Late stage assay for the probe development effort to identify inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): TRFRET-based biochemical dose response assay for inhibitors of Rin1-Abl interactions
Name: Late stage assay for the probe development effort to identify inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): TRFRET-based biochemical dose response assay for inhibitors of Rin1-Abl interactions. ..more
BioActive Compounds: 20
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: John Colicelli, UCLA
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA136699-01A1
Grant Proposal PI: John Colicelli, UCLA
External Assay ID: RIN1-ABL_INH_TRFRET_1536_3XIC50 MDRUN
Name: Late stage assay for the probe development effort to identify inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): TRFRET-based biochemical dose response assay for inhibitors of Rin1-Abl interactions.
Chromosome translocations that join the BCR and ABL1 (a.k.a. c-Abl) genes give rise to BCR-ABL1 fusion proteins causative in chronic myeloid leukemia (CML), some cases of acute lymphocytic leukemia (ALL) and occasionally other myeloproliferative disorders (1). In addition, ETV6 forms fusion oncogenes with ABL1 (2) and the closely related ABL2 (a.k.a. ARG) (3) in some leukemias. ABL proteins are non-receptor tyrosine kinases normally under tight regulation, but BCR-ABL1 fusions are constitutively active. The ABL kinase inhibitor imatinib mesylate (a.k.a. STI571 or Gleevec) is an effective treatment for CML (4), demonstrating that direct oncoprotein targeting can be used to manage cancer and perhaps eventually be part of a curative therapy. Some leukemias with activated ABL oncoproteins do not respond to imatinib, however, and for CML patients who do respond there is a significant risk of developing resistance due to strong selective pressure for BCR-ABL1 kinase domain mutations that block inhibitor action (5). Resistance and relapse are even more common in BCR-ABL1 positive ALL. Some attempts have been made to circumvent resistance by reducing BCR-ABL1 expression (6, 7) or stability (8, 9) or by targeting collaborative signaling pathways (10-12). A more direct approach for improving treatment would be to maintain focus on reducing tyrosine kinase activity by targeting oncogenic ABL outside the catalytic site.
RIN1 is a RAS effector protein that binds to and activates ABL tyrosine kinases (13, 14). Signaling is initiated by low affinity binding of a proline rich sequence on RIN1 to the SH3 domain of ABL. This interaction leads to phosphorylation of RIN1 on tyrosine 36, which subsequently associates with the ABL SH2 domain. The resulting stable divalent interaction (RIN1 proline-rich motif and phospho-Tyr36 bound to ABL SH3 and SH2 domains, respectively) relieves the ABL autoinhibitory fold and leads to activation of the ABL kinase through enhanced catalytic efficiency (13). Both ABL1 and ABL2 are activated by RIN1, and this requires only the ABL SH3, SH2 and kinase domains. Activation by RIN1 is independent of ABL trans-phosphorylation and is unaffected by an imatinib-resistance mutation (13). Silencing of RIN1 results in less tyrosine phosphorylation of the ABL substrate CRKL, and deletion of the mouse Rin1 gene causes reduction in basal levels of phospho-CRKL (14). These data demonstrate that RIN1 directly stimulates the tyrosine kinase activity of ABL proteins and is required for maintaining normal ABL kinase activity.
Because human ABL fusion oncoproteins consistently retain the autoinhibitory SH3 and SH2 domains, we reasoned that these constitutively active tyrosine kinases might still be subject to positive regulation by RIN1. Indeed, RIN1 binds to and enhances the catalytic, transforming and leukemogenic properties of BCR-ABL1. Deletion of RIN1 blocked transformation of bone marrow cells by BCR-ABL1 and ETV6-ABL1. Transformation was rescued by ectopic RIN1, indicating a cell autonomous mechanism. BCR-ABL1T315I, a drug resistant mutant found in CML patients, was also dependent on RIN1 for transformation. Silencing of RIN1 in human leukemia cells reduced phospho-tyrosine levels and sensitized cells to imatinib. The dependence of BCR-ABL1 on a directly binding regulator (RIN1) provides a unique point of vulnerability that could be exploited to treat kinase inhibitor-resistant leukemias. In addition, combining drugs that inhibit BCR-ABL1 activation by RIN1 with standard ABL kinase inhibitors could provide therapy that is more efficacious and less prone to resistance and disease relapse. Finally, the concept of interference with positive regulators may be applicable to the targeted therapy of other oncogenic kinases.
1. Wong, S. and O.N. Witte, The BCR-ABL story: bench to bedside and back. Annu Rev Immunol, 2004. 22: p. 247-306.
2. Papadopoulos, P., et al., The novel activation of ABL by fusion to an ets-related gene, TEL. Cancer Res, 1995. 55(1): p. 34-38.
3. Iijima, Y., et al., A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12)(q25;p13) translocation. Blood, 2000. 95(6): p. 2126-2131.
4. Deininger, M., E. Buchdunger, and B.J. Druker, The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood, 2005. 105(7): p. 2640-2653.
5. Gorre, M.E., et al., Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science, 2001. 293(5531): p. 876-880.
6. Bueno, M.J., et al., Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression. Cancer Cell, 2008. 13(6): p. 496-506.
7. Chen, R., V. Gandhi, and W. Plunkett, A sequential blockade strategy for the design of combination therapies to overcome oncogene addiction in chronic myelogenous leukemia. Cancer Res, 2006. 66(22): p. 10959-10966.
8. Kawano, T., et al., MUC1 oncoprotein regulates Bcr-Abl stability and pathogenesis in chronic myelogenous leukemia cells. Cancer Res, 2007. 67(24): p. 11576-11584.
9. Wu, L.X., et al., Disruption of the Bcr-Abl/Hsp90 protein complex: a possible mechanism to inhibit Bcr-Abl-positive human leukemic blasts by novobiocin. Leukemia, 2008. 22(7): p. 1402-1409.
10. Dierks, C., et al., Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation. Cancer Cell, 2008. 14(3): p. 238-249.
11. Hess, P., et al., Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts. Nat Genet, 2002. 32(1): p. 201-205.
12. Zhao, C., et al., Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature, 2009. 458(7239): p. 776-779.
13. Cao, X., et al., Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms. J Biol Chem, 2008. 283(46): p. 31401-31407.
14. Hu, H., et al., RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial-cell adhesion and migration. Curr Biol, 2005. 15(9): p. 815-823.
late stage, powders, purchased, synthesized, MDRUN, RIN1, Ras and Rab interactor 1, RIN1-ABL, JC99, ras interaction/interference protein 1, protein kinase, kinase, enzyme, Abl1, c-abl oncogene 1, BCR-ABL, non-receptor tyrosine kinase, bcr/c-abl; proto-oncogene c-Abl; proto-oncogene tyrosine-protein kinase ABL1, tyrosine-protein kinase ABL1, v-abl Abelson murine leukemia viral oncogene homolog 1, biochemical, HTRF, TR-FRET, FRET, terbium, Tb, fluor, fluorescence, inhibit, inhibitor, dose response, DRUN, titration, screen, triplicate, 1536, Scripps Florida, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine RIN1-ABL inhibitory dose response curves for powder samples of compounds identified as RIN1-ABL inhibitor probe candidates.
This biochemical TR-FRET-based assay employs RIN1-SBP and ABL1-GFP fusion proteins as binding partners, whose interaction is monitored using strep-Terbium (Tb). Preincubation of the fusion proteins allows tyrosine phosphorylation of RIN1-SBP by ABL1-GFP, and leads to formation of a stable complex. A streptavidin-complexed lanthanide (Tb) attaches to the streptavidin-binding-peptide (SBP) tag on RIN1. When excited, Tb transfers energy to a GFP tag on ABL1, now complexed with RIN1-SBP. As designed, a compound that acts as an inhibitor of the RIN1::ABL1 interaction will prevent either phosphorylation of RIN1-SBP and/or inhibit RIN-ABL complex formation, leading to reduced energy transfer and reduced well FRET (GFP emission / Tb emission). Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nominal concentration of 73.5 uM.
The assay was started by dispensing 5 uL of Control Mix in assay buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 500 uM ATP, 1 mM DTT, 100 uM Na3VO4 and 100 uM BSA) containing 100 nM of ABL-eGFP and 2.5 nM of Terbium-Streptavidin into columns 1 thru columns 3 of 1536 microtiter plates. Next, 5 uL of Experimental Mix containing 100 nM of ABL-eGFP, 2.5 nM Terbium-Streptavidin and 100 nM of RIN1-SBP in assay buffer were dispensed into columns 4 thru 48. Then, the plates were centrifuged and pinned with 37 nL of test compound in DMSO or DMSO alone (0.73% final concentration). The plates were incubated for 60 minutes at 25 C and TR-FRET was measured on a Viewlux microplate reader (Perkin Elmer, Turku, Finland). After excitation at 340 nm, well fluorescence was monitored at 495 nm and 525 nm.
For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I525nm / I495nm.
I525nm represents the measured fluorescence emission at 525 nm.
I495nm represents the measured fluorescence emission at 495 nm.
The percent inhibition for each compound was calculated using as follows:
%_Inhibition = 100 * (Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) )
Test_Compound is defined as wells containing the Experimental Mix in the presence of test compound.
High_Control is defined as wells containing the Control Mix and DMSO.
Low_Control is defined as wells containing the Experimental Mix and DMSO.
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. 73.5uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 73.5 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 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 for active compounds is 100-14, and for inactive compounds 8-1.
List of Reagents:
RIN1-SBP fusion protein (supplied by Assay Provider)
ABL1-eGFP fusion protein (supplied by Assay Provider)
LanthaScreen Terbium-Streptavidin (Invitrogen, part PV3966)
Tris (Amresco, part 0497)
NaCl (Sigma, part S6546)
MgCl2 (Fisher, part BP214)
Na3VO4 (Fisher, part S454)
ATP (Sigma, part A7699)
DTT (Fisher, part BP172)
BSA (EMD Biosciences, part 2910)
1536-well plates (Corning, part 7234)
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 located in or on wells of the microtiter plate, and compounds that modulate well fluorescence/FRET. 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: lead optimization
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: transferase: kinase
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target detail: binding reporter specification: interaction: protein-protein
BAO: assay design: conformation reporter: protein
BAO: detection technology: fluorescence: fret: tr-fret
BAO: bioassay specification: bioassay type: binding
BAO: bioassay specification: assay footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)