Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor.
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.
Merck Research Laboratories, Boston, Massachusetts 02115, USA.
Kumiko
K
Nagashima
Stuart D
SD
Shumway
Sriram
S
Sathyanarayanan
Albert H
AH
Chen
Brian
B
Dolinski
Youyuan
Y
Xu
Heike
H
Keilhack
Thi
T
Nguyen
Maciej
M
Wiznerowicz
Lixia
L
Li
Bart A
BA
Lutterbach
An
A
Chi
Cloud
C
Paweletz
Timothy
T
Allison
Youwei
Y
Yan
Sanjeev K
SK
Munshi
Anke
A
Klippel
Manfred
M
Kraus
Ekaterina V
EV
Bobkova
Sujal
S
Deshmukh
Zangwei
Z
Xu
Uwe
U
Mueller
Alexander A
AA
Szewczak
Bo-Sheng
BS
Pan
Victoria
V
Richon
Roy
R
Pollock
Peter
P
Blume-Jensen
Alan
A
Northrup
Jannik N
JN
Andersen
D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D004285:Dogs; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase
D009363:Neoplasm Proteins; C000631429:PDK1 protein, human; D047428:Protein Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017346:Protein Serine-Threonine Kinases