ABSTRACT
A genomics-based approach to identify pharmacodynamic biomarkers was used for a cyclin-dependent kinase inhibitory drug. R547 is a potent cyclin-dependent kinase inhibitor with a potent antiproliferative effect at pharmacologically relevant doses and is currently in phase I clinical trials. Using preclinical data derived from microarray experiments, we identified pharmacodynamic biomarkers to test in blood samples from patients in clinical trials. These candidate biomarkers were chosen based on several criteria: relevance to the mechanism of action of R547, dose responsiveness in preclinical models, and measurable expression in blood samples. We identified 26 potential biomarkers of R547 action and tested their clinical validity in patient blood samples by quantitative real-time PCR analysis. Based on the results, eight genes (FLJ44342, CD86, EGR1, MKI67, CCNB1, JUN, HEXIM1, and PFAAP5) were selected as dose-responsive pharmacodynamic biomarkers for phase II clinical trials.
Subject(s)
Biomarkers, Tumor/blood , Cyclin-Dependent Kinases/antagonists & inhibitors , Neoplasms/drug therapy , Pyrimidines/therapeutic use , Adult , Aged , Aged, 80 and over , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Neoplasms/blood , Neoplasms/enzymology , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Pyrimidines/pharmacologyABSTRACT
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 = 0.60 mumol/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.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Clinical Trials, Phase I as Topic , Cyclin-Dependent Kinases/metabolism , Female , G1 Phase/drug effects , G2 Phase/drug effects , Genes, MDR/drug effects , Humans , Mice , Mice, Nude , Phosphorylation/drug effects , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Rats, Inbred F344 , Retinoblastoma/drug therapy , Retinoblastoma/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/pharmacokinetics , Pyrimidines/pharmacology , Pyrimidines/pharmacokinetics , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Chemical Phenomena , Chemistry, Physical , Cyclin-Dependent Kinase 2/chemistry , Drug Design , Female , Humans , Indicators and Reagents , Kinetics , Mice , Mice, Nude , Models, Molecular , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.
Subject(s)
Cyclin-Dependent Kinase 4/chemistry , Animals , Caffeine/pharmacology , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Line , Cell Proliferation , Cyclin-Dependent Kinase 4/metabolism , DNA, Complementary/metabolism , Enzyme Inhibitors/pharmacology , G1 Phase , G2 Phase , HeLa Cells , Humans , Imidazoles/pharmacology , Inhibitor of Apoptosis Proteins , Inhibitory Concentration 50 , Kinetics , Microtubule-Associated Proteins/metabolism , Mitosis , Models, Chemical , Neoplasm Proteins/metabolism , Phenotype , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Pyridines/pharmacology , RNA/chemistry , RNA, Small Interfering/metabolism , Retinoblastoma Protein/metabolism , Reverse Transcriptase Polymerase Chain Reaction , S Phase , Survivin , Time Factors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4 position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives insight to their increased potency. The biochemical evaluation of a representative derivative is also described.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Cell Division/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinase 2 , DNA/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , Indoles/chemistry , Models, Molecular , Molecular Conformation , Paclitaxel/pharmacology , Structure-Activity RelationshipABSTRACT
A novel class of 3,5,6-trisubstituted naphthostyril analogues was designed and synthesized to study the structure-activity relationship for inhibition of cyclin-dependent kinase 2 (CDK2). These compounds, particularly molecules with side-chain modifications providing additional hydrogen bonding capability, were demonstrated to be potent CDK2 inhibitors with cellular activities consistent with CDK2 inhibition. These molecules inhibited tumor cell proliferation and G1-S and G2-M cell-cycle progression in vitro. The X-ray crystal structure of a 2-aminoethyleneamine derivative bound to CDK2, refined to 2.5A resolution, is presented.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Naphthalenes/chemical synthesis , Naphthalenes/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Antimetabolites , Bromodeoxyuridine , Cell Cycle/drug effects , Cell Line, Tumor , Crystallography, X-Ray , Cyclin-Dependent Kinase 2 , Drug Design , Drug Screening Assays, Antitumor , Humans , Indicators and Reagents , Models, Molecular , Molecular Conformation , Oxidation-Reduction , Tetrazolium Salts , ThiazolesABSTRACT
A series of oxindole CDK2 inhibitors was synthesized. These novel analogues have a saturated monosubstituted cyclic moiety at their C-4 position that mimics the ribofuranoside of ATP. This substitution afforded agents with increased potency relative to the parent indolinone and nanomolar range IC(50) against the CDK2 enzyme and two cancer cell lines.