Kinases function as molecular routers in the transmission of signaling events from the cell surface (e.g. the binding of a hormone or cytokine) to the cellular response in the nucleus (e.g. transcription). One kinase, Akt1, serves as a node in multiple downstream signaling pathways and has been shown to be overexpressed and/or hyperactivated in a wide variety of cancers. Ligands which bind to this kinase and its alternate activation states would enable the development of diagnostic platforms for the study and early detection of cancer. We propose to design a series of peptide-based bi-ligands that recognize the phosphorylation states of Akt with high affinity. By combining chemical one-bead, one-compound peptide libraries and in situ click chemistry with ATP analogs, we can rapidly generate a series of bi-ligands that recognize each of the common phosphorylation states of Akt1 and its isoforms. These ligands will be integrated into microarrays and used to quantitate the concentration and activation states of Akt in complex biological samples. To begin, small libraries of azide-modified Akt1 inhibitor peptides will be screened against fully active Akt1 in the presence of a second library of alkyne-modified ATP-mimics. The Akt1 target will direct the conjugation of the optimal peptide and small molecule to form a high-affinity triazole-linked bi-ligand. Following characterization of the """"""""hit"""""""" molecules, the phosphorylation state specificity and affinity of the lead compounds will be increased by re-screening with larger and more diverse peptide libraries. The final round of ligand maturation will employ in situ click chemistry in conjunction with a second library of alkyne-bearing small molecules to select multi-functionalized ligands with improved affinity and biostability. Bi-ligands against each of the phosphorylation states of Akt1, Akt2, and Akt3 will be generated in a similar manner. Following optimization, the affinity capture agents will be immobilized in a microarray and used to measure the concentrations of each phosphorylation state of Akt1, Akt2, and Akt3 in mammalian cell extracts. Increased Akt expression and/or activity has been observed in ovarian, breast, thyroid, prostate, lung, and colon cancers. The ligands generated in this study will be combined into an inexpensive, high-throughput microarray device to map the changes in the activation state of Akt1 in normal, pre-cancer, and cancer tissues, The ability to rapidly determine the concentration and activation state of Akt isoforms in tissue samples would be a major step toward the early diagnosis of many cancers and the determination of optimal therapeutic regimes for cancer patients.
| Millward, Steven W; Henning, Ryan K; Kwong, Gabriel A et al. (2011) Iterative in situ click chemistry assembles a branched capture agent and allosteric inhibitor for Akt1. J Am Chem Soc 133:18280-8 |
