CDK2 inhibitors have been proposed as effective anti-cancer therapeutics. We show here that CYC202 (R-roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC 50 ‫؍‬ 0.10 M) with an average cytotoxic IC 50 of 15.2 M in a panel of 19 human tumour cell lines, and we also demonstrate selectivity for rapidly proliferating cells over non-proliferating cells. A study of the cell cycle effects of CYC202 in Lovo colorectal carcinoma cells showed that the major effect was not the predicted arrest in one part of the cycle, but rather an induction of cell death from all compartments of the cell cycle. The maximum tolerated dose given intravenously to mice was in excess of 20 mg/kg. Doses up to 2,000 mg/kg were tolerated when administered orally in mice. Following repeated intraperitoneal administration (3 times daily for 5 days) of 100 mg/kg to nude mice bearing the Lovo human colorectal tumour, CYC202 induced a significant antitumour effect with a 45% reduction in tumour growth compared to controls. A second experiment using the human uterine xenograft MESSA-DX5 treated with orally administered CYC202 (500 mg/kg 3 times daily for 4 days) also exhibited a significant reduction in the rate of growth of the tumour (62%). These data, showing enzyme and cellular potency together with antitumour activity, confirm the potential of CDK2 inhibitors such as CYC202 as anticancer drugs. © 2002 Wiley-Liss, Inc. Key words: cyclin-dependent kinase inhibitors; CYC202; roscovitine; cell cycle; anti-tumour efficacyCyclin-dependent kinases (CDKs) are key regulators in the process of cell cycle progression. 1 These enzymes are activated by periodic formation of complexes with cyclins, which are proteins that are present only at specific stages of the cell cycle. CDK4 and CDK6, coupled with their partner cyclin D, are responsible for progression through G1, whereas CDK2 in combination with cyclin E is responsible for normal progression from G1 into S phase. CDK2/cyclin A is required for progression through S phase, and CDK1/cyclin B is necessary for mitosis to occur. 2 These CDK/ cyclin complexes are regulated in turn by stoichiometric association with small proteins, cyclin-dependent kinase inhibitors (CDKIs), such as p19, p16, p15, p21 and p27, which are members of the INK4 and WAF1/KIP1 class of CDK inhibitor. Mutation and/or deletion of some of these CDKIs has been shown in many human neoplasias. 3 Hence control of the cell cycle through CDKs, by means of small molecule CDK inhibitors, has been of great interest as a novel cancer treatment strategy. Questions remain, however, regarding the importance of CDK selectivity for this type of agent. Both CDK2 and CDK4 have been targeted for small molecule inhibitor development, and recent results suggest that CDK2 antagonists may induce apoptosis selectively in transformed cells regardless of p53 status, 4 while the function of CDK4 has now been linked to modulation of the rate of cellular growth and has been suggested to be (in Drosophila at least) dispensable for cel...

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

NDRG1 is phosphorylated by SGK1 (but not PKB) in vivo at three residues each contained within three nonapeptide repeats. Here, we demonstrate that this nonapeptide, like the NDRG1 protein, is phosphorylated by SGK1, but not by PKBa or RSK1 in vitro. The inability of PKBa and RSK1 to phosphorylate the nonapeptide was traced to residues n + 1, n + 2 and n À 4 (where n is the phosphorylation site). Changing them from Ser, Glu and Ser to Phe, Ala and Pro, respectively, transformed the nonapeptide into an excellent substrate for PKBa and RSK1. Our results identify a specific substrate for SGK1 and may facilitate detection of additional physiological substrates for this enzyme.