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A Novel Affinity Reagent To RAS Perturbs Its Function In Cells

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Title: A Novel Affinity Reagent To RAS Perturbs Its Function In Cells
Author(s): Smith, Russell S
Advisor(s): O'Bryan, John P
Contributor(s): McLachlan, Alan; Karginov, Andrei; Tyner, Angela L; Grippo, Paul J; O'Bryan, John P
Department / Program: Pharmacology
Degree Granting Institution: University of Illinois at Chicago
Degree: PhD, Doctor of Philosophy
Genre: Doctoral
Subject(s): NS1 monobody H-RAS K-RAS RAS chimera RAS inhibitor RAS dimerization RAS nanoclustering
Abstract: RAS GTPases are mutated to the constitutively active state in around 30% of human cancers. These mutations lock RAS in a GTP-bound ‘active’ state and lead to excessive and inappropriate activation of the pro-proliferative MEK/ERK pathway and pro-survival AKT pathway, both of which are markers of poor prognosis in human cancer. This has made discovery of RAS inhibitors a top priority of cancer research. However, due to its picomolar affinity for GTP and lack of deep binding pockets on its surface, RAS inhibitors have proven elusive, with previous attempts to isolate direct inhibitors of RAS falling short of the mark. Thus there are currently no RAS-inhibitors available in the clinic. Here I describe a monobody affinity reagent that binds to H-RAS and K-RAS, but not N-RAS, with nanomolar affinity resulting in potent inhibition of RAS-driven signaling, oncogenic transformation and tumor growth. NS1 primarily elicits these effects by binding to the α4-β6-α5 interface of RAS, thereby preventing RAS dimerization at the plasma membrane. This in turn blocks the ability of RAS to promote dimerization of RAF, a requirement for RAF activation. However, NS1 elicits isoform-specific effects on RAS in addition to blocking RAS dimerization. For example, NS1 reduces K-RAS association with the plasma membrane and interaction with RAF. Neither of these effects were observed with the H-RAS. Finally, NS1 also reduces the pool of wild type RAS that is GTP-loaded, without blocking nucleotide cycling in vitro. Structural studies suggest that these additional inhibitory effects are the result of NS1 protruding towards to the plasma membrane and altering the orientation or interaction of RAS with the plasma membrane. This work demonstrates the utility of targeting the α4-β6-α5 interface for blocking RAS function and outlines a promising region of RAS to target with therapeutics.
Issue Date: 2017-05-05
Type: Thesis
URI: http://hdl.handle.net/10027/21928
Date Available in INDIGO: 2017-10-31
Date Deposited: August 201
 

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