ABSTRACT
Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Checkpoint Kinase 1 , Cyclin-Dependent Kinases/metabolism , DNA Damage , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Oxazoles/pharmacology , Protein Kinases/biosynthesis , Protein Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering , Thiazoles/pharmacology , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2 , Drug Design , Humans , Inhibitory Concentration 50 , Models, Molecular , Structure-Activity RelationshipABSTRACT
A simple method for the derivatization of primary amines and carboxylic acids with tris(trimethoxyphenyl)phosphonium (TMPP) reagents to enhance their detection by electrospray mass spectrometry (ESI-MS) has been developed. The synthesis of novel TMPP reagents and their stable isotopically labelled analogues is described. Through the use of stable isotopically labelled TMPP "tags", incorporation of a doublet (1:1, (1)H/(2)H or (12)C/(13)C) into the target molecule can be achieved, enabling the use of isotopic target analysis to detect compounds of unknown molecular weight but with a characteristic isotope pattern and accurate mass difference.