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)
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). ..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: BCLXLBIM_INH_FP_1536_3XIC50 MDCSRUN SAR ROUND 0
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).
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.
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, BCL2-related protein long isoform, BCLXL, BCL-XL, BCL2L, BCL2L1, BCL2-like 1, BCLX, myeloma cell leukemia sequence 1, MCL1, MCL-1, Mcl1, cancer, anti-apoptotic protein, chronic lymphocytic leukemia, multiple myeloma, lymphoma, inhibitor, inhibition, counterscreen, dose response, uHTS, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to determine dose response curves for powder samples of compounds identified as active in a set of previous experiments entitled, "Primary biochemical high throughput screening assay to identify inhibitors of BCL2-related protein, long isoform (BCLXL)" (AID 2129), and that confirmed activity in a set of experiments entitled, "Counterscreen for MCL1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of BCL2-related protein, long isoform (BCLXL)" (AID 2166). This assay also serves as a counterscreen for a set of previous experiments entitled, "Fluorescence polarization primary biochemical high throughput screening assay to identify inhibitors of Myeloid cell leukemia-1 (Mcl-1)" (AID 2057) and confirms BH3 mimetic activity of compounds of interest. 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 in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 109 micromolar.
Prior to the start of the assay, 2.5 microliters of Assay Buffer (Dulbecco's PBS pH 7.2, Brij 35 0.001%) containing 12.5 nM BCL-XL 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 (0.9% 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
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 of inactive compounds is 0-0, there are no actives.
List of Reagents:
GST-BCL-XL 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)
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)