Counterscreen for inhibitors of Janus kinase 2 mutant JAK2V617F: Cell-based high throughput assay to identify inhibitors of parental Ba/F3 cell viability.
Name: Counterscreen for inhibitors of Janus kinase 2 mutant JAK2V617F: Cell-based high throughput assay to identify inhibitors of parental Ba/F3 cell viability. ..more
BioActive Compounds: 2410
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Ross Levine, Memorial Sloan Kettering Cancer Center (MSKCC)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH084174-01
Grant Proposal PI: Ross Levine, MSKCC
External Assay ID: BAF3CYTOX_INH_LUMI_1536_%INH
Name: Counterscreen for inhibitors of Janus kinase 2 mutant JAK2V617F: Cell-based high throughput assay to identify inhibitors of parental Ba/F3 cell viability.
Regulation of the differentiation and activity of inflammatory cells through cytokine signaling is essential for proper immune function and to prevent auto-immune disease and leukemias (1, 2). The protein tyrosine kinase Janus kinase 2 (JAK2) is critical for this process (3-6). Following activation of cytokine receptors, particularly erythropoietin and interferon receptors, JAK2 phosphorylates transcription factors of the signal transducers and activators of transcription (STAT) family, leading to expression of target genes involved in immune cell homeostasis (2, 3). The discovery of acquired genetic mutations in the JAK2 gene in hematopoetic cells from patients with Myeloid Proliferative Disorders (MPDs) characterized by excess proliferation of differentiated bone marrow myeloid cells (7-10), provided insights into the role of JAK2 in hematopoiesis. The identified JAK2 mutation (JAK2V617F) replaces guanine with thymine, resulting in a valine to phenylalanine amino acid substitution at codon 617 within the pseudokinase JH2 autoinhibitory domain (7-10). The resulting JAK2V617F variant is constitutively active, mediating JAK-STAT signaling efficiently even in the absence of ligand. Thus, the discovery of small molecules that can differentially inhibit JAK2V617F and not wildtype JAK2 may provide insights into JAK2 signaling in hematopoiesis and MPD pathogenesis (11, 12).
1. O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Immunity. 2008 Apr;28(4):477-87.
2. Ihle, JN. Cytokine receptor signalling. Nature. 1995 Oct 19;377(6550):591-4.
3. Yamaoka K, Saharinen P, Pesu M, Holt VE 3rd, Silvennoinen O, O'Shea JJ. The Janus kinases (Jaks). Genome Biol. 2004;5(12):253.
4. Stepkowski SM, Chen W, Ross JA, Nagy ZS, Kirken RA. STAT3: an important regulator of multiple cytokine functions. Transplantation. 2008 May 27;85(10):1372-7.
5. O'Sullivan LA, Liongue C, Lewis RS, Stephenson SE, Ward AC. Cytokine receptor signaling through the Jak-Stat-Socs pathway in disease. Mol Immunol. 2007 Apr;44(10):2497-506.
6. Parganas, E., Wang, D., Stravopodis, D., Topham, D.J., Marine, J., Teglund, S., Vanin, E.F., Bodner, S., Colamonici, O.R., van Deursen, J.M., Grosveld, G., and Ihle, J.N., Jak2 is essential for signaling through a variety of cytokine receptors. Cell, 1998. 93: p. 385-395.
7. Levine, R.L., Wadleigh, M., Cools, J., Ebert, B.L., Wernig, G., Huntly, B.J., Boggon, T.J., Wlodarska, I., Clark, J.J., Moore, S., Adelsperger, J., Koo, S., Lee, J.C., Gabriel, S., Mercher, T., D'Andrea, A., Frohling, S., Dohner, K., Marynen, P., Vandenberghe, P., Mesa, R.A., Tefferi, A., Griffin, J.D., Eck, M.J., Sellers, W.R., Meyerson, M., Golub, T.R., Lee, S.J., and Gilliland, D.G., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 2005. 7(4): p. 387-397.
8. Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., Scott, M.A., Erber, W.N., and Green, A.R., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 2005. 365(9464): p. 1054-61.
9. Kralovics, R., Passamonti, F., Buser, A.S., Teo, S., Tiedt, R., Passweg, J.R., Tichelli, A., Cazzola, M., and Skoda, R., A gain-of-function mutation of JAK2 in myeloproliferative disorders. New England Journal of Medicine, 2005. 352: p. 1779-90.
10. James, C., Ugo, V., Le Couedic, J.P., Staerk, J., Delhommeau, F., Lacout, C., Garcon, L., Raslova, H., Berger, R., Bennaceur-Griscelli, A., Villeval, J.L., Constantinescu, S.N., Casadevall, N., and Vainchenker, W., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature, 2005. 434(7037): p. 1144-8.
11. Levine RL, Gilliland DG. Myeloproliferative disorders. Blood. 2008 Sep 15;112(6):2190-8.
12. Vainchenker W, Dusa A, Constantinescu SN. JAKs in pathology: Role of Janus kinases in hematopoietic malignancies and immunodeficiencies. Semin Cell Dev Biol. 2008 Jul 17.
JAK2, JAK2 V617F, Janus Kinase 2, myeloproliferative disorder, MPD, mutant, cytotoxicity, Ba/F3, pro-B, counterscreen, HTS, high throughput screen, 1536, inhibitor, inhibition, luciferase, Scripps, 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 compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput assay for inhibitors of the mutant JAK2V617F"(PubChem AID 1446), were nonselective inhibitors due to cytotoxicity of the parental Ba/F3 cell line. In this assay, murine pro-B Ba/F3 cell viability is measured using the CellTiter-Glo luminescent reagent, which contains luciferase to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. As designed, compounds that inhibit cell viability will lead to decreased ATP levels, resulting in decreased luciferase activity and well luminescence.
The murine Ba/F3 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media was RPMI 1640 Glutamax-1 medium containing 10% FBS (v/v), 1ng/mL IL-3, and 1X penicillin-streptomycin-neomycin antibiotic mix.
Prior to the start of the assay, cells were suspended at a concentration of 200,000 cells per mL in phenol red free RPMI supplemented as above except with 2ng/mL IL-3. The assay was started by dispensing 5 microliters of cell suspension to each well. Next, 28 nL of test compound (5.60 micromolar final nominal concentration) in DMSO or DMSO alone (0.56 % final concentration, set as 0% inhibition), or JAK inhibitor I (5.6 micromolar final nominal concentration, set as 100% inhibition) in DMSO was added to the appropriate wells. After 72 hours incubation at 37 degrees C in 5% CO2, the plates were removed from the incubator and allowed to equilibrate to room temperature for 15 minutes. Next, 5 microliters of Cell-Titer Glo reagent was added to all wells and the plates were allowed to incubate for 10 minutes at room temperature. Well luminescence was recorded for 30 seconds per well using the ViewLux reader (PerkinElmer, Turku, Finland).
The percent inhibition for each compound was defined according to the following mathematical formula:
% Inhibition = 100* (Median_Negative_Control - Test_Compound) / (Median_Negative_Control - Median_Positive_Control)
Negative_Control is defined as wells containing cells treated with DMSO,
Test_Compound is defined as wells containing cells treated with test compound,
Positive_Control is defined as wells containing cells treated with the JAK1 inhibitor compound.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The inactive compounds of this assay have activity score range of 0 to 24 and active compounds range of activity score is 24 to 100.
List of Reagents:
RPMI 1640 Medium + Glutamax (Invitrogen, part 11835)
Fetal bovine serum (Hyclone, part SH 30088.03)
JAK Inhibitor I (Calbiochem, part 420099)
IL-3 (PeproTech, part 213-13)
CellTiter-Glo Reagent (Promega, part G7572)
1536-well plates (Greiner, part 789173)
T175 flasks (Corning, part 430828)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. In this assay Jak Inhibitor I had an IC50 of approximately 1 micromolar. 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 and compounds that modulate luciferase activity. 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 by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)