The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity

Abstract

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent.

Fig. 1

Computational analyses for design of CDK7 Inhibitors. (A) AMSOL solvation energies of six core motifs based on roscovitine (ΔG_solv, kcal/mol). R¹, R² and R³ are the same as those in roscovitine. (B) Lowest energy Glide XP pose for pyrazolopyrimidine 1. The orange space-filling Phe91 is the gatekeeper residue. Hydrogen bonds with Met94 and Asn141 are displayed as dashed red lines. (C) Glide XP pose for BS-181 with hydrogen bonds to Glu20, Met94 and the Thr170 phosphate. (D, E) Gatekeeper residue packing for CDK7 (D) and CDK2 (E) is shown. The packing is tighter for CDK7 relative to CDK2 asmeasured by surface exposure of the hydrophobic aggregation of Lys41 (blue) and Phe91 (green), 161 and 165 Ų, respectively.

Fig. 2

BS-181 inhibits phosphorylation of CDK7 substrates. Whole cell lysates were prepared from MCF-7 cells treated with BS-181 or Roscovitine for four hours, at the concentrations shown. (A) Immunoblotting was carried out using antibodies for RNA polymerase II, or Pol II phosphorylated at Ser2 or Ser5 in the C-terminal domain. The concentration at which apparent inhibition of PolII phosphorylation by 50% would be achieved was determined following densitometry of immunoblots from three experiments. (B-C) Immunoblotting was carried out as in part A, using the antibodies as labelled.

Fig 3

BS-181 treatment of MCF-7 cells leads to G1 arrest at and apoptosis. (A) MCF-7 cells were treated with BS-181 at the concentrations shown, or with vehicle (DMSO) for 24 hours, prior to fixation, staining with propidium iodide (PI) and flow cytometric analysis. The percentage of cells in the sub-G1 (apoptosis), G1, S-phase and G2/M, as determined from 3 independent experiments, are shown. Error bars represent the standard errors of the mean (SEM). (B) Cells treated with DMSO or BS-181 were stained with an antibody for Annexin V and with PI. The percentage of cells that stained positive for Annexin V following the addition of BS-181 or roscovitine are shown for three independent experiments. Error bars represent the standard errors of the mean (SEM). P-values were determined using Student’s t-test, in which comparison between the DMSO control and BS-181 treatment at each concentration was carried out. Asterisks (*) denote p < 0.05, whilst p < 0.005 is denoted by the symbol ¥.

Fig. 4

BS-181 inhibits the growth of MCF-7 tumors in nude mice. Randomized MCF-7 tumour bearing mice were injected intraperitoneally twice daily with 5mg/kg or 10 mg/kg BS-181, giving a total daily dose of 10 mg/kg/day or 20 mg/kg/day, respectively, over a period of 14 days. Mouse weights were determined daily, tumour volumes being measured every 2 days. (A) The change in tumour volume was determined for each animal, as tumour volume relative to the tumour volume of each animal at day 1. The line graphs show the mean tumour volumes for the animals in each treatment group. Error bars represent the standard error of the mean. Asterisks depict the statistical significance of the differences between the control group and each of the BS-181 treatment groups, carried out using Student’s t-test. (B) Shown are the animal weights, as percentage change relative to the animal weights at day 1. Error bars represent the standard error of the mean.