Development of chemical induced molecular traps for time resolved, in vivo studie

Beckman Research Institute/City Of Hope, Duarte CA

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Abstract

DESCRIPTION (provided by applicant): The ability to rapidly and selectively antagonize specific proteins and/or molecular complexes is essential to differentiate the underlying cellular processes active in healthy and diseased tissues. This type of control remains elusive for over 90% of the known genome. In part, this stems from the fact that most macromolecular interactions are spread over large interfaces and do not present sufficient cavities necessary to develop a high affinity, small molecular antagonist. On the other hand, distributing multiple weak interactions over a large interface can lead to a high apparent affinity and exquisite specificity, and appears to be a prevalent operational feature in biological systems (e.g., antigen-antibody interactions). In recognition of this principle, we hypothesized and developed a novel approach using bivalent peptide-based reagents that can be regulated by the addition of a small molecule to either block a macromolecular interface or strip an individual component from a designated complex. In analogy to the VEGF trap, we have coined these inducible molecular traps. Using these inducible molecular traps, we have shown for the first time that the dynein light chains act as allosteric regulators of the dynein motor complex. These reagents also provide the first insight to the timescales of dynein mediate processes (e.g., organelle transport). Such information enabled by this novel methodology is not currently available with other techniques (e.g., siRNA and microinjection). Based on strong preliminary data, we propose to extend this methodology by 1) developing a photocleavable analog to permit rapid dissociation of the molecular traps and 2) developing random inducible dimeric libraries for forward genetic screens against a designated target. The successful development of these reagents and the demonstration of their utility will lead to multiple applications in biology and therapeutic development of neurological and other disorders. Moreover, combining these reagents with specific promoters will permit reversible trapping in a tissue specific manner and provide molecular information within a whole organism. PUBLIC HEALTH RELEVANCE: While the completion of the human genome, rapid SNP analyses, and new imaging and computational techniques has lead to novel and significant insight to neurological and other pathogenic processes, there remains a significant gap in our ability to selectively target molecules of interest and interrogate their action in complex and entangled processes. Without this control, it is difficult to discern how point mutations in these molecules lead to disease. Herein, we propose a novel method to select and generate inducible, bivalent inhibitors that afford small molecule-like properties as well as a new reagent to afford spatial and temporal control of the activity of selected targets. The successful development of the reagents emanating from this proposal will provide researchers new tools that will ultimately lead to novel insight to the mechanism of disease under study.