Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram; Chen, Albert H.; Dolinski, Brian; Xu, Youyuan; Keilhack, Heike; Nguyen, Thi; Wiznerowicz, Maciej; Li, Lixia; Lutterbach, Bart A.; Chi, An; Paweletz, Cloud; Allison, Timothy; Yan, Youwei; Munshi, Sanjeev K.; Klippel, Anke; Kraus, Manfred; Bobkova, Ekaterina V.; Deshmukh, Sujal; Xu, Zangwei; Mueller, Uwe; Szewczak, Alexander A.; Pan, Bo-Sheng; Richon, Victoria; Pollock, Roy; Blume-Jensen, Peter; Northrup, Alan; Andersen, Jannik N.

doi:10.1074/jbc.M110.156463

Title: Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

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Abstract

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profilingmore »« less

Authors:

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram; Chen, Albert H.; Dolinski, Brian; Xu, Youyuan; Keilhack, Heike; Nguyen, Thi; Wiznerowicz, Maciej; Li, Lixia; Lutterbach, Bart A.; Chi, An; Paweletz, Cloud; Allison, Timothy; Yan, Youwei; Munshi, Sanjeev K.; Klippel, Anke; Kraus, Manfred; Bobkova, Ekaterina V.; Deshmukh, Sujal more »

Merck

Publication Date:: Wed Nov 20 00:00:00 EST 2013

Research Org.:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:: INDUSTRY

OSTI Identifier:: 1025623

Resource Type:: Journal Article

Journal Name:: J. Biol. Chem.

Additional Journal Information:: Journal Volume: 286; Journal Issue: (8) ; 02, 2011; Journal ID: ISSN 0021-9258

Country of Publication:: United States

Language:: ENGLISH

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; BENCHMARKS; COLONY FORMATION; CONVERGENCE; GENETICS; IN VITRO; NEOPLASMS; PHOSPHORYLATION; PHOSPHOTRANSFERASES; PROTEINS; TISSUE CULTURES; TUMOR CELLS

Citation Formats


                    Nagashima, Kumiko, Shumway, Stuart D., Sathyanarayanan, Sriram, Chen, Albert H., Dolinski, Brian, Xu, Youyuan, Keilhack, Heike, Nguyen, Thi, Wiznerowicz, Maciej, Li, Lixia, Lutterbach, Bart A., Chi, An, Paweletz, Cloud, Allison, Timothy, Yan, Youwei, Munshi, Sanjeev K., Klippel, Anke, Kraus, Manfred, Bobkova, Ekaterina V., Deshmukh, Sujal, Xu, Zangwei, Mueller, Uwe, Szewczak, Alexander A., Pan, Bo-Sheng, Richon, Victoria, Pollock, Roy, Blume-Jensen, Peter, Northrup, Alan, and Andersen, Jannik N. Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor.  United States: N. p., 2013. 
        Web.  doi:10.1074/jbc.M110.156463.

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                    Nagashima, Kumiko, Shumway, Stuart D., Sathyanarayanan, Sriram, Chen, Albert H., Dolinski, Brian, Xu, Youyuan, Keilhack, Heike, Nguyen, Thi, Wiznerowicz, Maciej, Li, Lixia, Lutterbach, Bart A., Chi, An, Paweletz, Cloud, Allison, Timothy, Yan, Youwei, Munshi, Sanjeev K., Klippel, Anke, Kraus, Manfred, Bobkova, Ekaterina V., Deshmukh, Sujal, Xu, Zangwei, Mueller, Uwe, Szewczak, Alexander A., Pan, Bo-Sheng, Richon, Victoria, Pollock, Roy, Blume-Jensen, Peter, Northrup, Alan, & Andersen, Jannik N. Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor.  United States.  https://doi.org/10.1074/jbc.M110.156463

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                    Nagashima, Kumiko, Shumway, Stuart D., Sathyanarayanan, Sriram, Chen, Albert H., Dolinski, Brian, Xu, Youyuan, Keilhack, Heike, Nguyen, Thi, Wiznerowicz, Maciej, Li, Lixia, Lutterbach, Bart A., Chi, An, Paweletz, Cloud, Allison, Timothy, Yan, Youwei, Munshi, Sanjeev K., Klippel, Anke, Kraus, Manfred, Bobkova, Ekaterina V., Deshmukh, Sujal, Xu, Zangwei, Mueller, Uwe, Szewczak, Alexander A., Pan, Bo-Sheng, Richon, Victoria, Pollock, Roy, Blume-Jensen, Peter, Northrup, Alan, and Andersen, Jannik N. 2013.  
        "Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor".  United States.  https://doi.org/10.1074/jbc.M110.156463.

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@article{osti_1025623,

  title        = {Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor},

  author       = {Nagashima, Kumiko and Shumway, Stuart D. and Sathyanarayanan, Sriram and Chen, Albert H. and Dolinski, Brian and Xu, Youyuan and Keilhack, Heike and Nguyen, Thi and Wiznerowicz, Maciej and Li, Lixia and Lutterbach, Bart A. and Chi, An and Paweletz, Cloud and Allison, Timothy and Yan, Youwei and Munshi, Sanjeev K. and Klippel, Anke and Kraus, Manfred and Bobkova, Ekaterina V. and Deshmukh, Sujal and Xu, Zangwei and Mueller, Uwe and Szewczak, Alexander A. and Pan, Bo-Sheng and Richon, Victoria and Pollock, Roy and Blume-Jensen, Peter and Northrup, Alan and Andersen, Jannik N.},

  abstractNote = {Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.},

  doi          = {10.1074/jbc.M110.156463},

  url          = {https://www.osti.gov/biblio/1025623},
  journal      = {J. Biol. Chem.},

  issn         = {0021-9258},

  number       = (8) ; 02, 2011,

  volume       = 286,

  place        = {United States},

  year         = {Wed Nov 20 00:00:00 EST 2013},

  month        = {Wed Nov 20 00:00:00 EST 2013}

}

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