Assay Development and High-Throughput Screening of Small Molecular c-Abl Kinase Activators

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC50 <10 µM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.

[1]  R. Bronson,et al.  Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene , 1991, Cell.

[2]  Bruce A. Roe,et al.  Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene , 1986, Cell.

[3]  E. Sargsyan,et al.  AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic beta-cells without changing triglyceride levels. , 2008, Journal of molecular endocrinology.

[4]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[5]  J. Wang,et al.  Abl tyrosine kinase in signal transduction and cell-cycle regulation. , 1993, Current opinion in genetics & development.

[6]  L. Serrano,et al.  Crystal structure of the abl-SH3 domain complexed with a designed high-affinity peptide ligand: implications for SH3-ligand interactions. , 1998, Journal of molecular biology.

[7]  Oliver Hantschel,et al.  Organization of the SH3-SH2 unit in active and inactive forms of the c-Abl tyrosine kinase. , 2006, Molecular cell.

[8]  R. V. van Etten,et al.  c-Abl Has High Intrinsic Tyrosine Kinase Activity That Is Stimulated by Mutation of the Src Homology 3 Domain and by Autophosphorylation at Two Distinct Regulatory Tyrosines* , 2000, The Journal of Biological Chemistry.

[9]  B. Viollet,et al.  Beyond AICA riboside: In search of new specific AMP‐activated protein kinase activators , 2009, IUBMB life.

[10]  R. A. Etten,et al.  Mutational analysis of the regulatory function of the c-Abl Src homology 3 domain , 2001, Oncogene.

[11]  B. Hansen,et al.  A-769662 activates AMPK beta1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle. , 2009, American journal of physiology. Cell physiology.

[12]  Shohei Koide,et al.  A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain , 2010, Nature Structural &Molecular Biology.

[13]  M. Baccarani,et al.  Chronic myeloid leukaemia , 2007, The Lancet.

[14]  D. Auld,et al.  Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection. , 2003, Assay and drug development technologies.

[15]  G. Superti-Furga,et al.  Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase , 2003, Cell.

[16]  Kristin E. D. Coan,et al.  Promiscuous Aggregate-Based Inhibitors Promote Enzyme Unfolding , 2009, Journal of medicinal chemistry.

[17]  Wolfgang Jahnke,et al.  Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site. , 2010, Biochimica et biophysica acta.

[18]  D. McDonald,et al.  Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  N. Narula,et al.  Targeting of the c-Abl Tyrosine Kinase to Mitochondria in Endoplasmic Reticulum Stress-Induced Apoptosis , 2001, Molecular and Cellular Biology.

[20]  B. Viollet,et al.  Targeting the AMPK pathway for the treatment of Type 2 diabetes. , 2009, Frontiers in bioscience.

[21]  Arthur Christopoulos,et al.  Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders , 2009, Nature Reviews Drug Discovery.

[22]  J. Mestan,et al.  Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay. , 2010, Journal of the American Chemical Society.

[23]  S. Goff,et al.  Mice homozygous for the abl m1 mutation show poor viability and depletion of selected B and T cell populations , 1991, Cell.

[24]  G. Neubauer,et al.  Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase , 2001, Oncogene.

[25]  R. Ames,et al.  Gene expression in mammalian cells using BacMam, a modified baculovirus system. , 2007, Methods in molecular biology.

[26]  Yoshiaki Washio,et al.  Direct small-molecule kinase activation: Novel approaches for a new era of drug discovery. , 2009, Current opinion in drug discovery & development.

[27]  D. Wasilko,et al.  The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. , 2009, Protein expression and purification.

[28]  C. Hudis,et al.  A phase II trial of imatinib mesylate monotherapy in patients with metastatic breast cancer , 2005, Breast Cancer Research and Treatment.

[29]  William P Schiemann,et al.  Activated Abl kinase inhibits oncogenic transforming growth factor‐β signaling and tumorigenesis in mammary tumors , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  Xuefei Gao,et al.  Inhibitory effects of A-769662, a novel activator of AMP-activated protein kinase, on 3T3-L1 adipogenesis. , 2009, Biological & pharmaceutical bulletin.

[31]  D. Baltimore,et al.  The mouse type IV c-abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization , 1989, Cell.

[32]  J. Mestan,et al.  Allosteric inhibitors of Bcr-abl–dependent cell proliferation , 2006, Nature chemical biology.

[33]  G. Superti-Furga,et al.  A Myristoyl/Phosphotyrosine Switch Regulates c-Abl , 2003, Cell.

[34]  L. Kifle,et al.  Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. , 2006, Cell metabolism.

[35]  James Inglese,et al.  Apparent activity in high-throughput screening: origins of compound-dependent assay interference. , 2010, Current opinion in chemical biology.

[36]  R. A. Etten Cycling, stressed-out and nervous: cellular functions of c-Abl , 1999 .