Annette Walter

The discovery of tetrahydro-β-carbolines as inhibitors of the kinesin Eg5

A series of tetrahydro-beta-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of removing potential metabolic liabilities and improving cellular potency.

4-(1H-indazol-5-yl)-6-phenylpyrimidin-2(1H)-one analogs as potent CDC7 inhibitors.

A series of 4-(4-hydroxyphenyl)-6-phenylpyrimidin-2(1H)-ones were identified by HTS as inhibitors of CDC7. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus on removing potential metabolic liabilities and improving cellular potency.

Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read‐outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read‐outs for other potential kinase cancer targets. J. Cell. Biochem. 104: 1075–1086, 2008.

3D Pharmacophore Model-Assisted Discovery of Novel CDC7 Inhibitors

A ligand-based 3D pharmacophore model for serine/threonine kinase CDC7 inhibition was created and successfully applied in the discovery of novel 2-(heteroaryl)-6,7-dihydrothieno[3,2-c]pyridin-4(5H)-ones. The pharmacophore model provided a hypothesis for lead generation missed by docking to a homology model. Medicinal chemistry exploration of the series revealed clear structure-activity relationships consistent with the pharmacophore model and pointed to further optimization opportunities.