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BioAssay: AID 2166

Counterscreen for MCL1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of BCL2-related protein, long isoform (BCLXL).

Name: Counterscreen for MCL1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of BCL2-related protein, long isoform (BCLXL). ..more
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 Tested Compounds
 Tested Compounds
All(1720)
 
 
Active(922)
 
 
Inactive(798)
 
 
 Tested Substances
 Tested Substances
All(1720)
 
 
Active(922)
 
 
Inactive(798)
 
 
AID: 2166
Data Source: The Scripps Research Institute Molecular Screening Center (BCLXLBIM_INH_FP_1536_3X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-01
Modify Date: 2010-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 922
Related Experiments
Show more
AIDNameTypeProbeComment
2057Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Screening depositor-specified cross reference: Primary screen (Mcl-1 inhibitors).
2090Summary of probe development efforts to identify inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Summary depositor-specified cross reference
2129Primary biochemical high throughput screening assay to identify inhibitors of BCL2-related protein, long isoform (BCLXL).Screening depositor-specified cross reference: Primary screen (BCL2 inhibitors).
2144Summary of probe development efforts to identify inhibitors of BCL2-related protein, long isoform (BCLXL).Summary depositor-specified cross reference: Summary AID.
2218Counterscreen for inhibitors of MCL1: fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of BCL2-related protein, long isoform (BCLXL).Confirmatory depositor-specified cross reference
2790Late stage counterscreen results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of BCL2-related protein, long isoform (BCLXL)Confirmatory depositor-specified cross reference
2791Late stage results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptideConfirmatory depositor-specified cross reference
493058Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: absorbance-based cell-based assay to identify compounds that are preferentially active in causing apoptosis in MCL-1 primed vs. Bax/Bak deficient DHL10 cellsConfirmatory depositor-specified cross reference
493059Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: fluorescence-based cell-based cytotoxicity assay using 4',6-diamidino-2-phenylindole (DAPI) to identify compounds that are pro-apoptotic in cancer cells grown in cultureOther depositor-specified cross reference
493062Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: fluorescence-based cell-based assay to identify compounds that preferentially activate caspase in 2B4/MCL-1 vs. 2B4/BCL-2 cellsOther depositor-specified cross reference
493086Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptideConfirmatory depositor-specified cross reference
602164Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: absorbance-based cell-based assay to identify compounds that are preferentially active in causing apoptosis in Mcl-1 primed (2B4/Mcl1) vs. Bcl-2 (2B4/Bcl-2) cell linesConfirmatory depositor-specified cross reference
602165Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide, Round 3Confirmatory depositor-specified cross reference
624393Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide (Probe)Confirmatory1 depositor-specified cross reference
624396Late stage assay provider results from the probe development effort to identify MCL1-BIM inhibitors: fluorescence-based cell-based cytotoxicity assay using 4',6-diamidino-2-phenylindole (DAPI) to identify compounds that are pro-apoptotic in cancer cells grown in culture (Probe)Other1 depositor-specified cross reference
624411Late stage counterscreen results from the probe development effort to identify MCL1-BIM inhibitors: Fluorescence polarization-based biochemical dose response assay for inhibitors of BCL2-related protein, long isoform (BCLXL) (Probe)Confirmatory depositor-specified cross reference
2168Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Screening same project related to Summary assay
2217Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Confirmatory same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Michael Cardone, Eutropics
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R43 CA135915-01 Fast Track
Grant Proposal PI: Michael Cardone, Eutropics
External Assay ID: BCLXLBIM_INH_FP_1536_3X%INH

Name: Counterscreen for MCL1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of BCL2-related protein, long isoform (BCLXL).

Description:

Cancer initialization and survival depends upon evasion of the programmed cell death (apoptosis) machinery that normally kills an unneeded or rogue cell (1). Although an effective mechanism for anti-cancer chemotherapeutics is apoptosis induction, cancer cells develop resistance to the pro-apoptotic proteins activated by these drugs (2). Multiple myeloma (MM) and chronic lymphoblastic leukemia (CLL) are two wellcharacterized lymphoid cancers (3). Bcl-2 is an oncoprotein activated in these lymphomas, and serves to inhibit apoptosis induced by many cytotoxic compounds. Members of the Bcl-2 protein family are regulated by protein-protein interactions, forming homo- and heterodimers (4, 5). One of these proteins, MCL1, is essential for survival of human MM cells (6). MCL1 and other Bcl-2 proteins such as Bcl-xl share Bcl-2's ability to oppose apoptosis, as well as sequence homology in 4 a-helical Bcl-2 homology (BH) regions, BH1-BH4 (3). As a result, these proteins are promising targets for studies on tumor initiation, progression and apoptosis resistance. Research showing that MCL1 opposes cell death (7), is highly expressed in hematopoetic stem cells and is regulated by growth factors (8), and that inhibiting Bcl-2 protein-protein interactions via the crucial BH3 domain is a valid approach to cancer drug development (2, 9, 10), suggest that targeted therapies for MCL1 are needed. The identification of selective inhibitors of MCL1 will provide useful tools for the study of lymphoid tumorigenesis, and elucidate mechanisms for apoptosis induction in resistant cancers.

References:
1. McConkey, DJ and Zhu, K, Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist Updat, 2008. 11(4-5): p. 164-79.
2. Reed, JC, Drug insight: cancer therapy strategies based on restoration of endogenous cell death mechanisms. Nat Clin Pract Oncol, 2006. 3(7): p. 388-98.
3. Cory, S and Adams, JM, Killing cancer cells by flipping the Bcl-2/Bax switch. Cancer Cell, 2005. 8(1): p. 5-6.
4. Petros, AM, Olejniczak, ET and Fesik, SW, Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta, 2004. 1644(2-3): p. 83-94.
5. Redzepovic, J, Weinmann, G, Ott, I and Gust, R, Current trends in multiple myeloma management. J Int Med Res, 2008. 36(3): p. 371-86.
6. Derenne, S, Monia, B, Dean, NM, Taylor, JK, Rapp, MJ, Harousseau, JL, Bataille, R and Amiot, M, Antisense strategy shows that MCL1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. Blood, 2002. 100(1): p. 194-9.
7. Kozopas, KM, Yang, T, Buchan, HL, Zhou, P and Craig, RW, MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3516-20.
8. Opferman, JT, Iwasaki, H, Ong, CC, Suh, H, Mizuno, S, Akashi, K and Korsmeyer, SJ, Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science, 2005. 307(5712): p. 1101-4.
9. Letai, A, Pharmacological manipulation of Bcl-2 family members to control cell death. J Clin Invest, 2005. 115(10): p. 2648-55.
10. Oltersdorf, T, Elmore, SW, Shoemaker, AR, Armstrong, RC, Augeri, DJ, Belli, BA, Bruncko, M, Deckwerth, TL, Dinges, J, Hajduk, PJ, Joseph, MK, Kitada, S, Korsmeyer, SJ, Kunzer, AR, Letai, A, Li, C, Mitten, MJ, Nettesheim, DG, Ng, S, Nimmer, PM, O'Connor, JM, Oleksijew, A, Petros, AM, Reed, JC, Shen, W, Tahir, SK, Thompson, CB, Tomaselli, KJ, Wang, B, Wendt, MD, Zhang, H, Fesik, SW and Rosenberg, SH, An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature, 2005. 435(7042): p. 677-81.

Keywords:

BCL2-related protein long isoform, BCLXL, BCL-XL, BCL2L, BCL2L1, BCL2-like 1, BCLX, myeloma cell leukemia sequence 1, MCL1, MCL-1, Mcl1, cancer, anti-apoptotic protein, chronic lymphocytic leukemia, multiple myeloma, lymphoma, inhibitor, inhibition, counterscreen, confirmation, uHTS, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this biochemical assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, "Primary biochemical high throughput screening assay to identify inhibitors of BCL2-related protein, long isoform (BCLXL)" (AID 2129). This assay also serves as a counterscreen for a set of previous experiments entitled, "Fluorescence polarization primary biochemical high throughput screening assay to identify inhibitors of Myeloid cell leukemia-1 (Mcl-1)" (AID 2057) and confirms BH3 mimetic activity of compounds of interest. This fluorescence polarization (FP)-based assay monitors binding of the BH3 domain of the Bcl-2 family member, Bim, to the binding pocket of Bcl-xL. GST-Bcl-xL fusion protein is incubated with FITC-BH3-Bim peptides, in the presence of test compounds. Binding of peptide to Bcl-xL target protein increases the effective molecular mass of the peptide, slowing its rotation and increasing millipolarization (mP) units in the well. As designed, compounds that inhibit Bcl-xL will prevent binding of peptide to Bcl-xL protein, and increase the ratio of free to bound peptides, thereby reducing mP in the well. Compounds are tested in triplicate at a final nominal concentration of 10.9 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters of Assay Buffer (Dulbecco's PBS pH 7.2, Brij 35 0.001%) containing 12.5 nM Bcl-XL were dispensed into a 1536 microtiter plate. Next, 55 nL of test compound in DMSO, unlabeled Bim-BH3 control peptide (0.770 micromolar final concentration), or DMSO alone (0.9% final concentration) were added to the appropriate wells.

The assay was started by dispensing 2.5 microliters of 8.0 nanomolar FITC-BH3-Bim peptide in assay buffer (Dulbecco's PBS pH 7.2, Brij 35 0.001%) into all wells. Plates were centrifuged and after 20 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a FITC FP filter set and a FITC dichroic mirror (excitation = 525nm, emission = 595nm). Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.

The percent inhibition for each compound was calculated as follows:

Percent inhibition = ( Test_Compound_mP - median_Negative_Control_mP ) / ( median_Positive_Control_mP - median_ Negative_Control_mP ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Negative_Control is defined as wells containing DMSO.
Positive_Control is defined as wells containing unlabeled Bim-BH3 peptide.

A mathematical algorithm was used to determine nominally inhibiting compounds in the screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter for the Primary screen was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-4, for inactive 4-0.

List of Reagents:

GST-Bcl-xL enzyme (supplied by Assay Provider)
FITC-BH3-Bim peptide (supplied by Assay Provider)
Unlabeled BH3-Bim peptide (supplied by Assay Provider)
1536-well plates (Corning, part 3864)
Dulbecco's PBS (Sigma-Aldrich, part D8537)
Brij 35 (Sigma-Aldrich, part B4184)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (10.9μM**)Normalized percent inhibition of the counterscreen at a compound concentration of 10.9 micromolar.Float%
2Standard DeviationStandard deviation of the counterscreen derived from the normalized percent inhibition of the triplicate data for each compound.Float

** Test Concentration.
Additional Information
Grant Number: 1 R43 CA135915-01

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