Development of 2-Aminoquinoline Derivatives with Improved Drug-Like Character as Small Molecule Inhibitors for a Protein-Protein Interaction Domain

Abstract

Protein-protein interactions (PPIs) involve the physical interaction between two distinct protein surfaces to form protein complexes, which propagate cell signalling pathways and control of biological function. If PPIs are dysregulated, disease often ensues; PPIs are therefore attractive therapeutic targets. Historically, PPIs have been considered “undruggable”; they generally contain few defined sites for selective inhibitor targeting, and PPI interfaces are often hydrophobic. Src Homology 3 (SH3) domains facilitate cell signalling pathways through PPIs with proline-rich regions in their native binding partners; signalling pathways controlled by SH3 domains have roles in the progression of diseases (including cancers). With over 200 proteins in the human proteome containing a SH3 domain, there is good scope for selective targeting of specific SH3 domains. The work described in this thesis contributes to a structure-based drug discovery program for SH3 domains. The small molecule 2-aminoquinoline was identified as a ligand for the murine Tec SH3 domain, and a ligand-protein binding model was proposed. Development of this ligand scaffold yielded identification of 6-position extended 2-aminoquinoline derivatives with improved binding affinity and SH3 domain selectivity over 2-aminoquinoline. However, the lead ligands were largely hydrophobic and presented limited opportunities for ligand modification. This thesis describes the development of novel small molecule ligands for the Tec SH3 domain with improved hydrophilicity (i.e., drug-like character) compared to those reported previously. As the strongest reported ligands for the Tec SH3 domain contain a benzylpiperidine substituent, the primary focus of this work investigated the synthesis (via general synthetic pathways) and binding activities of benzylpiperazinyl- and phenylamidopiperazinyl-analogues of previously reported 6- substituted 2-aminoquinoline ligands. Furthermore, the synthesis and binding activities of more complex ligands containing combinations of favourable structural features were investigated. A broad general synthetic pathway was developed for each complex ligand family, which was tailored as required for each derivative. A surface plasmon resonance (SPR) binding assay was previously used to determine ligand binding affinities for the Tec SH3 domain, however this assay derived binding affinities which were agnostic to the location of the ligand-protein binding interaction. A secondary focus of the work in this thesis involved validating the SPR binding assay by investigating the location of the measured ligandprotein binding interaction. Separately, efforts towards elucidation of the proposed ligand-protein binding model were made, but were unsuccessful. The binding affinities of all novel ligands in this work were investigated using SPR binding assays. None of the assayed ligands exhibited improved binding affinity for the Tec SH3 domain, although some benzylpiperazine-containing ligands displayed binding affinities comparable to the current lead compounds. Ligand solubilisation under biologically-relevant assay conditions was achieved in all but two cases, indicating the ligands had improved drug-like character. Importantly, the SPR binding assay was validated; it was conclusively shown to measure a binding interaction consistent with the previously proposed ligand-protein binding model. These results report a more drug-like, equipotent small molecule ligand scaffold, which can be derivatised further for identification of stronger binding SH3 domain ligands and supports the notion of targeting PPIs with small molecules generally.