Metal-Bis(2-picoryl)amihe Complexes as State 1(T) Inhibitors of Activated Ras Protein

摘要

The small guanine nucleotide binding (GNB) protein Ras is a central component in cellular signaling networks controlling proliferation, differentiation, and apoptosis. Alternating between an inactive GDP- and an active GTP-bound form, Ras acts as molecular switch, which is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (see, for example, Ref. ). Ras with GTP bound strongly interacts with effectors such as Raf kinase, RalGDS, and PI3 kinase that transmit the incoming activation signal to downstream targets. ~(31)P NMR spectroscopy was used to identify two distinct conformational states in Ras when a nucleoside triphosphate (T) is bound, called state 1(T) and state 2(T). These states were first defined by the spectroscopic properties: in state 1(T) the ~(31)P NMR resonance lines of the a- and γ-phosphate groups are shifted downfield relative to those corresponding to state 2(T). These states are in dynamic equilibrium, with exchange rates on the millisecond timescale at room temperature. Ras in state 1(T) has a more than 2 orders of magnitude lower affinity for effectors than Ras in state 2(T). The affinity of the two states for Raf was estimated as 7 μm and 0.012 μm, respectively, at 283 K. In contrast, the guanine nucleotide exchange factor Sos binds preferentially to state 1(T). State 2(T) becomes stabilized when Ras is complexed to the Ras-binding domains (RBDs) of its effectors such as Raf kinase, RalGDS, AF-6, and Byr2, and is consequently also referred to as the effector-binding state. For wild-type (wt) H-Ras(l-166) the equilibrium constant K_(12) for the two states is 11.3 in the presence of its natural ligand GTP.Partial loss-of-function mutants, such as Ras(T35A) and Ras(T35S), complexed to Mg~(2+)-GppNHp (GppNHp = guanosine-5'-(β,γ-imi-no)triphosphate) activate only a subset of the known Ras effectors and predominantly exist in the weak effector-binding conformational state l(T). In cancer research Ras represents a target of high interest owing to its critical involvement in about 30 of all human malignancies. Oncogenic Ras cannot be switched off by either by its intrinsic GTPase activity or by GAPs, leading to uncontrolled cell growth and thus to tumor formation.