Developing selective inhibitors of P70S6K from an understanding of the molecular determinants of intra-family AGC kinase inhibitor selectivity using PKA chimeric protein. (360G-Wellcome-090174_Z_09_Z)

The aim of the project is to construct, crystallise and determine the structures of a suite of PKAbased chimeric proteins: PKA, P70S6K-PKA, PKB-PKA, ROCKII-PKA with minimal changes to the PKA sequence in the ATP-binding site (3-5 residue) to make the chimeras representative of the non-PKA component, a strategy already proved successful for PKB-PKA [4,5] and ROCKPKA [6]. We will particularly focus on establishing a novel P70S6K-PKA chimera. We will establish back-soaking protocols for ligand binding to enable high-throughput determination of ligand-protein crystal structures. We will select a representative set of potential P70S6K inhibitors with varying chemotypes and kinase selectivities based on compounds from our own in-house projects, and from published compounds with AGC kinase activity, and will characterise the selectivity profiles using Caliper ProfilerPro assays recently established in the Centre. The crystallographic binding modes of suitable inhibitors in the different AGC chimeras, and wild type protein where available, will be determined and compared. Correlation of the key interactions and binding poses will establish the molecular determinants of small molecule selectivity for P70S6K against other AGC kinases.