INHIBITION OF CYCLIN-DEPENDENT KINASES BY PURINE ANALOGS

While testing purines related to the non-specific protein kinase inhib itors N-6-dimethylaminopurine and N-6-(Delta(2)-isopentenyl)adenine as potential inhibitors of the p34(cdc2)/cyclin B kinase, we discovered a compound with high specificity, (2-hydroxyethylamino)-6-benzylamino- 9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition rev eals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for bot h substrates). The kinase specificity of this inhibition was investiga ted for 35 highly purified kinases (including p34(cdk4)/cyclin D1, p40 (cdk6)/cyclin D3, cAMP-dependent and cGMP-dependent kinases, eight pro tein kinase C isoforms, calmodulin-dependent kinase II, myosin light-c hain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stra nded RNA-activated protein kinase, AMP-stimulated kinase, eight tyrosi ne kinases). Most kinases are not significantly inhibited. Only the ce ll-cycle regulating p34(cdc2)/cyclin B, p33(cdk2)/cyclin A and p33(cdk 2)/cyclin E kinases, the brain p33(cdk5)/ p35 kinase and the ERK1/MAP- kinase (and its starfish homologue p44(mpk)) are substantially inhibit ed by olomoucine (IC50 values are 7, 7, 7, 3 and 25 mu M, respectively ). The cdk4/cyclin D1 and cdk6/ cyclin D3 kinases are not significantl y sensitive to olomoucine (IC50 values greater than 1 mM and 150 mu M, respectively). N-6-(Delta(2)-Isopentenyl)adenine is confirmed as a ge neral kinase inhibitor with IC50 values of 50-100 mu M for many kinase s. The purine specificity of cyclin-dependent kinase inhibition was in vestigated: among 81 purine derivatives tested, only C2, N6 and N9-sub stituted purines exert a strong inhibitory effect on the p34(cdc2)/cyc lin B kinase. An essentially similar sensitivity to this olomoucine fa mily of compounds was observed for the brain-specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the inte ractions of olomoucine and its analogues with the kinase catalytic sub unit. Olomoucine inhibits in vitro M-phase-promoting factor activity i n metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synth esis in Xenopus interphase egg extracts and inhibits the licensing fac tor, an essential replication factor ensuring that DNA is replicated o nly once in each cell cycle. Olomoucine inhibits the starfish oocyte G 2/M transition in vitro. Through its unique selectivity olomoucine pro vides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.