Bookmark and Share
BioAssay: AID 1768

qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Texas Red Labeled MLL-derived Mutant Peptide

Translocations of the MLL (Mixed Lineage Leukemia) gene are frequently found in human leukemias affecting both children and adults. Fusion of MLL to one of more than 60 genes results in generation of oncogenic proteins upregulating Hox genes, which are vital to blood cell development. Patients harboring fusion of the MLL gene suffer from aggressive leukemias and respond poorly to available more ..
_
   
 Tested Compounds
 Tested Compounds
All(263428)
 
 
Active(615)
 
 
Inactive(231462)
 
 
Inconclusive(31694)
 
 
 Tested Substances
 Tested Substances
All(266676)
 
 
Active(615)
 
 
Inactive(234159)
 
 
Inconclusive(31902)
 
 
AID: 1768
Data Source: NCGC (MENN9466)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-05-15

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 615
Depositor Specified Assays
Show more
AIDNameTypeComment
2076qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Summarysummary
504537Inhibitor SAR for Compounds Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Fluorescein Labeled MLL-derived Peptideconfirmatory
504585Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Binding Confirmation with NMR Saturation Transfer Differenceother
2778Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Confirmation in an HTRF (commercial TR-FRET) Assay using His-tagged menin and biotin-labeled MLL peptideconfirmatory
2782Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Texas Red Labeled MLL-derived Mutant Peptideconfirmatory
2781Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: NMR spectroscopy to verify direct binding of compounds to meninscreening
2783Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Fluorescein Labeled MLL-derived Peptideconfirmatory
504613Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Confirmation in an MLL leukemia cell growth assayconfirmatory
743469On Hold
Description:
qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH084875-01A1
Assay Submitter (PI): Jolanta Grembecka, Tomasz Cierpicki, University of Virginia

Translocations of the MLL (Mixed Lineage Leukemia) gene are frequently found in human leukemias affecting both children and adults. Fusion of MLL to one of more than 60 genes results in generation of oncogenic proteins upregulating Hox genes, which are vital to blood cell development. Patients harboring fusion of the MLL gene suffer from aggressive leukemias and respond poorly to available therapies. All of the oncogenic fusion proteins have a preserved N-terminal fragment of MLL that has been identified to interact with menin. It has been recently discovered that association with menin is critical to the leukemogenic activity of the MLL fusion oncoproteins. Selective targeting of the menin-MLL interaction could provide an attractive therapeutic approach to develop novel drugs for MLL-related leukemias. Small molecules blocking the menin-MLL interaction should reverse the oncogenic potential of MLL fusion proteins.

We have developed and optimized a fluorescence polarization-based assay for the identification of Menin-MLL inhibitors. The assay was run employing two different MLL-derived peptides. The present assay uses a 12 amino acid peptide labeled with fluorescein at its N-terminus. This peptide consists of the menin high affinity binding motif from MLL and is potently bound by menin. Another peptide with a mutated sequence, labeled with Texas Red, was used in parallel as a less stringent screen for inhibitors.
Protocol
The Menin protein, the MLL-wild type (w.t.) derived peptide (N-terminal 12 amino acids), the MLL-derived peptide labeled with Texas Red and the MLL-w.t. peptide labeled with Fluorescein were kindly supplied by laboratory of Dr. Jolanta Grembecka, University of Virginia. All protein and peptide solutions were stored in single use aliquots at -80C. The Eu3+ Cyrptate-conjugated mouse monoclonal antibody anti-6 Histidine (Eu3+-AB) and the Streptavidin-XL665 conjugate (XL665) were purchased from Cisbio International (Bedford, MA). All antibody and XL665 conjugate solution stocks were stored in single use aliquots at -20C. The Buffer components for all assays were purchased from Sigma-Aldrich (St. Louis, MO) and Invitrogen (Carlsbad, CA).

FP Assay Reagents:

1. FP reaction buffer: 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1mM DTT.
Before use, 0.05% BSA is added.
2. Menin protein, MLL-w.t. labeled with fluorescein, MLL-mutant peptide labeled with texas red.
Stored in singe use aliquots at -80C.
3. MLL-w.t. unlabeled peptide as positive control: 10 uM final in FP buffer.

FP Assay Procedure:
1. Prepare Menin at 200 nM, MLL-w.t.-fluorescein at 25 nM, and MLL-mutant-texas red at 50 nM mixture in FP buffer.
2. Prepare Menin at 200 nM, MLL-w.t.-fluorescein at 25 nM, MLL-mutant-texas red at 50 nM, and 10 uM MLL-w.t. unlabeled mixture in FP buffer.
3. Prepare MLL-w.t.-fluorescein at 25 nM and MLL-mutant-texas red at 50 nM mixture in FP buffer.
3. Dispense 4 ul per well of reagent in step 1 in columns 2, 4 - 48 in a black 1536 well plate, dispense 4 ul per well of reagents from steps 2 and 3 in columns 1 and 3 respectively. Incubate plate at ambient for 15 minutes.
4. Transfer 23 nl per well of compound to plate and incubate at ambient for 1 hr.
5. Read fluorescence polarization for each label using the PerkinElmer Envision.

Envision protocol for fluorescein: FITC FP Dual Enh Mirror, Excitation filter 480, Emission filter (1) P-pol 535, Emission filter (2) S-pol 535
Envision protocol for texas red: FP Dual Enh Mirror, Excitation filter 555, Emission filter (1) P-pol 632, Emission filter (2) S-pol 632
Comment
Compound Ranking:

Two readouts are measured in this assay, fluorescence polarization (FP) and total fluorescence. FP gives a measure of inhibition of the protein-peptide interaction. Total fluorescence is used to flag fluorescent molecules that might otherwise interfere with an accurate determination of FP.

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive for both FP and total fluorescence readouts. See data field "FP Curve Description" and "Total Fluorescence Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Total_fluorescence-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Total_fluorescence-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Total_fluorescence-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Total_fluorescence-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Total_fluorescence-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Total_fluorescence-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Total_fluorescence-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Total_fluorescence-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Total_fluorescence-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Total_fluorescence-Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
15Total_fluorescence-Activity at 0.00823 uM (0.00823μM**)% Activity at given concentration.Float%
16Total_fluorescence-Activity at 0.00920 uM (0.0092μM**)% Activity at given concentration.Float%
17Total_fluorescence-Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
18Total_fluorescence-Activity at 0.042 uM (0.0422738μM**)% Activity at given concentration.Float%
19Total_fluorescence-Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
20Total_fluorescence-Activity at 0.212 uM (0.211754μM**)% Activity at given concentration.Float%
21Total_fluorescence-Activity at 0.417 uM (0.417345μM**)% Activity at given concentration.Float%
22Total_fluorescence-Activity at 0.503 uM (0.503478μM**)% Activity at given concentration.Float%
23Total_fluorescence-Activity at 0.802 uM (0.801988μM**)% Activity at given concentration.Float%
24Total_fluorescence-Activity at 1.158 uM (1.15786μM**)% Activity at given concentration.Float%
25Total_fluorescence-Activity at 2.284 uM (2.28425μM**)% Activity at given concentration.Float%
26Total_fluorescence-Activity at 3.000 uM (2.99983μM**)% Activity at given concentration.Float%
27Total_fluorescence-Activity at 4.424 uM (4.42428μM**)% Activity at given concentration.Float%
28Total_fluorescence-Activity at 6.430 uM (6.42983μM**)% Activity at given concentration.Float%
29Total_fluorescence-Activity at 11.71 uM (11.7091μM**)% Activity at given concentration.Float%
30Total_fluorescence-Activity at 16.74 uM (16.7415μM**)% Activity at given concentration.Float%
31Total_fluorescence-Activity at 24.77 uM (24.7684μM**)% Activity at given concentration.Float%
32Total_fluorescence-Activity at 44.53 uM (44.5264μM**)% Activity at given concentration.Float%
33Total_fluorescence-Activity at 61.65 uM (61.6486μM**)% Activity at given concentration.Float%
34Total_fluorescence-Activity at 94.63 uM (94.6342μM**)% Activity at given concentration.Float%
35Total_fluorescence-Activity at 137.7 uM (137.685μM**)% Activity at given concentration.Float%
36Total_fluorescence-Activity at 188.0 uM (188μM**)% Activity at given concentration.Float%
37FP-Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
38FP-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
39FP-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
40FP-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
41FP-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
42FP-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
43FP-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
44FP-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
45FP-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
46FP-Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
47FP-Activity at 0.00823 uM (0.00823μM**)% Activity at given concentration.Float%
48FP-Activity at 0.00920 uM (0.0092μM**)% Activity at given concentration.Float%
49FP-Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
50FP-Activity at 0.042 uM (0.0422738μM**)% Activity at given concentration.Float%
51FP-Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
52FP-Activity at 0.212 uM (0.211754μM**)% Activity at given concentration.Float%
53FP-Activity at 0.417 uM (0.417345μM**)% Activity at given concentration.Float%
54FP-Activity at 0.503 uM (0.503478μM**)% Activity at given concentration.Float%
55FP-Activity at 0.802 uM (0.801988μM**)% Activity at given concentration.Float%
56FP-Activity at 1.158 uM (1.15786μM**)% Activity at given concentration.Float%
57FP-Activity at 2.284 uM (2.28425μM**)% Activity at given concentration.Float%
58FP-Activity at 3.000 uM (2.99983μM**)% Activity at given concentration.Float%
59FP-Activity at 4.424 uM (4.42428μM**)% Activity at given concentration.Float%
60FP-Activity at 6.430 uM (6.42983μM**)% Activity at given concentration.Float%
61FP-Activity at 11.71 uM (11.7091μM**)% Activity at given concentration.Float%
62FP-Activity at 16.74 uM (16.7415μM**)% Activity at given concentration.Float%
63FP-Activity at 24.77 uM (24.7684μM**)% Activity at given concentration.Float%
64FP-Activity at 44.53 uM (44.5264μM**)% Activity at given concentration.Float%
65FP-Activity at 61.65 uM (61.6486μM**)% Activity at given concentration.Float%
66FP-Activity at 94.63 uM (94.6342μM**)% Activity at given concentration.Float%
67FP-Activity at 137.7 uM (137.685μM**)% Activity at given concentration.Float%
68FP-Activity at 188.0 uM (188μM**)% Activity at given concentration.Float%
69Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084875-01A1

Data Table (Concise)
Classification
PageFrom: