DEVELOPMENT OF THERAPEUTICS TARGETING THE HEPATOCYTE GROWTH FACTOR(HGF)/MET SYSTEM

Abstract

In this dissertation I will describe the development of prototype small molecule therapeutics directed at the HGF/Met system. In addition I will introduce a new approach for quantitatively localizing Met at the sub-cellular level, which should aid in identifying physiological and pathophysiological processes that may be impacted by these new therapeutic agents. According to the National Institute of Health; cancer is responsible for nearly 25% of all deaths in United States, making it the second leading cause of death in the nation. Despite the enormity of the problem and the massive expenditures that have been made to improve treatment only modest progress has been made. Clearly there is a need for novel and effective treatment options focused on new molecular targets. One particularly attractive anti-cancer target is HGF/ Met system, which is hyperactive in many human cancers. Not-surprisingly, the development of HGF/Met blockers is currently a major focus of the pharmaceutical industry. Here I present a new therapeutic approach to block the HGF/Met system, which has intrinsic specificity, low cost, and exploits a semi-unique property of this system: the need for HGF to pre-dimerize before it can activate Met. Previous efforts by the Harding laboratory demonstrated that Norleual (an AngIV analog) exhibits anti-Met and anti-cancer activities. Homology between Norleual and the dimerization domain (Hinge region) of HGF led to the hypothesis that Norleual acts by interfering with HGF dimerization/multimerization and functions as a dominant negative Hinge-region mimic. To further validate the idea the that Norleual was acting as a Hinge region mimic, we synthesized a hexapeptide representing the Hinge sequence and established its capacity to similarly block HGF-dependent activation of Met and HGF-dependent cellular functions. Despite their impressive anti-cancer profile Norleual and the Hinge peptide are highly unstable making their direct transition to clinical use problematic. Thus another family of AngIV analogs (6AH family) has been developed with improved metabolic stability and pharmacokinetic properties, which has a 6-amniohexanoic amide substituted at the C-terminal position. Here I show that the 6AH family members demonstrate a range of biological and therapeutic activities against the HGF/Met system and that this activity is correlated to their ability to bind HGF and block its dimerization. In last part of these dissertation studies I use atomic force microscopy (AFM) to ascertain the sub-cellular localization of Met on hippocampal neurons and to distinguish between activated and non-activated receptors. I show that aggregated/active Met is localized to the dendrites where it exerts its role as a determinant of synaptic connectivity by regulating the density and structure of dendritic spines.