Elucidating the mode of action of a typical Ras state 1(T) inhibitor.

Abstract

The small GTPase Ras is an essential component of signal transduction pathways within the cell, controlling proliferation, differentiation, and apoptosis. Only in the GTP-bound form does Ras interact strongly with effector molecules such as Raf-kinase, thus acting as a molecular switch. In the GTP-bound form, Ras exists in a dynamic equilibrium between at least two distinct conformational states, 1(T) and 2(T), offering different functional properties of the protein. Zn2+-cyclen is a typical state 1(T) inhibitor; i.e., it interacts selectively with Ras in conformational state 1(T), a weak effector binding state. Here we report that active K-Ras4B, which is prominently found to be mutated in human tumors, exhibits a dynamic equilibrium like H-Ras, which can be modulated by Zn2+-cyclen. The titration experiments of Ras with Zn2+-cyclen indicate a cooperatively coupled binding of the ligands to the two interaction sites on Ras that could be identified for H-Ras previously. Our data further indicate that as in state 2(T) where induced fit produces the substate 2(T)* after effector binding, a corresponding substate 1(T)* can be detected at the state 1(T) mutant Ras(T35A). The interaction of Zn2+-cyclen with Ras not only shifts the equilibrium toward the weak effector binding state 1(T) but also perturbs the formation of substate 1(T)*, thus enhancing the inhibitory effect. Although Zn2+-cyclen shows an affinity for Ras in only the millimolar range, its potency of inhibition corresponds to a competitive state 2 inhibitor with micromolar binding affinity. Thus, the results demonstrate the mode of action and potency of this class of allosteric Ras inhibitors.

• Authors: Ina C Rosnizeck, Daniel Filchtinski, Rui Pedro Lopes, Bärbel Kieninger, Christian Herrmann, Hans Robert Kalbitzer, Michael Spoerner
• Journal: Biochemistry (Biochemistry) Vol. 53 Issue 24 Pg. 3867-78 (Jun 24 2014) ISSN: 1520-4995 [Electronic] United States
• PMID: 24866928 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Coordination Complexes
• Cyclams
• Heterocyclic Compounds
• Heterocyclic Compounds, 1-Ring
• Ligands
• Guanosine Triphosphate
• cyclen
• raf Kinases
• ras Proteins

Topics

• Coordination Complexes (pharmacology)
• Cyclams
• Guanosine Triphosphate (metabolism)
• Heterocyclic Compounds (pharmacology)
• Heterocyclic Compounds, 1-Ring (pharmacology)
• Humans
• Ligands
• Models, Molecular
• Nuclear Magnetic Resonance, Biomolecular
• Protein Binding
• Protein Conformation
• raf Kinases (metabolism)
• ras Proteins (antagonists & inhibitors, chemistry, genetics, metabolism)