ABSTRACT
This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.
Subject(s)
Adenosine Triphosphate/physiology , Antineoplastic Agents/chemical synthesis , Imidazoles/chemical synthesis , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyridines/chemical synthesis , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Catalytic Domain , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Mice , Microsomes, Liver/metabolism , Models, Molecular , Phosphorylation , Protein Binding , Protein Conformation , Pyridines/chemistry , Pyridines/pharmacology , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.
