title: 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.forename: Ina C
authors.initials: IC
authors.lastname: Rosnizeck
authors.forename: Daniel
authors.initials: D
authors.lastname: Filchtinski
authors.forename: Rui Pedro
authors.initials: RP
authors.lastname: Lopes
authors.forename: Bärbel
authors.initials: B
authors.lastname: Kieninger
authors.forename: Christian
authors.initials: C
authors.lastname: Herrmann
authors.forename: Hans Robert
authors.initials: HR
authors.lastname: Kalbitzer
authors.forename: Michael
authors.initials: M
authors.lastname: Spoerner
mesh_terms: D056831:Coordination Complexes / Q000494:pharmacology*; D000080027:Cyclams; D006160:Guanosine Triphosphate / Q000378:metabolism; D006571:Heterocyclic Compounds / Q000494:pharmacology; D006573:Heterocyclic Compounds, 1-Ring / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D048490:raf Kinases / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D056831:Coordination Complexes; D000080027:Cyclams; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D008024:Ligands; D006160:Guanosine Triphosphate; C038072:cyclen; D048490:raf Kinases; D018631:ras Proteins