Bookmark and Share
BioAssay: AID 2781

Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: NMR spectroscopy to verify direct binding of compounds to menin

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(63)
 
 
Active(13)
 
 
Inactive(50)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(13)
 
 
Inactive(50)
 
 
AID: 2781
Data Source: NCGC (MENN9470)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-04-27
Hold-until Date: 2011-04-26
Modify Date: 2011-04-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 13
Related Experiments
Show more
AIDNameTypeComment
1766qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Fluorescein Labeled MLL-derived PeptideConfirmatorydepositor-specified cross reference
1768qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Texas Red Labeled MLL-derived Mutant PeptideConfirmatorydepositor-specified cross reference
2076qHTS Assay for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: SummarySummarydepositor-specified cross reference
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 peptideConfirmatorysame project related to Summary assay
2782Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Texas Red Labeled MLL-derived Mutant PeptideConfirmatorysame project related to Summary assay
2783Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Fluorescein Labeled MLL-derived PeptideConfirmatorysame project related to Summary assay
504537Inhibitor SAR for Compounds Targeting the Menin-MLL Interaction in MLL Related Leukemias: Competition With Fluorescein Labeled MLL-derived PeptideConfirmatorysame project related to Summary assay
504585Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Binding Confirmation with NMR Saturation Transfer DifferenceOthersame project related to Summary assay
504613Confirmation of Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leukemias: Confirmation in an MLL leukemia cell growth assayConfirmatorysame project related to Summary assay
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.

The present assay was developed to confirm hits from this primary screen, examining saturation transfer differences in NMR spectra upon incubation of the menin protein with compound.
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 Buffer components for all assays were purchased from Sigma-Aldrich (St. Louis, MO) and Invitrogen (Carlsbad, CA).
Assay Reagents:
1. Menin protein
2. MLL w.t.
3. NMR buffer: 50mM phosphate, 50mM NaCl, 1mM DTT, pH = 7.5
NMR protocol:
1. Prepare 450 uL of menin (2.5 uM) in complex with 100uM compound in NMR buffer and run STD (Saturation Transfer Difference) on NMR Bruker 600 MHz
2. Prepare 450 uL of menin (2.5 uM) in complex with 100 uM compound and 25uM of MLL w.t. in NMR buffer and run STD (Saturation Transfer Difference) on NMR Bruker 600 MHz
Comment
Compound Ranking:
Compounds positive for NMR STD are given an activity score of 50. Inactive compounds negative for NMR STD are given an activity score of 0.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1NMR STD (100μM**)Whether saturation transfer difference was observed with 100uM compound - positive or negative.String

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: