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Published on: November 11, 2018
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.
Insights
Zn2+-cyclen inhibits Ras signaling by stabilizing a weak effector-binding state. This allosteric inhibitor modulates Ras conformational dynamics, enhancing its anti-cancer potential.
Area of Science:
- Molecular biology
- Cell signaling
- Biochemistry
Background:
- Ras GTPases are crucial regulators of cell signaling, controlling proliferation, differentiation, and apoptosis.
- Ras functions as a molecular switch, active in its GTP-bound form, interacting with effectors like Raf-kinase.
- Ras exists in distinct conformational states (1(T) and 2(T)) influencing its interactions and function.
Purpose of the Study:
- To investigate the effect of Zn2+-cyclen on the conformational dynamics of active K-Ras4B.
- To elucidate the mechanism of action of Zn2+-cyclen as an allosteric inhibitor of Ras.
- To assess the potency and therapeutic potential of Zn2+-cyclen in modulating Ras signaling.
Main Methods:
- Utilized titration experiments to study the interaction between Zn2+-cyclen and Ras.
- Analyzed Ras conformational states and dynamics using biophysical techniques.
- Investigated the formation of specific Ras substates (1(T)* and 2(T)*) upon inhibitor binding.
Main Results:
- Zn2+-cyclen modulates the dynamic equilibrium of active K-Ras4B, similar to H-Ras.
- Cooperative binding of Zn2+-cyclen to Ras was observed, involving two identified interaction sites.
- A novel substate 1(T)* was detected in the Ras(T35A) mutant, analogous to the effector-induced 2(T)* substate.
- Zn2+-cyclen shifts the equilibrium towards the weak effector-binding state 1(T) and perturbs 1(T)* formation, enhancing inhibition.
- Despite millimolar affinity, Zn2+-cyclen exhibits micromolar potency, comparable to competitive state 2 inhibitors.
Conclusions:
- Zn2+-cyclen acts as an allosteric inhibitor by stabilizing the weak effector-binding state 1(T) of Ras.
- The inhibitor perturbs Ras conformational substates, leading to enhanced inhibitory effects.
- Zn2+-cyclen demonstrates significant potency, highlighting its potential as a therapeutic agent targeting Ras-driven cancers.
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