In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials

Abstract

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K i = 1 -3 nmol/L) and was inactive (K i > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC 50 s V 0.60 Mmol/L. The growth-inhibitory activity is characterized by a cell cycle block at G 1 and G 2 phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials.

Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines

“…Similarly, flavopiridol administered on an optimized scheduled caused sustained tumor growth inhibition of f60% without evidence of regression in the HN12 xenograft model (34). Newergeneration compounds, such as R547 (35) and P276-00 (36) from Hoffmann-La Roche and Nicholas Piramal, exhibited maximal in vivo growth inhibitions of 60% and 80%, respectively, in several models; but again, no regressions were reported. Similarly, CDKi-277 from Amgen was reported to give cytostasis with regressions observed only at nontolerated doses (37).…”

Preclinical biomarkers for a cyclin-dependent kinase inhibitor translate to candidate pharmacodynamic biomarkers in phase I patients

“…This gene product forms a complex with p34 (cdc2) creating maturation promoting factor driving the G 2 -M transition (31). CDC2, also known as CDK1, is a direct target of R547 (13). Interestingly, although there was no change in cdc2 at the mRNA level, there was a decrease in transcription of CCNB1 both in the preclinical Affymetrix studies and in the phase I qRT-PCR analyses.…”

“…Cell Culture HCT116 (Rb + /p53 + ) and DU145 (Rb − /p53 − ) cells were maintained as described (13). MTT assays were used to determine dosing: for HCT116 cells, IC 50 Human peripheral blood mononuclear cells (PBMC) were separated from whole blood of seven healthy donors and cultured in six T75 flasks per donor at 10 7 cells per flask in 10% heat-inactivated fetal bovine serum in RPMI.…”

“…12,13). It has a strong antiproliferative effect at pharmacologically relevant doses in 19 tested cell lines regardless of tissue type, p53, multidrug resistance, or retinoblastoma status.…”

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine

“…5), (R)-roscovitine (CYC202/Seliciclib; refs. 11, 12), R547 (13,14), SNS-032 (15), PD-0332991 (16,17), AZD5438 (18,19), ZK 304709 (20,21), AG-024322, AT7519, and P276-00 (21).…”