Multi-pathway cellular analysis of compound selectivity.

Signaling pathways and their protein target constituents (e.g. kinases) have become important therapeutic targets in many disease areas. Traditional selectivity profiling for kinase inhibitors has relied upon screening panels of recombinant enzymes in biochemical assay formats. Recent studies have highlighted the importance of using cellular assays to better approximate true biological selectivity. We have developed a portfolio of CellSensor beta-lactamase transcriptional reporter gene assays that can be used to screen for perturbagens of various endogenous signaling pathways. Here we describe a multi-pathway profiling approach for generating compound-pathway selectivity maps. To demonstrate the utility of this approach, we have screened 32 known compounds across a diverse panel of 12 key signaling pathways and generated the first comprehensive cellular pathway selectivity profiles of several clinically approved kinase and other well-known bioactive inhibitors. Selectivity score comparisons identified several kinase inhibitors that were more promiscuous than predicted by traditional biochemical profiling methods. For example, we identified effects of sorafenib on the JAK/STAT pathway and demonstrated the potential therapeutic indication of sorafenib in treating leukemia/myeloproliferative disorder patients harboring TEL-JAK2 or JAK2V617F mutations. Our results indicate that multi-pathway profiling can efficiently characterize both on and off-pathway compound activities, revealing potential novel pathways and opportunities for drug repositioning purposes and/or safety liabilities in one profiling campaign.

[1]  Xin-Hua Feng,et al.  Critical regulation of TGFβ signaling by Hsp90 , 2008, Proceedings of the National Academy of Sciences.

[2]  Ronald Breslow,et al.  Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug , 2007, Nature Biotechnology.

[3]  Sandra A. Moore,et al.  Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. , 2005, Cancer cell.

[4]  N. Tsuyama,et al.  IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3. , 2005, Biochemical and biophysical research communications.

[5]  P. Marks,et al.  Histone Deacetylase Inhibitors: Overview and Perspectives , 2007, Molecular Cancer Research.

[6]  S. Wilhelm,et al.  Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. , 2006, Methods in enzymology.

[7]  Jenny Bain,et al.  BIRB796 Inhibits All p38 MAPK Isoforms in Vitro and in Vivo* , 2005, Journal of Biological Chemistry.

[8]  Shadan Ali,et al.  Exploitation of protein kinase C: a useful target for cancer therapy. , 2009, Cancer treatment reviews.

[9]  P. Bork,et al.  Drug Target Identification Using Side-Effect Similarity , 2008, Science.

[10]  P. Hozák,et al.  Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. , 2007, Blood.

[11]  C. Horvath,et al.  Unexpected roles for deacetylation in interferon- and cytokine-induced transcription. , 2005, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[12]  L. Luo,et al.  Heat shock protein 90 (Hsp90) regulates the stability of transforming growth factor beta-activated kinase 1 (TAK1) in interleukin-1beta-induced cell signaling. , 2009, Molecular immunology.

[13]  M. Tremblay,et al.  Selective regulation of tumor necrosis factor–induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase , 2005, Nature Immunology.

[14]  T. Suga,et al.  c-Src and Hydrogen Peroxide Mediate Transforming Growth Factor-β1–Induced Smooth Muscle Cell–Gene Expression in 10T1/2 Cells , 2004, Arteriosclerosis, thrombosis, and vascular biology.

[15]  Kevan M Shokat,et al.  Features of selective kinase inhibitors. , 2005, Chemistry & biology.

[16]  G. Zlokarnik,et al.  Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. , 1998, Science.

[17]  M. Maa,et al.  Functional implication of the interaction between EGF receptor and c-Src. , 2003, Frontiers in bioscience : a journal and virtual library.

[18]  L. Neckers,et al.  Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1 alpha-degradative pathway. , 2002, The Journal of biological chemistry.

[19]  R Berger,et al.  A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. , 1997, Science.

[20]  L. Neckers,et al.  Hsp90 Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-1α-degradative Pathway* , 2002, The Journal of Biological Chemistry.

[21]  K. Vogel,et al.  Developing assays for kinase drug discovery – where have the advances come from? , 2008, Expert opinion on drug discovery.

[22]  R. Roskoski,et al.  Src kinase regulation by phosphorylation and dephosphorylation. , 2005, Biochemical and biophysical research communications.

[23]  G. Piraino,et al.  Mitogen-Activated Protein Kinases and NF-κB Are Involved in TNF-α Responses to Group B Streptococci1 , 2002, The Journal of Immunology.

[24]  M. Vieth,et al.  Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-beta type I receptor kinase domain. , 2003, Journal of medicinal chemistry.

[25]  G. Parmigiani,et al.  Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses , 2008, Science.

[26]  T. Machleidt,et al.  Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors. , 2008, Assay and drug development technologies.

[27]  Ignacio Wistuba,et al.  Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion , 2007, Molecular Cancer Therapeutics.

[28]  James E. Thompson and.  Photochemical Preparation of a Pyridone Containing Tetracycle: A Jak Protein Kinase Inhibitor. , 2010 .

[29]  Y. Li,et al.  Raf inhibitor stabilizes receptor for the type I interferon but inhibits its anti-proliferative effects in human malignant melanoma cells , 2007, Cancer biology & therapy.

[30]  Zhi-wei Li,et al.  NF-κB in the pathogenesis and treatment of multiple myeloma , 2008, Current opinion in hematology.

[31]  Mindy I. Davis,et al.  A quantitative analysis of kinase inhibitor selectivity , 2008, Nature Biotechnology.

[32]  Paul A Clemons,et al.  The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease , 2006, Science.

[33]  G. Demetri,et al.  Molecular basis for sunitinib efficacy and future clinical development , 2007, Nature Reviews Drug Discovery.

[34]  T. Machleidt,et al.  HTS assays using a disease-relevant cell model for interrogating the MAP kinase pathway initiated by multiple receptors. , 2008, Assay and drug development technologies.

[35]  F. Sams-Dodd.  Target-based drug discovery: is something wrong? , 2005, Drug discovery today.

[36]  P. Cohen,et al.  The selectivity of protein kinase inhibitors: a further update. , 2007, The Biochemical journal.

[37]  A. Maity,et al.  EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms. , 2006, Cancer research.

[38]  L. Ellis,et al.  HIF-1α, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas , 2005, Oncogene.