Late 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)
Name: Late 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). ..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: APOPTOSIS_ACT_DAPI_FLUOR_3X%ACT MCSRUN (Probe)
Name: Late 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).
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 BXL-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.
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.
late stage, probe, ML311, Bcl2, BCL-xL, NCI-H929, DHL-10, DHL-6, cells, cell-based, powders, purchased, synthesized, chemistry, SAR, myeloma cell leukemia sequence 1, MCL, MCL1, MCL-1, Mcl1, cancer, anti-apoptotic protein, chronic lymphocytic leukemia, multiple myeloma, lymphoma, inhibitor, inhibition, fluorescence, DAPI, chromatin, condensation, morphology, mitochondria, membrane potential, cytochrome c, caspase, cleavage, pycnotic, staurosporine, 2B4, 2B4/MCL-1, 2B4/BCL-2, counterscreen, BH3, Noxa, assay provider, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
§ Panel component ID.
The purpose of this assay is to identify compounds that activate caspase 3 and 7 resulting in apoptotic nuclear morphology. As activators of apoptosis, the compounds should increase caspase activity due to loss of mitochondrial membrane potential and cytochrome c release. To visualize the percentage of cells in which there is caspase activity treated cells will be fixed with methanol and stained with the DNA binding DAPI (4',6-diamidino-2-phenylindole) and observed using a fluorescence microscope. The DNA bound dye emits at 456 nm when excited by 350 nm light. Caspase-mediated cleavage of nucleosomal proteins cause condensed, pycnotic nuclear morphology that is easily scored. As designed, compounds that induce apoptosis will cause an increase in the number of cells exhibiting high dye uptake and the abnormal pycnotic nuclear morphology. Compounds were tested in triplicate at 25 uM, or in a dose response series at concentrations of 5.0 uM, 2.5 uM , 1.25 uM, 625 nM, 313 nM, 156 nM, and 78 nM. This assay tested batches of the ML311 probe against the following cancer cell lines: MCL1/1780 (MCL1-dependent, murine), BCLxL/1863 (BCL2), NC1-H929 (human myeloma), DHL-6 (human B cell lymphoma), and DHL-10 (human B cell lymphoma) cells.
The MCL1 1780 and BCL2 1863 cell lines are B220+ murine lymphocytic leukemia cell. The NCI-H929 cell line was established from a malignant effusion in a patient with myeloma. The cells are positive for plasma cell antigen 1 (PCA-1), transferrin receptor, CD38 (T10), but are negative for HLA DR, CALLA and markers of early B cell development. They are negative for Epstein-Barr virus nuclear antigen (EBNA). The cells have a rearrangement of the c-myc proto oncogene and express c-myc RNA. There is also an activated ras allele. DHL-6 possesses a t(14;18)(q32;q21) translocation. DHL-10 cells were derived from a patient with B-cell non-Hodgkin lymphoma, and are deficient in the proapoptotic Bax/Bak proteins. The probe should be inactive in the DHL-10 cells, and active in all other cells.
The lymphoid-derived cell lines DHL-6 (Bax/Bak functional, on-target indicator) and DHL-10 (Bax/Bak deficient, anti-target indicator) were obtained from Anthony Letai of the Dana Farber Cancer Research Institute, Boston, MA20. The myeloid derived cell line NCI-H929 (Bax/Bak functional, on-target indicator) was obtained from the NIH/NCI cell repository. Cells were grown in RPMI 1640 medium (GIBCO-BRL) with 2 mM L-glutamine, 4.5 g/L glucose, 1.0 mM sodium pyruvate and 5% fetal bovine serum. Cells were expanded in tissue culture in appropriate media and then sub-cultured into 96-well plates at a seeding density of 20,000 cells per well. After incubation for 24 hours, cells were treated with compounds that are titrated into appropriate medium with FCS.
Prior to the start of the assay, cell lines tested were plated at 2E5 cells/mL in 24-well plates in 1 mL of growth media per well, in an atmosphere of 5% CO2 and 95% relative humidity, with or without compound in DMSO, or DMSO alone. Cells treated with 10 uM N-benzoyl staurosporine were also analyzed as a positive control. Cells were treated for 48 hours with 25 uM compound diluted directly into the media from a 10 mM stock solution (2.5 uL added). Following treatment, cells were washed and fixed with ethanol and stained with DAPI following standard protocol. DAPI stock was prepared at 3 uM in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40). The cell suspension was collected from each well and placed in a 1.5 mL Eppendorf tube. The cells were pelleted by centrifugation (700xg, 5 min). The supernatant was discarded and 1 mL of cold ethanol (-20 C) was added to the cell pellets and incubated on ice for 10 min. The cells were pelleted by centrifugation (700 g, 5 min) and the ethanol was discarded. PBS buffer (10 mM Sodium phosphate, pH 7.4, 150 mM NaCl) was added to the fixed cell pellet to rehydrate the cells for 15 min. The cells were pelleted again by centrifugation, and the supernatant discarded. The cells were resuspended in 0.5 mL of 3 uM DAPI solution, and incubated at room temperature for 15 min. The samples were stored at 4 C until analyzed. Stained cells were mounted and visualized using a florescence microscope (excitation 350 nm, emission 470 nm), and scored (minimum of 100 cells examined) for condensed/pycnotic nuclei. The percent apoptosis was expressed relative to cells treated with DMSO only (0%).
ML311 has robust on-target activity in biochemical assays. The primary criteria of the probe report have been achieved: ML311 induces cell death in a highly active Mcl-1 cell line (Mcl-1/1780, EC50 = 0.3 uM). The compound does not induce appreciable cell death in the anti-target cell based assay (DHL-10) even at the highest dose tested (5 uM), and in other runs for a previous batch (SID 131465452) this lack of anti-target effect was verified at higher concentrations (to 25 uM). There is thus at least 8-fold selectivity for the DHL-6 cell line versus the DHL-10 cell line, meeting the criteria established for probe in the CPDP of greater than 3-fold selectivity. ML311 is also approximately 4-fold more active in Mcl-1-1780 cells than vs. the highly Bcl-2 active cell line Bcl-2-1863 (EC50 = 1.1 uM), meeting another probe development objective. Batch-to-batch consistency in activity is seen in all assay formats.
For SID 135383441, the percent apoptosis (%_Apoptosis) was calculated for each cell line (MCL1 target and BclxL anti-target), determined as the fraction of apoptotic cells observed at 25 uM.
PubChem Activity Outcome and Score:
For each apoptosis panel assay:
An active compound induces > 15% apoptosis of cells.
For each dose response panel assay:
Dose response values were determined using GraphPad Prism software. Any Compounds with an IC50 value equal to or less than 10 uM were considered active. Compounds with an IC50 value greater than 10 uM were considered inactive.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
1780 MCl1: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
1863 BCL-XL: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
1780 MCl1: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
NCI-H929: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
DHL-6: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
DHL-10: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
MCL1 1780 Cells (Dr Tony Letai, Dana Farber Cancer Research Center)
BCL2 1863 Cells (Dr Tony Letai, Dana Farber Cancer Research Center)
Control: N-Benzoyl Staurosporine (Calbiochem 539648)
RPMI 1640 (GIBCO 11875 or 11835)+ 1% Pen-Streptomycin-Glutamine (GIBCO 10378) +5% FBS (Hyclone SH30396.02, Thermo Scientific)
4', 6 diamidino-2-phenylindole dihydrochloride (DAPI) dye (Invitrogen, D1306)
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. ** Test Concentration. § Panel component ID.