Late 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 lines
Name: Late 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 lines. ..more
BioActive Compounds: 8
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: 2B4-MCL1-APOP_ACT_ABS_0096_3XEC50 Round 3
Name: Late 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 lines.
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.
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, 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, absorbance, MTS, formazan, dye, reduction, cytox, cytotoxicity, viability, cell growth, Bcl-2/MCL-1 primed, counterscreen, dose response, 96, assay provider, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether powder samples of compounds identified as possible MCL1 inhibitor probes are preferentially active in lymphoma-derived cell lines that are Mcl-1 dependent (2B4/Mcl-1) compared to Bcl-2 dependent (2B4/Bcl-2) as well as in cells that are deficient in Bax/Bak (SUDHL-10). This colorimetric cytotoxicity assay employs the MTS method, in which MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of the electron coupling agent phenazine methosulfate (PMS), is reduced by viable cells to produce a formazan dye that has an absorbance maximum at 490-500 nm in phosphate-buffered saline. As designed compounds thast reduce viable cell numbers will reduce the conversion of the MTS dye, leading to lower well absorbance.
Lymphoma-derived cell lines were grown in RPMI with 10% FBS. Treatments were performed at 37 C. Cells (2 x10^5 per sample) were grown in a 96 well plate and treated with compounds (concentration range 0.390625 uM to 25 uM) for 48 hr. Viability was measured by the MTS assay.
For each test compound, percent inhibition was plotted against compound concentration. 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. 25 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 25 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 100-1. There are no inactive compounds.
List of Reagents:
Reagent list not provided by assay provider.
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. 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 or compounds that modulate absorbance. 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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: confirmatory
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: meta target: molecular target: protein target: enzyme: oxidoreductase
BAO: meta target: biological process target: cell death
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: viability reporter: cell number
BAO: detection technology: spectrophotometry: absorbance
* Activity Concentration. ** Test Concentration.
Data Table (Concise)