Article: Genetic and pharmacological inhibition of PDK1 in cancer cells: Characterization of a selective allosteric kinase inhibitor

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TitleGenetic and pharmacological inhibition of PDK1 in cancer cells: Characterization of a selective allosteric kinase inhibitor
AuthorsNagashima, K1
Shumway, SD1
Sathyanarayanan, S1
Chen, AH1
Dolinski, B1
Xu, Y1
Keilhack, H1
Nguyen, T1
Wiznerowicz, M1
Li, L1
Lutterbach, BA1
Chi, A1
Paweletz, C1
Allison, T2
Yan, Y2
Munshi, SK2
Klippel, A1
Kraus, M1
Bobkova, EV1
Deshmukh, S1
Xu, Z1
Mueller, U1
Szewczak, AA1
Pan, BS1
Richon, V1
Pollock, R1
BlumeJensen, P1
Northrup, A1
Andersen, JN1
Issue Date2011
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
CitationJournal Of Biological Chemistry, 2011, v. 286 n. 8, p. 6433-6448 [How to Cite?]
DOI: http://dx.doi.org/10.1074/jbc.M110.156463
AbstractPhosphoinositide-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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
ISSN0021-9258
2011 Impact Factor: 4.773
2011 SCImago Journal Rankings: 0.793
DOIhttp://dx.doi.org/10.1074/jbc.M110.156463
ISI Accession Number IDWOS:000287476400054
ReferencesReferences in Scopus
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dc.contributor.authorNagashima, K
dc.contributor.authorShumway, SD
dc.contributor.authorSathyanarayanan, S
dc.contributor.authorChen, AH
dc.contributor.authorDolinski, B
dc.contributor.authorXu, Y
dc.contributor.authorKeilhack, H
dc.contributor.authorNguyen, T
dc.contributor.authorWiznerowicz, M
dc.contributor.authorLi, L
dc.contributor.authorLutterbach, BA
dc.contributor.authorChi, A
dc.contributor.authorPaweletz, C
dc.contributor.authorAllison, T
dc.contributor.authorYan, Y
dc.contributor.authorMunshi, SK
dc.contributor.authorKlippel, A
dc.contributor.authorKraus, M
dc.contributor.authorBobkova, EV
dc.contributor.authorDeshmukh, S
dc.contributor.authorXu, Z
dc.contributor.authorMueller, U
dc.contributor.authorSzewczak, AA
dc.contributor.authorPan, BS
dc.contributor.authorRichon, V
dc.contributor.authorPollock, R
dc.contributor.authorBlumeJensen, P
dc.contributor.authorNorthrup, A
dc.contributor.authorAndersen, JN
dc.date.accessioned2012-08-08T08:39:27Z
dc.date.available2012-08-08T08:39:27Z
dc.date.issued2011
dc.description.abstractPhosphoinositide-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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
dc.description.natureLink_to_subscribed_fulltext
dc.identifier.citationJournal Of Biological Chemistry, 2011, v. 286 n. 8, p. 6433-6448 [How to Cite?]
DOI: http://dx.doi.org/10.1074/jbc.M110.156463
dc.identifier.doihttp://dx.doi.org/10.1074/jbc.M110.156463
dc.identifier.epage6448
dc.identifier.isiWOS:000287476400054
dc.identifier.issn0021-9258
2011 Impact Factor: 4.773
2011 SCImago Journal Rankings: 0.793
dc.identifier.issue8
dc.identifier.pmid21118801
dc.identifier.scopuseid_2-s2.0-79953181999
dc.identifier.spage6433
dc.identifier.urihttp://hdl.handle.net/10722/156005
dc.identifier.volume286
dc.languageeng
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
dc.publisher.placeUnited States
dc.relation.ispartofJournal of Biological Chemistry
dc.relation.referencesReferences in Scopus
dc.subject.meshAllosteric Regulation - Drug Effects - Genetics
dc.subject.meshAnimals
dc.subject.meshCatalytic Domain - Genetics
dc.subject.meshCell Line, Tumor
dc.subject.meshCell Proliferation - Drug Effects
dc.subject.meshCrystallography, X-Ray
dc.subject.meshDogs
dc.subject.meshDrug Screening Assays, Antitumor - Methods
dc.subject.meshHumans
dc.subject.meshNeoplasm Proteins - Antagonists & Inhibitors - Genetics - Metabolism
dc.subject.meshNeoplasms - Drug Therapy - Enzymology - Genetics
dc.subject.meshPhosphorylation - Drug Effects - Genetics
dc.subject.meshProtein Kinase Inhibitors - Chemistry - Pharmacology
dc.subject.meshProtein-Serine-Threonine Kinases - Antagonists & Inhibitors - Genetics - Metabolism
dc.titleGenetic and pharmacological inhibition of PDK1 in cancer cells: Characterization of a selective allosteric kinase inhibitor
dc.typeArticle
Author Affiliations
  1. Merck Research Laboratories
  2. Merck & Co.