Late 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 cells
Name: Late 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 cells. ..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: CASPASE-2B4_ACT_FLUOR_96_3XEC50 MDCSRUN Round 0
Name: Late 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 cells.
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, MCL-1, 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 MCL-1 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.
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.
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The purpose of this assay is to identify compounds that induce caspase activation in a MCL-1 dependent fashion and that exhibit selectivity for MCL-1 over BCL-2 as reflected by enhanced potency in MCL-1 dependent cell line compared to a BCL-2 dependent cell line. The BCL-2 specific compound ABT-737 (this compound does not bind to MCL-1) will be used as a control to measure BCL-2 mediated caspase activation.
Cells are treated with compound in microtiter plate wells. Compounds are tested in triplicate in a 8-point, 1:3 dilution series starting at a nominal test concentration of 100 uM. The Apo-ONE(R) Homogeneous Caspase-3/7 Assay is used to detect caspase-3/7 activity based on the cleavage of a profluorescent DEVD peptide-rhodamine 110 substrate [(Z-DEVD)2-R110]. The Apo-ONE(R) Reagent (Promega) is added directly to culture wells using a 1:1 ratio of reagent to culture medium. The contents are mixed and incubated for 1-2 or more hours, and the fluorescent signal is measured. For a detailed protocol and background information about this system, please see Promega Technical Bulletin #TB295 (www.promega.com/tbs/tb295/tb295.html).
All cells (2B4/MCL-1, 2B4/BCL-2, and 2B4/Bax/Bak(-/-) are cultured in flasks in growth media consisting of RPMI 1640 supplemented with 5% FBS and 1% pen-strep-glutamine antibiotic mix.
Cells are treated for 48 h with compound added to the media at 25 uM concentration. Cells are transferred to a black 384-well microtiter plate and the Apo-ONE(R) Caspase-3/7 Reagent is added 1:1 (v/v) to the cells according to the manufacturer instructions.
For each test compound, caspase activity is plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve is then fit to the data (Prism Software) to define an EC50. EC50 values are generated from fitted curves by solving for the X-intercept value at the 50% activation level of the maximum activation value.
Compounds that activate caspases in the 2B4/Bax/Bak(-/-) cell line are considered to act via a non-MCL-1 directed mechanism. Compounds exhibiting a lower EC50 in the 2B4/MCL-1 cell lines as compared to the 2B4/Bcl-2 cell line are considered to be selective MCL-1 inhibitors.
PubChem Activity Outcome and Score:
Overall Activity Outcome and Score:
Compounds with an EC50 greater than 25 uM in 2B4/MCL-1 cell type are considered inactive. Compounds with an EC50 equal to or less than 25 uM in 2B4/MCL-1 cell type were considered active. Compounds exhibiting caspase activation at a level less than or equal to 2-fold of the vehicle control are considered inactive. Compounds exhibiting greater than 2-fold caspase activation compared to vehicle are considered active.
Activity score was then ranked by the potency of EC50 in 2B4/MCL1-1 cell type, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-38, and for inactive compounds 1-0.
List of Reagents:
2B4/MCL-1 cells (Dr Tony Letai, Dana Farber Cancer Research Center)
2B4/BCL-2 cells (Dr Tony Letai, Dana Farber Cancer Research Center)
2B4/Bax/Bak(-/-)(Dr Tony Letai, Dana Farber Cancer Research Center)
96-well black flat bottom plate (Corning 3915)
Control: Bim BH3 peptide (Tufts New England Medical Center Peptide Facility) sequence: Ac-Met-Arg-Pro-Glu-Ile-Trp-Ile-Ale-Gln-Glu-Leu-Arg-Arg-Ile-Gly-Asp-Glu-Phe-Asn-Ala-NH2
RPMI 1640 (GIBCO 11875 or 11835)+ 1% Pen-Streptomycin-Glutamine (GIBCO 10378) +5% FBS (Hyclone SH30396.02, Thermo Scientific)
Control compound ABT-737 (Selleck Chemicals, S1002)
Apo-ONE(R) Homogeneous Caspase-3/7 Assay and protocol (Promega Cat. G7790, G7791, G7792)
This assay was performed by the assay provider. Replicate values and raw data were not provided. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to: dust or lint and compounds that modulate 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. § Panel component ID.