Over 60 Rab GTPases serve as regulators of individual membrane transport steps and a growing subset underlie human diseases from neurologic disorders to cancer. Rab7 and its interacting partners regulate early to late endocytic membrane transport and play pivotal roles in intracellular signaling, growth control, autophagy and macromolecular degradation. Rab7 or late endocytic trafficking defects have been demonstrated to underlie Charcot-Marie-Tooth type2B disease, thyroid adenoma, Niemann-Pick type C disease, Alzheimer's disease and Down's syndrome. To further dissect the functions of Rab7 in normal and diseased cell physiology, we are interested in identifying small molecule probes that will enable the acute and specific manipulation of Rab7 activity. It is our hypothesis that small molecules that alter protein-protein interactions will target unique sites on the surface of the GTPase and thereby provide probes that can specifically modulate the function of a GTPase of interest. A previous high throughput screen (HTS) based on a GTP-binding assay was conducted in collaboration with the New Mexico Molecular Libraries Screening Center, giving us relevant experience. Thus, we identified a novel class of small molecules that non-selectively activates GTP-binding on small Ras-like GTPases by binding to an allosteric site. New classes of inhibitors were found that are Rho family selective and Cdc42 specific and likely act competitively by binding in the nucleotide pocket. However, we did not identify any probes that were selective or specific for Rab GTPases using GTP-binding as our screen. The importance of Rab GTPases in disease prompts us to pursue this important goal by screening directly for small molecules that alter protein-protein interactions between the endosomal Rab7 GTPase and its interacting partners as a test case. Our laboratory first characterized the function of Rab7 and several interacting partners, has established biochemical assays for monitoring protein-protein interactions and unique experience in adapting assays for flow cytometry and high throughput screens. During year one we will adapt our protein-protein interaction assays for quantitative measurement by flow cytometry. We will work closely with the New Mexico Molecular Libraries Specialty Center to ensure suitability of assays to microtiter plate, small volume format to allow implementation of a multiplex screen in year two. Small molecules that selectively affect Rab7 regulatory or effector protein interactions are expected to provide specific probes for altering Rab7 activity and likely to have downstream applications in a number of disease models. Success of the approach will also provide a paradigm for targeting other small GTPase targets.
The Rab GTPases represent novel and as yet under-explored therapeutic targets for the treatment of cancer, and Alzheimer's disease, as well as for hereditary disorders affecting vision, immune and nerve cell function. The proposed work seeks to analyze a library of over 300,000 chemicals to identify those that modulate the associations between the Rab7 GTPase and its partner proteins. Chemicals identified by the proposed survey may be used to control cell growth and fate and ultimately offer new therapeutic potential.
