ABSTRACT
The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms' distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.
Subject(s)
Antineoplastic Agents/pharmacology , Lymphocytes/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Allosteric Regulation , Allosteric Site , Animals , Antineoplastic Agents/chemical synthesis , Cell Line , Drug Design , Gene Expression , HEK293 Cells , Humans , Inhibitory Concentration 50 , Lymphocytes/cytology , Lymphocytes/enzymology , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , Small Molecule Libraries/chemical synthesis , Spodoptera , Structure-Activity RelationshipABSTRACT
Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers, which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive structure-activity relationship and broke ground in the development of selective probes to delineate the isoform-specific functions of Akt kinases.
