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

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.

Metal-bis(2-picolyl)amine complexes as state†1(T) inhibitors of activated Ras protein.

Allosteric interactions: Metal(II) cyclens inhibit Ras-effector interactions by stabilizing a weak effector-binding state of Ras, state 1(T), and binding directly in the active site. The novel state (1T) inhibitor Zn(2+)-BPA (BPA = bis(2-picolyl)amine) binds outside the nucleotide binding pocket but nevertheless allosterically stabilizes state 1(T) and thus inhibits the Ras-Raf interaction.

Cu2+-cyclen as probe to identify conformational states in guanine nucleotide binding proteins.

(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran.

Time-resolved fluorescence-based assay for the determination of alkaline phosphatase activity and application to the screening of its inhibitors.

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu(3)TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu(3)TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 micromol L(-1). Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 micromol L(- 1) of theophylline.