Late 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)
Name: Late 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). ..more
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: BCLXL_INH_FP_0096_3XIC50 (Probe)
Name: Late 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).
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 alpha-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.
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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.
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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.
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The purpose of this assay is to determine dose response curves for powder samples of the MCL probe ML311. This assay serves as a counterscreen to determine probe ML311 activity against the anti-target BCL-xL. The probe should not be active in this assay. 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 by the assay provider in a dilution series starting at a maximum concentration of 40 uM (40 uM - 0.0390625 uM).
A nineteen amino acid peptide, corresponding to the BH3 domain of Bim, with the sequence FITC-GGGIAQELRRIGDEFNAY (SEQ ID NO: 1) was labeled with the fluorophore FITC according to the manufacturer's instructions (Molecular Probes, Eugene, OR). In addition, recombinant GST-Bcl-xL fusion proteins were generated in E.coli and purified using glutathione-sepharose beads. Binding of the recombinant proteins to the fluorescent Bim BH3 domain was confirmed by titration of increasing concentrations of the recombinant proteins against a constant amount of labeled Bim peptide (16.65 nM). Quantitation of binding was accomplished by FP assay with mP measurements made on the Analyst-GT reader (Molecular Devices, Sunnyvale, CA).
The Bim peptide (in solution at 4 nM) and GST-Bcl-xL (in solution at 12.5 nM) were first combined together in phosphate buffered saline buffer (1x PBS/0.001% Brij). The compound solution in DMSO was then added in concentrations ranging from 20 uM to 0.1 nM and incubated for 20 minutes at room temperature. Milli polarization (mp) units were then measured using a polarizer plate reader.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = ( Test_Compound_mP - median_Negative_Control_mP ) / ( median_ Positive_Control_mP - median_ Negative_Control_mP ) * 100
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. Contact the assay provider for details. 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. 40 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 40 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with an IC50 value greater than 10 uM was assigned an activity score of zero. Any compound with an IC50 value equal to or less than 10 uM 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 0-0. There are no active compounds.
List of Reagents:
GST-Bcl-xL enzyme in DMSO (supplied by Assay Provider)
FITC-BH3-Bim peptide in DMSO (supplied by Assay Provider)
Unlabeled BH3-Bim peptide (supplied by Assay Provider)
1x PBS/0.001% Brij
Brij 35 (Sigma-Aldrich, part B4184)
This assay was run by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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
* Activity Concentration.
Data Table (Concise)