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BioAssay: AID 2791

Late 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 peptide

Name: Late 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 peptide. ..more
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 Tested Compounds
 Tested Compounds
All(20)
 
 
Active(11)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(20)
 
 
Active(11)
 
 
Inactive(9)
 
 
AID: 2791
Data Source: The Scripps Research Institute Molecular Screening Center (MCL1BIM_INH_FP_1536_3XIC50 MDRUN SAR ROUND 0)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-04-28
Hold-until Date: 2011-04-23
Modify Date: 2011-04-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
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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 (MCL1, 1X%INH)
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: Summary (MCL1 inhibitors)
2129Primary biochemical high throughput screening assay to identify inhibitors of BCL2-related protein, long isoform (BCLXL).Screening depositor-specified cross reference: Primary screen (BCLXL, 3X%INH)
2166Counterscreen for MCL1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of BCL2-related protein, long isoform (BCLXL).Screening depositor-specified cross reference: Counterscreen (BCLXL, 3X%INH)
2168Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Screening depositor-specified cross reference: Confirmation screen (MCL1, 3X%INH)
2217Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide.Confirmatory depositor-specified cross reference: Dose response screen (MCL1, 3XIC50)
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: Dose response counterscreen (BCLXL, 3XIC50)
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
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
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 same project related to Summary assay
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 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: MCL1BIM_INH_FP_1536_3XIC50 MDRUN SAR ROUND 0

Name: Late 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 peptide.

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 well-characterized 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 α-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:

late stage, powders, purchased, SAR, myeloma cell leukemia sequence 1, MCL, MCL1, MCL-1, Mcl1, cancer, anti-apoptotic protein, chronic lymphocytic leukemia, multiple myeloma, lymphoma, inhibitor, inhibition, dose response, 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 assay is to determine dose response curves for powder samples of compounds identified as active in a set of previous experiments entitled, "Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide" (AID 2057), and that confirmed activity in a set of experiments entitled, "Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of myeloid cell leukemia sequence 1 (MCL1) interactions with BIM-BH3 peptide" (AID 2168). This biochemical assay monitors binding of the BH3 domain of the BCL-2 family member, Bim, to the binding pocket of human MCL1. In this fluorescence polarization (FP)-based assay, GST-MCL1 fusion protein is incubated with FITC-BH3-Bim peptides, in the presence of test compounds. Binding of peptide to MCL1 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 MCL1 will prevent binding of Bim peptide to MCL1, and increase the proportion of free to bound peptides, thereby reducing mP in the well. Compounds are tested in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 109 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 9.2 nM GST-MCL1 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 (0.45% 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.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 109 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 109 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar were considered active.

PubChem Activity Outcome and Score:

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-57, for inactive 36-0.

List of Reagents:

GST-MCL-1 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
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this assay, unlabeled Bim-BH3 peptide had an IC50 of 12 nanomolar. 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 108.8 uM (108.8μM**)Value of %inhibition at 108.8 micromolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 36.3 uM (36.3μM**)Value of %inhibition at 36.3 micromolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 12.1 uM (12.1μM**)Value of %inhibition at 12.1 micromolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 4.0 uM (4μM**)Value of %inhibition at 4.0 micromolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 1.3 uM (1.3μM**)Value of %inhibition at 1.3 micromolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 447.7 nM (0.4477μM**)Value of %inhibition at 447.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 149.2 nM (0.1492μM**)Value of %inhibition at 149.2 nanomolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 49.7 nM (0.0497μM**)Value of %inhibition at 49.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 16.6 nM (0.0166μM**)Value of %inhibition at 16.6 nanomolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 5.5 nM (0.0055μM**)Value of %inhibition at 5.5 nanomolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** 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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