Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.
BACKGROUND
Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified.


RESULTS
To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries.


CONCLUSIONS
That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.
Knockaert
M
M
Station Biologique de Roscoff, CNRS, Roscoff cedex, 29682, France.
Gray
N
N
Damiens
E
E
Chang
Y T
YT
Grellier
P
P
Grant
K
K
Fergusson
D
D
Mottram
J
J
Soete
M
M
Dubremetz
J F
JF
Le Roch
K
K
Doerig
C
C
Schultz
P
P
Meijer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D056890:Eukaryota / Q000201:enzymology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D013215:Starfish / Q000166:cytology; D013379:Substrate Specificity; D013552:Swine; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D018844:Cyclin-Dependent Kinases