Characterising the impact of tropomyosin targeting compounds on the actin cytoskeleton

Abstract

Targeting specific actin filament populations involved in tumour cell function represents a long-standing challenge in the area of anti-cancer drug development. The tropomyosin (Tm) family of actin-binding proteins, define functionally distinct actin filament populations, and as such represent attractive candidates for drug targeting. As cells transform they become increasingly reliant on a restricted repertoire of Tms, including the isoform Tm5NM1. To test the hypothesis that targeting Tm5NM1 can perturb actin-filament function, small molecules were developed using computational docking models against the C- and N-terminus of Tm5NM1. A biochemical and cell-based approach was taken to investigate the nature and specificity of the drug-target interaction. Small molecules were first screened for activity against a panel of neuroblastoma and melanoma cell lines. Several lead compounds were identified that reduced cell viability and disrupted Tm5NM1-containing actin filament bundles in a dose-dependent manner. To delineate a molecular mechanism of action, select compounds were investigated for activity in biochemical assays using recombinant human Tm5NM1. Anti-Tm compound was found to reduce the binding affinity of Tm5NM1 for filamentous actin. This effect was specific to Tm5NM1, with no respective impact on the binding affinity of muscle Tm for actin. The compounds were also found to alter Tm5NM1-regulated actin filament kinetics, resulting in a slower rate of actin polymerisation and enhanced actin filament depolymerisation. To investigate if structural changes were mediating the impact on Tm5NM1 function, N- and C-terminal peptides were employed to model the intermolecular junction between two Tm5NM1 dimers. The conformational stability of the intermolecular junction was unchanged in the presence of anti-Tm compound, which would suggest that these small molecules perturb Tm5NM1 function by interfering with the weak electrostatic interactions involved in binding actin and not by disrupting core stability. Taken together, the work presented in this thesis describes a novel class of compounds which act to modulate the function of Tm5NM1. Importantly, this data supports targeting distinct Tm isoforms to perturb actin filament function, with significant clinical implications for the treatment of cancer.