While testing purines related to the non-specific protein kinase inhibitors M-dimethylaminopurine and N6-(A'-isopenteny1)adenine as potential inhibitors of the p34'd'z/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34"dk4/~y~lin D1, p4Wdk6/cyclin D3, CAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase 11, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded EWA-activated protein kinase, AMPstimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34'dc2/cyclin B, p33cdk2/~y~lin A and p33cdk2/~y~lin E kinases, the brain ~3 3 '~~' / p35 kinase and the ERKlMAP-kinase (and its starfish homologue ~44"'~~) are substantially inhibited by olomoucine (I& values are 7, 7, 7, 3 and 25 pM, respectively). The cdk4/cyclin D1 and cdk6/ cyclin D3 kinases are not significantly sensitive to olomoucine (I& values greater than 1 mM and 150 pM, respectively). M-(A2-1sopentenyl)adenine is confirmed as a general kinase inhibitor with IC,, values of 50-100 pM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34'd'2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdkYp35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G 2 M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.Phosphorylation of serine, threonine and tyrosine residues by protein kinases represents one of the most common post-translational regulatory modifications of proteins. More than 200 protein kinases have been described, following either purification to homogeneity or molecular cloning [1 -41. As much as 2 -3 % of eukaryotic genes may encode protein kinases. The importance of protein kinases in physiological processes has stimulated an active search for specific inCorrespondence to L. Meijer, Centre National de la Recherche Scientifique, Station Biologique, BP ...

Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-( 1 -ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], was identified by screening a series of C2,P,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A,, Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, (1997) Eur: J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rf,,n,, of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (&isomer is about twice as potent in inhibiting cdc2kyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2hoscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.Keywords: cell cycle ; cyclin-dependent kinase; purine ; protein-kinase inhibitor; anti-tumor agent.Protein kinases are involved in essentially all intracellular regulatory pathways. In view of their essential role in the regulation of the cell division cycle, cyclin-dependent kinases (cdk) have been the object of considerable investigation (reviews in Numerous examples of cdk deregulation in human primary tumours and in tumour cell lines have been described recently (reviews in [12, 131). These observations encourage the search for selective chemical inhibitors of cdk proteins and the use of natural inhibitors in potential gene therapy [14, 151. Prompted by the abundance of cdc2kyclin B kinase in starfish oocytes, its relatively easy purification by affinity chromatography on p9c7Ks/ p13'""-Sepharose and the rapid assay of its activity with histone H1 and ["PIATP, a few years ago we set up a simple screening test using purified p34'd"2/cyclin Bcdc13 as a target [16]. F-29682 Roscoff cedex, FranceThe first compound to be identified as a cdc2 inhibitor was 6-dimethylaminopurine (IC50: 120 pM) [17, 181. This compound was initially synthesized as a puromycin analogue and found to inhibit mitosis of sea urchin embryos without inhibiting protein synthesis [ 191. By structural analogy we identified N6-(d2-isopenteny1)adenine as a slightly more potent kinase inhibitor (ICs,,: 55 pM) [16]. Following extensive screening of a series of substituted purines designed for other purposes, olomoucine [2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine] was identified as a potent cdc2 inhibitor (ICso: 7 pM) [20]. ...

Cyclin-dependent kinases (cdk) have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle. The structure-activity relationships of cdk inhibition showed that the 1, 3; and 7 positions of the purine ring must remain free, probably for a direct interaction, in which it behaves as a hydrogen bond acceptor. Olomoucine (6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, OC), roscovitine (6-(benzylamino)-2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpur ine), and other N6,2,9-trisubstituted adenines were found to exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. Removal or change of the side chain at position 2 or the hydrophobic group at position 9 dramatically decreased the inhibitory activity of olomoucine or roscovitine. Inhibition of cdk with OC and related compounds clearly arrests cell proliferation of many tumor cell lines at G1/S and G2/M transitions and also triggers apoptosis in the target tumor cells in vitro and in vivo. Thus, from a pharmacological point of view, OC may represent a model compound for a new class of antimitotic and antitumor drugs.