Counterscreen for inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): Fluorescence-based biochemical high throughput assay to identify GFP inhibitors and fluorescence quenchers
Name: Counterscreen for inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): Fluorescence-based biochemical high throughput assay to identify GFP inhibitors and fluorescence quenchers. ..more
BioActive Compounds: 109
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: GFP_INH_QFRET_1536_3X%INH CSRUN
Name: Counterscreen for inhibitors of the interaction of the Ras and Rab interactor 1 protein (Rin1) and the c-abl oncogene 1, non-receptor tyrosine kinase (Abl): Fluorescence-based biochemical high throughput assay to identify GFP inhibitors and fluorescence quenchers.
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.
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The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, TR-FRET-based biochemical primary high throughput screening assay 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) (AID 588664) are nonselective due to quenching of eGFP and/or well FRET. This biochemical assay employs the ABL1-eGFP fusion construct incubated in the presence of test compounds, but in the absence of its binding partner RIN1-SBP. Since no interaction can occur between RIN1 and ABL in the absence of RIN1, compounds active in this assay act directly on the ABL1-eGFP protein alone or interfere with well fluorescence, and thus are not true inhibitors of the interaction between the RIN1 and ABL proteins. Compounds active in this assay are considered artifacts and will not be pursued. Compounds are tested in triplicate at a final nominal concentration of 6.75 uM.
The assay was started by dispensing 5 uL of Assay Buffer (1X Phosphate Buffered Saline) into columns 1 thru columns 3 of 1536 microtiter plates. Next, 5 uL of assay buffer containing 100 nM of ABL-eGFP were dispensed into columns 4 thru 48. Then, the plates were centrifuged and pinned with 34 nL of test compound in DMSO or DMSO alone (0.68% final concentration). The plates were incubated for 10 minutes at 25 C. Fluorescence was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a FITC filter set (excitation = 480 nm, emission = 540 nm) and a FITC dichroic mirror for 1 second.
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 ABL-eGFP in the presence of test compound.
High_Control is defined as wells containing the no ABL-eGFP protein.
Low_Control is defined as wells containing the ABL-eGFP + DMSO.
PubChem Activity Outcome and Score:
The average percent inhibition and standard deviation of each compound tested were calculated. In addition, two values were calculated: (1) the average percent inhibition of the wells containing DMSO only, and (2) three times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited an average percent inhibition greater than the calculated hit cutoff was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. % Inhibition values of greater than or equal to 100 are reported as activity score 100. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 10-0.
List of Reagents:
ABL1-eGFP fusion protein (supplied by Assay Provider)
PBS (Fisher, part BP399)
1536-well plates (Corning, part 7234)
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. 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 by the MLSMR. The MLSMR was unable to provide all compounds selected for testing.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay biosafety level: bsl1
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: bioassay specification: assay stage: secondary: counter screening
BAO: meta target detail: binding reporter specification: interaction: protein-protein
BAO: version: 1.4b1090
Assay Format: Biochemical
** Test Concentration.
Data Table (Concise)