pre-treatedâ withâ10â ÎŒg/mLâ MEL23â orâ MEL24,â followedâ byâ treatmentâ withâ10â ÎŒMâ MG132.â Ubiquitinationâ ofâ p53â wasâ analyzedâ byâWesternâblotting.âCellsâtreatedâwithâMG132âshowedâaâsignif-icantâincreaseâinâp53âubiquitinationâthatâwasâeliminatedâbyâpre-treatmentâwithâMEL23âorâMEL24â(âFig.â5Câ).âAdditionally,âtreatmentâwithâMEL23âorâMEL24âaloneâdemonstratedâthatâ
Figure 5. MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination in cells. A , MEL compounds inhibit Mdm2 ubiquitination in cells. Mdm2(wt)-luciferase cells were transfected with HA-ubiquitin (5 ÎŒg) and treated 8 hours later with 10 ÎŒg/mL (28 ÎŒM) MEL23, 10 ÎŒg/mL (31 ÎŒM) MEL24, or a DMSO control. After 16 hours, cells were additionally treated with 10 ÎŒM MG132 or a DMSO control. Three hours later cells were lysed and immunoprecipitated for 3 hours with an anti-HA affinity matrix. Total lysates and immunoprecipitated samples were analyzed by Western blotting using an anti-Mdm2 antibody. B , MEL23 increases the half-life of p53 and Mdm2 proteins. RKO cells were treated with 5 ÎŒg/mL (14 ÎŒM) MEL23, 10 ÎŒM MG132, or NT control for 4 hours, followed by treatment with 5 ÎŒg/mL (18 ÎŒM) cycloheximide for 30 minutes or 1, 2, 4, or 6 hours. Cell lysates were analyzed by Western blotting with anti-Mdm2, anti-p53 and anti-eIF4E antibodies. Protein levels were measured by densitometry using ImageJ and normalized to eIF4E. Half-life was calculated by plotting the protein levels in the treated cells to the protein level in the cells not treated with cycloheximide. C , MEL compounds inhibit p53 ubiquitination in cells. H1299 cells were transfected with Flag-Mdm2 (5 ÎŒg), p53 (0.5 ÎŒg), and HA-ubiquitin (2 ÎŒg). After 8 hours, cells were treated with 10 ÎŒg/mL (28 ÎŒM) MEL23, 10 ÎŒg/mL (31 ÎŒM) MEL24, or a DMSO control. After 16 hours cells were additionally treated with 10 ÎŒM MG132 or a DMSO control. Three hours later cells were lysed and analyzed by Western blotting using an anti-p53 antibody. Brackets indicate the slower migrating species of the protein that are the ubiquitin-conjugated forms. NT, no-treatment control.
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+ M
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EL2
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A C
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SEPTEMBERâ2011â CANCER DISCOVERYâ |â 319
Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay rEsEArCh ArtICLE
theâ factâ thatâ theâ Mdm2-MdmXâ hetero-complexâ isâ prefer-entiallyâ inhibited,â theâ overallâ extentâ ofâ theâ inhibitionâ byâMEL23â wasâ lowerâ inâ thisâ formatâ thanâ withâ theâ full-lengthâhetero-complex.â
âInâorderâ toâ furtherâ testâ theâspecificityâofâ theâMELâcom-pounds,â weâ analyzedâ theirâ effectâ onâ Roc1-Cul1â E3â RINGâdomainâligaseâactivity,âaâmulti-subunitâ ligaseâ(â30â).âWeâsawânoâinhibitionâwhenâtheâcomplexâwasâtreatedâwithâMEL23âorâMEL24â(SupplementaryâFig.âS13).âAdditionally,âweâsawânoâeffectâ ofâ theâ MELâ compoundsâ onâ theâ E3â ligaseâ activityâ ofâtheâ BRCA1/BARD1â proteinâ complexâ (â31â)â (SupplementaryâFig.âS14).âOfânote,âRoc1-Cul1âandâBRCA1/BARD1âshareâtheâsameâ ubiquitinâ conjugatingâ enzymeâ asâ Mdm2,â UbcH5C,âprovidingâanâadditionalâspecificityâcontrolâ(â32â).â
compoundsâ isâ theâ interfaceâ betweenâ theâ Mdm2â RINGâ andâtheâ MdmXâ RINGâ domains.â Weâ testedâ theâ activityâ ofâ theâcompoundsâ onâ theâ purifiedâ RINGâ domainsâ ofâ theâ pro-teins.â Theâ compoundsâ inhibitedâ theâ ubiquitinationâ ofâ theâMdm2(RING)-MdmX(RING)â hetero-complex,â butâ notâthatâ ofâ theâ Mdm2â RINGâ homo-complexâ (SupplementaryâFig.â S12).â However,â unlikeâ theâ full-lengthâ complex,â whichâwasâproducedâthroughâco-expressionâofâMdm2âandâMdmXâinâ SF9â cellsâ followedâ byâ 2â consecutiveâ purificationâ stepsâ(SupplementaryâFig.âS10),âtheâRINGâdomainâubiquitinationâreactionsâwereâperformedâbyâmixingâ individuallyâexpressedâandâpurifiedâMdm2âRINGâandâMdmXâRINGâproteins.âThus,âitâisâlikelyâthatâbothâactiveâhetero-âandâhomo-RINGâdomainâcomplexesâ wereâ presentâ inâ theâ reactions.â Asâ predictedâ byâ
Figure 6. MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination through the Mdm2-MdmX hetero-complex. A , MEL23 inhibits Mdm2-MdmX E3 ligase activity in vitro . Bottom panel: Ubiquitination assays in the presence of increasing concentrations of MEL compounds were performed with 100 ng of full-length Flag-Mdm2 or Flag-Mdm2/HA-MdmX complexes, 32 P-labeled ubiquitin, UbcH5C, and E1. Following 30 minutesâ incubation at 37°C, reactions were terminated and resolved on a SDS-PAGE gel followed by autoradiography. Top panel, the gels were quantified by phosphorimaging using ImageQuant software. PC, positive control; NC, negative control with UbcH5C eliminated from the reaction. B , MdmX is needed for efficient MEL23 activity in cells. Top panel, RKO cells were transfected with siRNAs against MdmX or luciferase, as a control. Forty-eight hours after transfection, cells were treated with 10 ÎŒg/mL (28 ÎŒM) MEL23, 0.5 ÎŒg/mL (0.9 ÎŒM) doxorubicin, or DMSO for 4 hours before harvesting. Lysates were analyzed by Western blotting using anti-MdmX, anti-p53, and anti-eIF4E antibodies. Bottom left panel, fold increase in p53 levels of NT and MEL23 treated cells normalized to the NT control under two different siRNA co-treatment conditions. Bottom right panel, raw p53 levels in each condition. Protein levels were measured by densitometry using ImageJ and graphed using GraphPad Prism. Error bars represent the SEM of 3 samples. C , MEL23 does not inhibit Mdm2-p53 and Mdm2-MdmX complex formation. MCF7 cells were transfected with FLAG-Mdm2 (12 ÎŒg) and p53 (2.5 ÎŒg). After 24 hours, cells were treated with 10 ÎŒg/mL (28 ÎŒM) MEL23, 5.3 ÎŒg/mL (10 ÎŒM) Nutlin-3a, or DMSO for 6 hours. Cell lysates were incubated with anti-FLAG M2 antibody for 2 hours, followed by incubation with Protein-A/G sepharose beads for 1 hour at 4°C. Cells were lysed and analyzed by Western blotting using anti-Mdm2, anti-MdmX, and anti-p53 antibodies. NT, no-treatment control.
FL-Mdm2 FL-Mdm2-MdmX
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ary
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7953_CD-11-0104_pp312-325.indd 319 8/30/11 12:23 PMCancer Research. on March 18, 2021. © 2011 American Association forcancerdiscovery.aacrjournals.org Downloaded from
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320â|â CANCER DISCOVERYâ SEPTEMBERâ2011â www.aacrjournals.org
Herman et al.rEsEArCh ArtICLE
ToâconfirmâthatâtheâMELâcompoundsâactâonâtheâMdm2-MdmXâ complexâ inâ cells,â weâ decreasedâ MdmXâ levelsâ viaâsiRNAâ transfectionâ inâ RKOâ cells.â Cellsâ transfectedâ withâ aânontargetingâsiRNAâandâthenâtreatedâwithâMEL23âshowedâanâ increaseâ ofâ greaterâ thanâ 20-foldâ inâ p53â proteinâ levelsâcomparedâ toâ dimethylsulfoxideâ (DMSO)-treatedâ controlâcells.âByâcontrast,âsiMdmX-transfectedâcellsâthatâwereâthenâtreatedâwithâMEL23âshowedâanâincreaseâofâapproximatelyâ7-foldâ inâ p53â proteinâ levelsâ comparedâ toâ DMSOâ controlâcellsâ(Fig.â6B).âCellsâwereâalsoâtreatedâinâparallelâwithâdoxo-rubicinâ toâ ensureâ weâ wereâ notâ saturatingâ p53â levelsâ withâtheâMdmXâknockdownâand/orâMELâtreatment.âWeâfoundâthatâ p53â levelsâ increasedâ moreâ (albeitâ modestlyâ so)â withâdoxorubicinâ thanâ MEL23â treatmentâ inâ cellsâ transfectedâwithâsiMdmX.âThisâresultâdemonstratesâthatâwithâreducedâMdmXâlevels,âtheâMELâcompoundsâareâlessâefficientâatâpre-ventingâp53âdegradation.
Inâ orderâ toâ furtherâ validateâ theâ mechanismâ ofâ actionâ ofâtheâ MELâ compounds,â weâ overexpressedâ p53,â Mdm2,â and/orâMdmXâinâU2OSâcells,âtreatedâwithâMEL23âandâanalyzedâp53âlevels.âWeâusedâMdm2âandâMdmXâatâconcentrationsâatâwhichâ synergyâ wasâ observedâ inâ theâ destabilizationâ ofâ p53â(19).âWeâmonitoredâp53â levelsâ inâcellsâunderâ4âDNAâtrans-fectionâ conditionsâ treatedâ withâ MEL23;â nontransfected,âp53â only,â p53â andâ Mdm2,â andâ p53,â Mdm2â andâ MdmXâtransfections.â Theâ mostâ dramaticâ stabilizationâ occurredâwhenâallâ3âproteinsâ(p53,âMdm2,âandâMdmX)âwereâoverex-pressedâ(SupplementaryâFig.âS15).âAnâincreaseâinâp53âinâtheânontransfectedâ cellsâ occurred,â althoughâ significantlyâ lessâthanâ inâ theâ transfectedâ cells,â asâ expectedâ sinceâ U2OSâ cellsâhaveâwild-typeâp53.âThisâresultâsuggestsâthatâtheâMELâcom-poundsâworkâmostâefficientlyâinâtheâpresenceâofâbothâMdm2âandâMdmX.
Althoughâ theâ dataâ suggestâ thatâ theâ MELâ compoundsâ in-terfereâ withâ theâ Mdm2-MdmXâ E3â ligaseâ activity,â theyâ wereânotâ foundâ toâ inhibitâ formationâ ofâ theâ complexâ betweenâMdm2â andâ MdmXâ inâ co-immunoprecipitationâ experimentsâ(Fig.â 6C;â Supplementaryâ Fig.â S16).â MEL23â alsoâ didâ notâ in-hibitâ p53-Mdm2â complexâ formation.â Theseâ resultsâ suggestâthatâ theâ MELâ compoundsâ haveâ aâ differentâ modeâ ofâ actionâthanâNutlin-3,âanâinhibitorâofâp53-Mdm2âinteractionâ(23).
Takenâ together,â theseâ resultsâ suggestâ thatâ whileâ theâ MELâcompoundsâ mayâ inhibitâ Mdm2â aloneâ toâ someâ extent,â theirâprimaryâtargetâinâcellsâisâtheâE3âligaseâactivityâofâtheâMdm2-MdmXâ hetero-complex.â Additionally,â theâ compoundsâfunctionâ viaâ aâ mechanismâ otherâ thanâ interferingâ withâ theâformationâofâthisâproteinâcomplex.
MEL23 Leads to Cell Death that Is p53-dependent and synergistic with DNA Damaging Agents
Toâ testâ theâ physiologicalâ outcomesâ andâ potentialâ thera-peuticâefficacyâofâtheâMELâcompounds,âweâexaminedâtheâsur-vivalâofâRKOâandâRKO-E6âcellsâafterâtreatmentâwithâMEL23.âBecauseâ treatmentâ ofâ RKOâ cellsâ withâ theâ MELâ compoundsâledâtoâanâincreaseâinâp53âlevelsâandâactivityâ(Fig.â3A,â3C,âandâ4D),âweâexpectedâaâcorrespondingâincreaseâinâp53-dependentâcellâdeath.âConsistentâwithâthis,âRKOâcellâsurvivalâdecreasedâinâ aâ dose-dependentâ mannerâ followingâ MEL23â treatment,â
whileâ RKO-E6â cellsâ remainedâ largelyâ unaffectedâ (Fig.â 7A).âMEL23â treatmentâ increasedâ theâ sub-G1â populationâ andâcausedâaâslightâG2âcell-cycleâarrestâinâRKO,âmouseâembryonicâfibroblastâ (MEF),â andâ HT-1080â cellsâ (Supplementaryâ Fig.âS17;âdataânotâshown).âTheseâcellsâstainedâpositiveâforâTUNELâandâ exhibitedâ caspaseâ activationâ (Supplementaryâ Fig.â S17),âsuggestingâanâapoptoticâcellâdeathâprocess.
Toâfurtherâtestâtheâp53-âandâMdm2-dependenceâofâMEL23-inducedâ cellâ death,â weâ analyzedâ 3â MEFâ cellâ lines:â wild-type,âp53-/-,â andâ p53-/-; mdm2-/-â (Fig.â 7B;â Supplementaryâ Fig.â S18).âConsistently,âMELâcompoundsâshowedâp53-dependentâactiv-ity,â asâ theâ survivalâ ofâ theâ wild-typeâ cellsâ wasâ decreasedâ inâaâ dose-dependentâ mannerâ byâ MEL23.â Theâ survivalâ ofâ theââp53-/-âMEFsâwasâalsoâdecreasedâbyâMEL23âtreatment,âindicat-ingâ someâ p53-independent,â Mdm2-dependentâ activity.â Thisâisâ consistentâwithâ reportsâ thatâMdm2âhasâoncogenicâeffectsâindependentâ ofâ p53â (33).â Onlyâ aâ smallâ decreaseâ inâ cellâ sur-vivalâwasâseenâinâtheâp53-/-; mdm2-/-âMEFsâtreatedâwithâMEL23.âTherefore,âconsistentâwithâtheâMdm2-MdmXâcomplexâbeingâtheâ targetâ ofâ theâ MELâ compounds,â theâ majorityâ ofâ growthâinhibitionâwasâdependentâonâtheâpresenceâofâMdm2âandâp53.
Weâ testedâ theâ abilityâ ofâ MEL23â toâ cooperateâ withâ DNA-damagingâ agentsâ toâ potentlyâ induceâ cellâ death.â U2OSâ (p53âwild-type)â andâ H1299â (p53-null)â cellsâ wereâ treatedâ withâ in-creasingâamountsâofâMEL23âandâeitherâcamptothecinâorâeto-posideâ inâ combinationâ forâ 48â hoursâ andâ cellâ viabilityâ wasâmeasured.â Theâ combinationâ ofâ MEL23â withâ theseâ DNA-damagingâagentsâledâtoâaâsynergisticâdecreaseâinâviabilityâ(Fig.â7C;âSupplementaryâFig.âS19).âTheâresultsâareâpresentedâasâaâdecreaseâinâcellâviabilityâoverâthatâpredictedâbyâtheâBlissâinde-pendenceâmodel.âBlissâ independenceâwasâdeterminedâbyâtheâformulaâCâ=âAâ+âBâââA*B,âwhereâAâisâtheâtreatmentâwithâoneâcompoundâ aloneâ andâ Bâ isâ treatmentâ withâ theâ secondâ com-poundâ aloneâ (34).â Theâ excessâ overâ Blissâ independenceâ wasâdeterminedâbyâsubtractingâtheâpredictedâBlissâeffectâfromâtheâexperimentallyâdeterminedâeffect,âyieldingâaâsynergisticâeffectâofâtheâcombinedâtreatment.
Becauseâ MEL23â canâ inhibitâ p53â ubiquitination,â andâNutlin-3â canâ preventâ Mdm2â fromâ inhibitingâ p53â transcrip-tionalâ activation,â weâ alsoâ testedâ theâ abilityâ ofâ MEL23â andâNutlin-3â toâ cooperativelyâ decreaseâ cellâ viability.â Synergyâ be-tweenâ MEL23â andâ Nutlin-3â occurred,â butâ toâ aâ lesserâ extentâthanâtheâcombinationâofâMEL23âandâDNA-damagingâagentsâ(Fig.â7C;âSupplementaryâFig.âS19).
Interestingly,âMEL23âalsoâsynergizedâwithâDNA-damagingâagentsâ inâp53-nullâH1299âcells.âNutlin-3,âonâtheâotherâhandâexhibitedâ synergyâ withâ camptothecinâ andâ etoposideâ onlyâ inâwild-typeâp53âcells.âThisâmayâbeâbecauseâtheâMELâcompoundsâareâ inhibitingâ additionalâ functionsâ ofâ Mdm2â thatâ areâ p53-âindependent,âbutâstillâpotentiallyâoncogenic.âTheseâsynergisticâeffectsâofâMEL23âwithâlowâconcentrationsâofâDNAâdamagingâagentsâthatâdoânotâcauseâdeleteriousâeffectsâonâtheirâownâmayâincreaseâtheâtherapeuticâutilityâofâtheâMELâcompounds.
DiscussionInâthisâstudy,âweâconductedâaâscreenâtoâidentifyâinhibitorsâ
ofâMdm2âE3â ligaseâactivity.âWeâfoundâ2âcloselyâ relatedâana-logsâoutâofâ270,080âtestedâcompoundsâMEL23âandâMEL24.âUsingâfull-lengthâMdm2âallowedâusâtoâtakeâadvantageâofâtheâ
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Figure 7. MEL23 decreases cell viability alone and in combination with DNAâdamaging agents. A , MEL23 specifically decreased RKO cell viability. RKO and RKO-E6 cells were seeded in 384-well plates and treated with a dilution series of MEL23 for 48 hours as indicated. Alamar Blue was added for 16 hours and fluorescence determined on a Victor3 Plate Reader. The median percent inhibition of 5 replicates was determined and normalized to a no-treatment control. One standard deviation is shown. B , percent survival of wild-type, p53 -/- , and p53 -/- ;mdm2 -/- MEFs. Cells were seeded in 384-well plates and treated with a dilution series of MEL23 for 48 hours at the indicated concentrations. Alamar Blue was added for 16 hours and fluorescence determined on a Victor3 Plate Reader. The median percent inhibition of 5 replicates was determined and normalized to a no-treatment control. One standard deviation is shown. C , MEL23 synergizes with DNA-damaging agents. U2OS (p53 wild-type) and H1299 (p53 null) cells were seeded in 384-well plates and treated with compound(s) for 48 hours. The starting concentrations of the compounds diluted vertically in the plate are MEL23 at 10 ÎŒg/mL (28 ÎŒM) and Nutlin-3 at 10 ÎŒg/mL (17 ÎŒM). The starting concentrations of the compounds diluted horizontally in the plate are camptothecin at 25 ÎŒg/mL (72 ÎŒM), Nutlin-3 at 50 ÎŒg/mL (86 ÎŒM), and etoposide at 100 ÎŒg/mL (170 ÎŒM). Compounds were diluted in a 2-fold, 7-point dilution vertically down the plate or in a 2-fold, 11-point dilution horizontally across the plate. After the incubation period, Alamar Blue was added for 16 hours, fluorescence was measured on a Victor3 Plate Reader, and the percent growth inhibition determined. Bliss independence was determined by the formula C = A + B â A*B, where A is the treatment with one compound alone and B is the treatment with the second compound alone. The excess over Bliss independence was determined by subtracting the predicted Bliss effect from the experimentally determined effect, yielding the synergistic effect of the combined treatment. Highlighted values in red have > 50% growth inhibition over that predicted by Bliss independence, values in orange have > 25%, and values in yellow have > 15%.
presenceâ ofâ allâ domainsâ ofâ Mdm2â inâ orderâ toâ improveâ theâlikelihoodâofâdiscoveringâinhibitorsâ.ââAdditionally,âtheâuseâofâaâcell-basedâassayâenabledâtargetingâofâpotentialâcellularâco-fac-torsânecessaryâforâligaseâactivity.âTheâhigh-throughputâscreenâweâpresentâhereâcouldâbeâmodifiedâ toâdiscoverâ inhibitorsâofâotherâ E3â ligases.â Theâ easeâ ofâ thisâ typeâ ofâ high-throughputâ
screenâ andâ itsâ adaptabilityâ mayâ thereforeâ beâ valuableâ inâ theâidentificationâ ofâ newâ small-moleculeâ inhibitorsâ directedâagainstâdifferentâE3âligases,âwhichâhaveâpreviouslyâbeenâcon-sideredâchallengingâtargetsâforâdrugâdiscovery.â
âMdm2â inhibitorsâ describedâ toâ dateâ includeâ peptideâ in-hibitorsâ(â35â)âandâsmallâmoleculesâsuchâasâNutlin-3â(â23â)âandâ
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23 % Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
A B
C
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
Excess over Bliss independence: U2OS cells Excess over Bliss independence: H1299 cells
Camptothecin
ME
L23 Camptothecin
Nutlin-3
Nutlin-3
ME
L23
Camptothecin
ME
L23
Camptothecin
Nutlin-3
Nutlin-3
ME
L23
% Survival
0
RKORKO-E6
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
% Survival
0
wt MEFsp53-/-p53-/-Mdm2-/-
10
40
20 30
5060
90
70 80
100110
[MEL23] uM10 0.5 10 1.0 10 1.5 10 2.0
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Herman et al.rEsEArCh ArtICLE
RITAâ (36).â Mdm2â E3â ligaseâ inhibitorsâ identifiedâ byâ in vitroâscreens,âsuchâasâtheâHLIâseriesâofâcompounds,âhaveâalsoâbeenâdescribedâ(24).âAlthoughâtheâHLIâcompoundsâwereâableâtoâin-duceâp53-dependentâapoptosis,âtheyâlackâspecificityâtowardsâMdm2â (24).â Otherâ Mdm2â E3â ligaseâ inhibitorsâ identifiedâ inâin vitroâubiquitinationâassays,âsuchâasâsempervirineâandâlisso-chlinidineâB,âhaveânotâbeenâshownâtoâinhibitâMdm2-MdmXâE3âligaseâactivityâ(37,â38).âMoreârecently,âtheâfirstâsmall-mol-eculeâinhibitorâofâMdmXâbindingâtoâp53âwasâidentifiedâ(39).âUnlikeâotherâMdm2âandâMdmXâinhibitorsâidentified,âMEL23âandâMEL24âareâaâuniqueâclassâofâMdm2âinhibitorsâidentifiedâinâaâcell-basedâassayâthatâareâableâtoâinhibitâtheâE3âligaseâactiv-ityâofâtheâMdm2-MdmXâcomplex.
TheseâdataâshowâthatâMEL23âandâMEL24âpreferentiallyâinhibitâ theâ E3â ligaseâ activityâ ofâ theâ Mdm2-MdmXâ com-plex,â althoughâ theyâ doâ haveâ someâ activityâ againstâ Mdm2âalone.âTheâinhibitionâofâtheâMdm2-MdmXâcomplexâin vitroâandâ stabilizationâ ofâ p53â andâ Mdm2â inâ cellsâ indicateâ thatâtheâ Mdm2-MdmXâ complexâ isâ centrallyâ involvedâ inâ regu-latingâ theâ degradationâ ofâ theseâ proteins.â Theseâ observa-tionsâareâinâagreementâwithâmultipleâstudiesâthatâdescribeâtheâimportanceâandâactivityâofâtheâMdm2-MdmXâcomplex.âWhileâMdm2âcanâcatalyzeâp53âubiquitinationâin vitroâ(40),âinâ cellsâ MdmXâ isâ neededâ alongâ withâ Mdm2â forâ efficientâp53âdegradationâ(19).âMdmXâcanâlowerâtheâconcentrationâofâ Mdm2â neededâ forâ bothâ p53â ubiquitinationâ andâ auto-ubiqutination,â andâ theâ Mdm2-MdmXâ complexâ hasâ beenâshownâtoâbeâaâbetterâligaseâforâp53âthanâMdm2âaloneâ(41).âAdditionallyâ mouseâ studiesâ haveâ shownâ thatâ theâ interac-tionâbetweenâMdm2âandâMdmXâisâessentialâforâregulatingâp53âduringâearlyâembryogeneisisâ(42,â43).âEnhancementâofâactivityâ ofâ RINGâ domainâ hetero-oligomersâ hasâ alsoâ beenâdemonstratedâ forâ BRCA1-BARD1â complexes,â whereâ theâpresenceâofâBARD1âinâtheâcomplexâincreasesâtheâligaseâac-tivityâofâBRCA1â(44).âThereforeâourâresultsâsupportâstudiesâsuggestingâthatâalthoughâMdm2âisâableâtoâfunctionâasâanâE3âligaseâonâitsâown,âMdmXâaugmentsâtheânatureâandâac-tivityâofâtheâligaseâwithâprofoundâfunctionalâconsequencesâinâcells.
Oneâcaveatâinâtheseâmechanisticâstudiesâwasâthatâhighâcon-centrationsâofâMELâcompoundsâwereânecessaryâtoâseeâ inhibi-tionâofâubiquitinationâin vitro.âItâisâpossibleâthatâtheâenzymaticâactivityâ ofâ theâ Mdm2-MdmXâ ligaseâ isâ difficultâ toâ inhibitâ in vitro;â theâ well-knownâ Nutlin-3â compoundâ doesâ notâ inhibitâMdm2âubiquitinationâ(45).âPerhapsâtheâMELâcompoundsâonlyâpartiallyâinhibitâE3âligaseâactivityâinâcells,âandâthisâisâsufficientâtoâcauseâp53âaccumulation,âconsistentâwithâmouseâdataâshow-ingâthatâjustâaâ20%âtoâ30%âreductionâinâMdm2âcanâleadâtoâp53âactivationâ (46).â Itâ isâ temptingâ toâ speculateâ thatâ compoundsâthatâ completelyâ inhibitedâ Mdm2â orâ Mdm2-MdmXâ E3â ligaseâactivityâwouldâactivateâp53âinâbothâcancerâandânormalâcellsâtoâsuchâ aâ largeâ extentâ thatâ theâ compoundsâ wouldâ notâ beâ toler-atedâ andâ wouldâ thereforeâ notâ beâ usefulâ asâ therapeutics.â TheâMELâcompounds,âonâtheâotherâhand,âhaveâaâsmallâbutâconsis-tentâdifferentialâactivityâbetweenâtumorâderivedâandânontrans-formedâcellâlines,âdecreasingâtheâsurvivalâofâtheâtumor-derivedâcellsâtoâaâgreaterâextentâ(SupplementaryâFig.âS20).
Becauseâ Mdm2â canâ inhibitâ p53â throughâ 2â independentâmechanismsâE3â ligase-mediatedâ degradationâ andâ binding-mediatedâfunctionalâinactivationâitâisâformallyâpossibleâthatâ
inhibitorsâofâMdm2âE3âligaseâactivityâwouldânotâbeâsufficientâtoâ activateâ p53.â Inâ thisâ model,â Mdm2â E3â ligaseâ inhibitorsâwouldâ increaseâ bothâ p53â andâ Mdm2â levels,â butâ wouldâ notâpreventâMdm2âfromâbindingâp53âandâtherebyâ inhibitingâ itsâactivity.â Basedâ onâ qRT-PCRâ dataâ (Fig.â 3D;â SupplementaryâFig.â S7)â andâ alsoâ co-immunoprecipitationâ dataâ (Fig.â 6C;âSupplementaryâ Fig.â S16),â itâ isâ likelyâ thatâ Mdm2â andâ p53âareâ stillâ interactingâ toâ someâ extentâ uponâ MELâ compoundâtreatmentâ andâ thatâ Mdm2â isâ partiallyâ inhibitingâ p53â activ-ity.â However,â weâ observedâ increasedâ p53â activityâ uponâ MELâtreatmentâ comparedâ toâ proteasomeâ inhibition,â whichâ alsoâincreasesâMdm2âandâp53âlevels.âInâRKOâcells,âsuchâincreasesâinâ p53â activityâ causedâ byâ MELâ compoundâ treatmentâ ap-pearâ toâ beâ sufficientâ forâ p53-dependentâ cellâ death,â albeitâ atâhigherâconcentrationsâofâMEL23âthanâareâneededâforâp53âsta-bilization.â Indeed,âHLIâcompounds,âanotherâclassâofâMdm2âE3â ligaseâ inhibitors,â alsoâ induceâ p53-dependentâ cellâ death,âsuggestingâ thatâ completeâ disruptionâ ofâ theâ Mdm2-p53â in-teractionâ mayâ notâ beâ necessaryâ forâ activationâ ofâ p53â (24).âAdditionally,â mutantâ knock-inâ miceâ studiesâ haveâ demon-stratedâ thatâ Mdm2-p53â binding,â withoutâ Mdm2-mediatedâp53â ubiquitination,â isâ notâ sufficientâ toâ controlâ p53â activityâ(47).âFurthermore,âalthoughâMELâcompoundsâdoânotâinduceâp53âpost-translationalâmodifications,âsomeâcancerâcellsâmayâalreadyâ haveâ suchâ modificationsâ inâ placeâ asâ aâ consequenceâofâoncogenicâstress.âNevertheless,âweâpredictâthatâtheâcombi-nationâ ofâ theâ MELâ compoundsâ andâ DNA-damagingâ agentsâwouldâenhanceâtheirâtherapeuticâpotential.âThisâcouldâalsoâal-lowâforâuseâofâDNA-damagingâagentsâatâconcentrationsâthatâdoânotâcauseâsomeâofâtheirânormalâdeleteriousâsideâeffects.
MEL23â cooperatedâ withâ DNA-damagingâ agentsâ inâ p53-nullâcellsâtoâaâsmall,âyetâreproducible,âextent.âMdm2âhasâbeenâshownâtoâhaveâp53-independentâoncogenicâeffects,â forâ exam-ple,â(1)âoverexpressionâofâMdm2âinâmiceâcausesâtumorsâinde-pendentâofâp53âstatusâ (33);â (2)â spliceâvariantsâofâMdm2âthatâcannotâbindâtoâp53âhaveâbeenâshownâtoâbeâoncogenicâ(48);â(3)âMdm2âdestabilizationâofâRbâ(49,â50)âandâp21â(51)âmayâcontrib-uteâ toâ tumorâ growth;â andâ (4)â Nutlin-3-mediatedâ disruptionâofâp73-Mdm2âbindingâenhancesâp73âfunctionâ(52).âTherefore,âtheâ MELâ compoundsâ andâ otherâ specificâ Mdm2â E3â ligaseâ in-hibitorsâmayâbeâbeneficialâinâp53-nullâorâp53-mutantâtumors.âThisâhypothesis,âalthoughâintriguing,ârequiresâfurtherâstudy.
AsâMdm2-MdmXâligaseâinhibitors,âMELâcompoundsâmayâprovideâ furtherâ insightâ intoâ theâ functionâ ofâ theâ Mdm2-MdmXâ E3â ligaseâ andâ allowâ forâ investigationâ ofâ theâ differ-encesâinâactivityâbetweenâtheâhetero-complexâandâtheâMdm2âhomo-complex.âItâwillâbeâinformativeâtoâultimatelyâdetermineâtheâ preciseâ biophysicalâ mechanismâ ofâ actionâ ofâ theâ MELâcompounds,â asâ wellâ asâ theâ bindingâ siteâ ofâ theâ compounds.âAdditionally,â theâ MELâ compoundsâ mayâ beâ usedâ asâ molecu-larâ toolsâ toâ validateâ novelâ targetsâ ofâ Mdm2-MdmX.â Finally,âalthoughâ theseâ compoundsâ wereâ notâ suitableâ forâ testingâ inâmice,â weâ haveâ demonstratedâ thatâ targetingâ Mdm2-MdmXâwithâsmallâmoleculesâisâfeasibleâandâfutureâstudiesâcouldâei-therâoptimizeâtheseâcompoundsâorâuseâthisâhigh-throughputâassayâ toâ discoverâ additionalâ drug-likeâ scaffoldsâ withâ similarâactivity.âAnâimprovedâunderstandingâofâtheâmechanismâofâac-tionâofâtheâMELâcompounds,âand/orâfutureâinhibitorsâofâthisâpathway,â mayâ leadâ toâ newâ waysâ toâ inhibitâ E3â ligases,â whichâcouldâbeâbeneficialâinâdiverseâapplications.
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Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay rEsEArCh ArtICLE
baculovirusâwasâaâkindâgiftâfromâDr.âS.âGrossmanâatâtheâUniversityâofâMassachusettsâMedicalâSchool.âBothâFlag-Mdm2âandâHA-MdmXâbaculovirusesâwereâpreparedâaccordingâtoâtheâBac-to-Bacâbaculovirusâexpressionâsystemâ(Invitrogen).
Compound LibrariesAâtotalâofâ51,356âcompoundsâwereâscreenedâinâtheâStockwellâLab.â
Theseâ includedâ syntheticâ compoundsâ andâ naturalâ productsâ fromâInterBioScreen,âTimtec,âChembridge,âMicroSource,âandâLifeâchemi-calsâ (detailsâavailableâuponârequest).âTheseâcompoundsâwereâstoredâinâDMSOâinâ384-wellâplatesâatâ4âmg/mL.âMEL23âandâMEL24âwereâidentifiedâ fromâ InterBioScreenâ andâ theâ analogsâ wereâ alsoâ obtainedâfromâInterBioScreen.
Aâ totalâ ofâ 218,724â compoundsâ wereâ screenedâ throughâ theâ NIHâMLSCNâatâtheâUniversityâofâPennsylvania.
Screening (Stockwell Lab)Theâ Mdm2(wt)-luciferaseâ cellâ lineâ wasâ seededâ atâ 7,500â cellsâ perâ
well,â inâ 384-wellâ whiteâ platesâ (PerkinElmer,â 6007688),â inâ 27â ÎŒLâ ofâmediaâ (DMEM,â FB,â P/S).â Assayâ platesâ withâ cellsâ wereâ incubatedâ atâ37°Câovernightâtoâallowâcellsâtoâadhere.âAfterâ24âhours,â2âÎŒLâfromâtheââmotherââplateâ(4âmg/mL)âwasâtransferredâtoâaââdaughterâplateââcon-tainingâ148âÎŒLâmediaâinâorderâtoâdiluteâtheâcompoundâ1:75.âNext,â3âÎŒLâfromâeachâwellâofâtheâdaughterâplatesâwasâaddedâtoâtriplicateâassayâplatesâforâaâfinalâconcentrationâofâ5.33âÎŒg/mL.âAssayâplatesâwereâin-cubatedâatâ37°Câforâ2âhours.âThen,âtheâcellsâwereâlysedâwithâtheâaddi-tionâofâ30âÎŒLâofâluminescenceâbufferâ(PerkinElmer,â6016989)âandâtheâplatesâwereâincubatedâatâRTâforâ30âminutesâbeforeâbeingâanalyzedâonâaâVictor3âPlateâReaderâ(PerkinElmer)âforâluminescence.âCompoundsâthatâcausedâanâ increaseâ inâ luminescenceâ.â30%âwereâre-orderedâforâfurtherâanalysis.âAllârepurchasedâcompoundsâwereâtestedâinâaâ2-foldâdilutionâ seriesâ viaâ theâ sameâ protocolâ asâ theâ primaryâ screening.â AllâtransfersâwereâconductedâusingâaâBioMekâ(BeckmanâCoulter).âTheâfi-nalâoptimizedâscreenâhasâaâZ'âvalueâofâ0.63â(57).âThisâwasâdeterminedâbyâcomparingâtheânegativeâcontrol,ânoâtreatment,âtoâtheâpositiveâcon-trol,â10âÎŒMâMG132.
Screening (MLSCN library)DescribedâinâPubchemâ(BioAssayâAIDâ1442,âAIDâ1230,âAIDâ1444,â
andâAIDâ1394).âTheâfinalâoptimizedâscreenâhasâaâZ'âvalueâofâ0.49â(57).
Western Blot Analysis Cellsâ wereâ seededâ andâ treatedâ withâ theâ indicatedâ compound(s).â
Cellsâwereâlysedâafterâeachâtimeâpointâinâaâbufferâofâ50âmMâTrisHClâpHâ 7.5,â 200â mMâ NaCl,â 0.5%â NP-40,â andâ 1â completeâ miniâ EDTA-freeâ proteaseâ inhibitorâ cocktailâ tabletâ perâ 10â mLâ ofâ bufferâ (Roche,â8360170).â Proteinâ contentâ wasâ quantifiedâ usingâ aâ Bio-Radâ proteinâassayâreagentâ(Bio-Rad,â500-0006).âSDSâsampleâbufferâwasâaddedâtoâtheâsamplesâandâtheyâwereâboiledâ forâ5âminutes.âEqualâamountsâofâproteinâwereâresolvedâonâaâ1DâSDS-PAGE.âFollowingâseparation,âpro-teinsâwereâtransferredâtoâaânitrocelluloseâmembrane,âblockedâwithâ5%âmilk,â andâ thenâ incubatedâ withâ primaryâ antibodyâ overnightâ atâ 4°C.âTheâmembraneâwasâincubatedâwithâsecondary-HRPâantibodyâforâ45âminutesâatâRTâandâdevelopedâwithâSuperSignalâWestâPicoâSubstrateâ(Pierce,â34080).
ImmunoprecipitationCellsâ wereâ lysedâ inâ 150â mMâ NaCl,â 10â mMâ TrisHClâ pHâ 7.5,â 0.5%â
NP-40,â10%âglycerol,â1âmMâEDTA,â1âmMâDTT,âandâ1âcompleteâminiâEDTA-freeâ proteaseâ inhibitorâ cocktailâ tabletâ perâ 10â mLâ ofâ buffer.âFLAG-taggedâ Mdm2â immunoprecipitationâ experimentsâ wereâ per-formedâ byâ incubatingâ cellâ lysatesâ withâ anti-M2â (FLAG)â antibodyâ(Sigma,âF1804)âforâ2âhoursâatâ4°CâandâwithâProtein-A/Gâsepharoseâbeadsâ(Pierce,â20421)âforâ1âhourâatâ4°C.âHA-ubiquitinâimmunopre-cipitationâ experimentsâ wereâ performedâ byâ incubatingâ cellâ lysatesâwithâ HA-affinityâ matrixâ (Roche,â 11815016001)â forâ 3â hoursâ atâ 4°C.â
MethoDsReagents
MG132â (Sigma,â C2211),â ALLNâ (Calbiochem,â 208719),â cyclohexi-mideâ (Sigma,â C7698),â doxorubicinâ hydrochlorideâ (Sigma,â D1515),âetoposideâ (Sigma,â E1383),â camptothecinâ (Sigma,â C9911),â andâNutlin-3â (Sigma,â N6287)â wereâ usedâ forâ cellâ treatmentsâ atâ theâ indi-catedâconcentrations.
Antibodiesâ toâ p53â (Calbiochem,â OP43â forâ Westernâ blotting,âandâ Calbiochem,â OP03â forâ co-immunoprecipitation),â Mdm2â(Calbiochem,âOP115),âanti-M2â(FLAG)â(Sigma,âF1804),âphospho-p53âser15â (Cellâ Signaling,â 9284s),â phospho-histoneâ H2A.Xâ (Millipore,â05-636),â histoneâ H2A.Xâ (Millipore,â 07-627),â MdmXâ (Bethyl,â A300-287A),âc-Mycâ(5605S,âCellâSignal),âc-junâ(9165S,âCellâSignal),âBRCA1â(Calbiochem,âOP92),âeIF4Eâ(BDâTransductionâLaboratories,â610270),âandâ actinâ (Santaâ Cruzâ Biotech,â sc-1616-R)â wereâ used.â Anti-Mdm2âSMP14,â2A10,â3G5âmix,âusedâasâsupernatantsâfromâhybridomaâcul-tures,âwasâusedâforâin vitroâMdm2âubiquitinationâassays.
Cell Lines and Transfections293Tâ (humanâ embryonicâ kidney),â H1299â (nonâsmall-cellâ
lungâ carcinoma),â MCF7â (mammaryâ glandâ adenocarcinoma),âwild-typeâ MEFs,â p53-/-â MEFs,â andâ p53-/-mdm2-/-â MEFsâ wereâ main-tainedâ inâ Dulbecco'sâ modifiedâ Eagle'sâ mediumâ (DMEM)â (Fisher,âMT-15-018-CM)â withâ 10%â fetalâ bovineâ serumâ (FBS)â andâ 100â ÎŒg/mLâ penicillin/streptomycinâ (P/S).â Mdm2(wt)-luciferaseâ andâMdm2(C464A)-luciferaseâ stableâ cellâ linesâ (inâ 293Tâ cells)â wereâ ad-ditionallyâtreatedâwithâ300âÎŒg/mLâofâzeocinâ(Invitrogen,âR250-05).âWild-typeâ MEFsâ wereâ aâ kindâ giftâ fromâ Craigâ B.â Thompsonâ atâ theâUniversityâofâPennsylvania.âP53-/-MEFsâandâp53-/-;mdm2-/-âMEFSâwereâaâ kindâ giftâ fromâ Guillerminaâ Lozanoâ atâ theâ Universityâ ofâ Texas.âRKOâ(colonâcarcinoma)âandâRKOâE6â(colonâcarcinoma)âcellsâwereâmaintainedâ inâ Minimumâ Essentialâ Mediumâ (MEM)â Eagleâ (Sigma,âM5650)â withâ 10%â FBâ andâ 100â ÎŒg/mLâ P/S.â U2OSâ (osteosarcoma)âandâ HCT116â (colorectalâ carcinoma)â wereâ maintainedâ inâ McCoyâsâ5aâMediumâ(Invitrogen,â16600-108)âwithâ10%âFBSâandâ100âÎŒg/mLâP/S.âHT-1080âcellsâwereâmaintainedâinâDMEM,â10%âFB,â100âÎŒg/mLâP/S,âandâ1%âNon-essentialâaminoâacidsâ(Invitrogen,â11140-050).âAllâcellsâwereâgrownâatâ37°Câinâ5%âCO2.
Cellsâ wereâ transfectedâ usingâ FuGENEâ 6â (Roche,â 814-442-001)â inâaccordanceâ withâ theâ manufacturerâsâ protocol.â Transfectionsâ wereâperformedâ inâ 6-wellâ dishes,â exceptâ forâ theâ co-immunoprecipitationâexperimentâperformedâinâ15-cmâdishesâorâasânoted.
Toâ knockâ downâ MdmXâ expression,â cellsâ wereâ transfectedâ withâ 20ânMâcontrolâsiRNAâ(AACTTACGCTGAGTACTTCGA)âorâMdmXâsiRNAâ(AGAGATTCAGCTGGTTATTAA)âusingâDharmaFECT1â(Dharmacon,âT-2001-01)âaccordingâtoâmanufacturer'sâinstructions.âAfterâ48âhoursâofâknockdown,âcellsâwereâtreatedâwithâtheâindicatedâcompounds.
PlasmidsMdm2(wt)-luciferaseâ andâ Mdm2(C464A)-luciferaseâ plasmidsâ
wereâ clonedâ intoâ theâ pcDNA3.1â vectorâ (Invitrogen,â P/Nâ 35-0574)âusingâ theâ Nheâ 1â andâ Xhoâ 1â restrictionâ sites.â Luciferaseâ isâ onâ theâN-terminusâ ofâ Mdm2â withâ aâ linkerâ (Glyâ Serâ Glyâ Glyâ Glyâ Serâ GlyâGlyâ Glyâ Glyâ Serâ Glyâ Glyâ Glyâ Glyâ Serâ Glyâ Glyâ Thrâ Glyâ Ser)â clonedâbetweenâ theâ Mdm2â andâ theâ luciferaseâ DNAâ sequence.â Thisâ wasâconfirmedâbyâsequencing.âPrimerâsequencesâ forâcloningâareâ inâ theâSupplementaryâData.
Flag-Mdm2âinâpcDNA3â(53),âHA-UbiquitinâinâpcDNA3â(53),âandâp53â inâ pcDNA3â (54)â haveâ beenâ previouslyâ described.â PK-ubiquitinâ(55)âandâHis-Ubch5Câ(55)âinâpET-15a,âHis-p53âinâpRSETBâ(56),âGST-Mdm2â(400-491)â(55),âandâGST-MdmXâ(410-491)â(55)âinâpGEX-4T1âhaveâbeenâpreviouslyâdescribed.âForâconstructingâHA-MdmXâbaculo-virus,âPCRâwasâperformedâtoâinsertâanâHAâtagâupstreamâofâMdmX.âTheâ PCRâ productâ wasâ purifiedâ andâ digestedâ withâ RsrIIâ andâ KpnIâandâclonedâintoâtheâpFastBacâHTaâplasmidâ(Invitrogen).âFlag-Mdm2â
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Herman et al.rEsEArCh ArtICLE
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Allâ lysatesâwereâwashedâ3âtimesâwithâ lysesâbufferâbeforeâelutingâtheâproteinâwithâSDSâsampleâbuffer.
In Vitro Ubiquitination AssayFull-lengthâ Mdm2â in vitroâ ubiquitinationâ reactionsâ wereâ per-
formedâinâ15âÎŒlâreactionsâmixturesâcontainingâ50âmMâTrisHClâpHâ7.5,â5âmMâMgCl2,â2âmMâNaF,â2âmMâATP,â0.6âmMâDTT,â50ângâE1â(Bostonâ Biochem,â E-305),â 1â ÎŒgâ PK-ubiquitin,â 50â ngâ His-UbcH5C,âandâ 100â ngâ Flag-Mdm2.â 50â ngâ His-p53â wasâ usedâ forâ in vitroâ p53âubiquitinationâ assay.â 100â ngâ Flag-Mdm2â andâ 100â ngâ Ha-MdmXâwereâ usedâ forâ ubiquitinationâ reactionsâ withâ theâ Mdm2-MdmXâcomplex.â Afterâ incubationâ forâ 30â minutesâ atâ 37°C,â SDSâ sampleâbufferâwasâaddedâtoâtheâsamplesâandâtheyâwereâboiledâforâ5âminutes.âTheâproductsâwereâresolvedâbyâSDS-PAGEâandâanalyzedâbyâWesternâblotâ withâ anti-p53,â anti-Mdm2â SMP14,â 2A10,â 3G5â mix,â orâ anti-MdmXâantibodies.
Radiolabeledâ in vitroâ ubiquitinationâ assaysâ wereâ doneâ inâ 30âÎŒlâ reactionâ mixturesâ withâ 32P-labeledâ ubiquitin.â 500â ngâ GST-Mdm2(400-491)âandâGST-MdmX(410-490)âwereâusedâforâtheâRING-RINGâ reactions.â Theâ productsâ ofâ theâ reactionâ wereâ resolvedâ onâ aâSDS-PAGEâgelâandâvisualizedâbyâautoradiography.
Roc1-Cul1â proteinâ wasâ aâ kindâ giftâ fromâ Zhen-Qiangâ Panâ atâ theâMountâSinaiâSchoolâofâMedicine;â3âÎŒgâofâproteinâwasâusedâperâubiq-uitinationâreaction.
Purifiedâ His-BARD1/BR304â (31)â wasâ incubatedâ withâ 5â pmolâUbch5Câandâ0.2âpmolâE1âenzymeâ(BostonâBiochem)âalongâwithâ300âpmolâubiquitinâinâreactionâbufferâ(withoutâBSA)âforâ2âhoursâatâ37°C.âTheâproductsâwereâresolvedâbyâSDS-PAGEâandâanalyzedâbyâWesternâblottingâwithâanti-BRCA1.âBRCA1/BARD1âwasâaâkindâgiftâfromâDr.âRichardâBaer.
Additional Materials and MethodsAdditionalâ informationâonâmethodsâandâmaterialsâ isâavailableâ inâ
theâSupplementaryâData,âincludingâAlamarâBlueâviabilityâassay,âBlissâindependenceâmodel,âproteinâpurification,âRNAâextractionâandâqRT-PCRâ analysis,â glutathioneâ reactivityâ assay,â TUNELâ stain,â cellâ cycleâprofiling,âandâcaspaseâactivity.
Disclosure of Potential Conflicts of InterestB.R.âStockwellâhasâequityâinâandâservesâasâaâconsultantâtoâSolarisâ
Therapeutics,â aâ startupâ companyâ thatâ hasâ anâ optionâ toâ licenseâ theâMdm2â inhibitorsâ describedâ inâ thisâ manuscript.â Noâ potentialâ con-flictsâofâinterestâwereâdisclosedâbyâtheâotherâauthors.
Grant supportB.R.âStockwellâisâanâEarlyâCareerâScientistâofâtheâHowardâHughesâ
Medicalâ Institute.â Thisâ researchâ wasâ alsoâ supportedâ byâ aâ BeckmanâYoungâ Investigatorâ Awardâ fromâ theâ Arnoldâ andâ Mabelâ BeckmanâFoundationâ (toâ B.R.â Stockwell),â byâ theâ NIHâ 5R01CA097061,â5R01GM085081,â 1R03MH082369â (toâ B.R.â Stockwell),â andâ5R01CA058316-17â (toâ C.â Prives),â byâ NYSTARâ (toâ B.R.â Stockwell),âbyâ theâ Trainingâ Programâ inâ Molecularâ Biophysicsâ T32GM008281â(toâ A.G.â Herman),â andâ byâ theâ Trainingâ Programâ inâ Cancerâ BiologyâT32CA009503â(toâM.âHayano).
Receivedâ Mayâ 10,â 2011;â revisedâ Julyâ 27,â 2011;â acceptedâ Julyâ 27,â2011;âpublishedâOnlineFirstâJulyâ28,â2011.
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Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay rEsEArCh ArtICLE
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