Inhibition of Cyclin‐Dependent Kinases by Purine Analogues

Abstract

While testing purines related to the non‐specific protein kinase inhibitors N 6‐dimethylaminopurine and N 6‐(2‐isopentenyl)adenine as potential inhibitors of the p34 cdc2/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 cdk4/cyclin D1, p40 cdk6/cyclin D3, CAMP‐dependent and cGMP‐dependent kinases, eight protein kinase C isoforms, calmodulin‐dependent kinase II, myosin light‐chain kinase, mitogen‐activated S6 kinase, casein kinase 2, double‐stranded RNA‐activated protein kinase, AMP‐stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell‐cycle regulating p34 cdc2/cyclin B, p33 cdk2/cyclin A and p33 cdk2/cyclin E kinases, the brain p33 cdk5/p35 kinase and the ERK1/MAP‐kinase (and its starfish homologue p44 mpk) are substantially inhibited by olomoucine (IC 50 values are 7, 7, 7, 3 and 25 μM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC 50 values greater than 1 mM and 150 μM, respectively). N 6‐(2‐Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC 50, values of 50–100 μM 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 cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain‐specific cdk5/p35 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 G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti‐mitotic reagent that may preferentially inhibit certain steps of the cell cycle.