A novel mechanism for the modulation of the Ras-effector interaction by small molecules

Spoerner, Michael; Graf, Thorsten; König, Burkhard; Kalbitzer, Hans Robert

doi:10.1016/j.bbrc.2005.06.144

Cited by 49 publications

(52 citation statements)

References 17 publications

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“…[140] In conformational state 1, Ras reveals unique surface pockets, which can be exploited for allosteric stabilization to shift the equilibrium towards the low affinity conformation and impair downstream signaling.B oth Zn 2+ -a nd Cu 2+ -cyclen complexes bind H-Ras-"GTP" in its state 1c onformation with millimolar affinity (Figure 11). [141,142] 31 PNMR shift analysis suggests two distinct binding sites,o ne with the metal ion coordinating the g-phosphate of the bound nucleotide,a nd the other located close to the C-terminus and loop 7. However,b inding site 2s eems to have al ow impact on the stabilization of the state 1conformation.…”

Section: Allosteric Inhibitorsmentioning

confidence: 99%

Direct Modulation of Small GTPase Activity and Function

et al. 2015

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Small GTPases are a family of GDP-/GTP-binding proteins that serve as biomolecular switches inside cells to control a variety of essential cellular processes. Aberrant function and regulation of small GTPases is associated with a variety of human diseases, thus rendering these proteins highly interesting targets in drug discovery. However, this class of proteins has been considered "undruggable", as intensive decade-long efforts did not yield clinically relevant direct modulators of small GTPases. Recently, the targeting of small GTPases has gained fresh impetus through the discovery of novel transient cavities on the protein surfaces and the application of new targeting strategies. Besides Ras proteins, other small GTPases have attracted increased attention since improved biological insight in combination with novel targeting strategies identified them as promising targets in drug discovery. This Review gives an overview of relevant aspects of the superfamily of small GTPases and summarizes recent progress and perspectives for the direct modulation of these challenging targets.

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Section: Allosteric Inhibitorsmentioning

confidence: 99%

Direct Modulation of Small GTPase Activity and Function

et al. 2015

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“…A small molecule that is able to shift the equilibrium towards Ras off might work in a molecular cancer therapy. [42] Our assay can screen for such small molecules. The approach can be A C H T U N G T R E N N U N G applied to other GTPases and ATPases.…”

Section: Ftir Assay For the Switchmentioning

confidence: 99%

Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

et al. 2007

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Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP-to-GTP exchange. This modifies the Ras surface (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to "off" (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)-to-Ras(on) transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time-resolved FTIR (trFTIR) spectroscopy. In the transition from Ras(off) to Ras(on) a GTP-bound intermediate is now identified, in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP was directly monitored by the C=O vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s(-1) at 260 K. A small molecule that shifted the equilibrium from the Ras(on)GTP state towards the Ras(off)GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.

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“…Binding studies using NMR spectroscopy as well as calorimetric or fluorescence-based methods show that these conformational equilibria directly influence the interaction between Ras and its effectors (3,10,11). Stabilizing state 1 by small ligands is a novel approach to reduce the affinity of Ras for its effectors (12,13). Very recent results lead to the assumption that state 1(T) of Ras is recognized by guanine nucleotide exchange factors and thus could represent an important conformational state in the Ras activation/inactivation cycle (14).…”

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confidence: 99%

Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis

Spoerner

Hozsa

Poetzl

et al. 2010

Journal of Biological Chemistry

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129

187

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The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH 2 p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer 31 P NMR experiments and effector binding studies we show that Ras(wt)⅐Mg 2؉ ⅐GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K 12 is 11.3. K 12 of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k ex ) for Ras(wt)⅐Mg 2؉ ⅐GTP is 7 s ؊1 and thus 18-fold lower compared with that found for the Ras⅐GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/ C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.The guanine nucleotide-binding protein Ras is involved in cellular signal transduction pathways inducing proliferation, differentiation, or apoptosis of cells. It functions as a molecular switch, cycling between an inactive GDP-bound state and an active GTP-bound state. In the active conformation Ras is able to bind different effector proteins such as Raf kinase or RalGDS with nanomolar affinity by interacting with its switch I region. Thus signals can be transmitted resulting in the corresponding cellular response.When bound to guanosine triphosphates Ras exists in two different conformational states, defined as states 1 and 2, which are in chemical equilibrium in solution (1-3). These conformational states are actually only defined for Ras with guanosine triphosphate bound (T), a state that may be different from a state with guanine diphosphate bound (D). Therefore, we will denote the two states in the following as state 1(T) and 2(T) whenever the nucleotide ligand is of concern. The equilibrium between the two states is strongly influenced by the nature of the guanine nucleotide bound to Ras and can be shifted by interaction with effector proteins or regulators such as GTPase activation proteins (GAPs).2 It was found that GTP analogs GppNHp and GppCH 2 p partially shift the equilibrium toward state 1. For the complex of wild-type Ras with physiological GTP itself the existence of the two states could not be...

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A novel mechanism for the modulation of the Ras-effector interaction by small molecules

Cited by 49 publications

References 17 publications

Direct Modulation of Small GTPase Activity and Function

Direct Modulation of Small GTPase Activity and Function

Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis

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