We report the discovery, synthesis, and crystallographic binding mode of novel furanopyrimidine and pyrrolopyrimidine inhibitors of the Chk1 kinase, an oncology target. These inhibitors are synthetically tractable and inhibit Chk1 by competing for its ATP site. A chronological account allows an objective comparison of modeled compound docking modes to the subsequently obtained crystal structures. The comparison provides insights regarding the interpretation of modeling results, in relationship to the multiple reasonable docking modes which may be obtained in a kinase-ATP site. The crystal structures were used to guide medicinal chemistry efforts. This led to a thorough characterization of a pair of ligand-protein complexes which differ by a single hydrogen bond. An analysis indicates that this hydrogen bond is expected to contribute a fraction of the 10-fold change in binding affinity, adding a valuable observation to the debate about the energetic role of hydrogen bonding in molecular recognition.

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Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins andChk1, 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.