Structural basis for the interaction of Ras with RaIGDS

Huang, Li-Shar; Hofer, Franz; Martin, G S; Kim, S H

doi:10.1038/nsb0698-422

Cited by 238 publications

(283 citation statements)

References 30 publications

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“…Calculations were done under the same ionic strength as the relevant measurements. Coordinates for calculations of the RalGDS-RBD͞Ras complex and for the individual proteins were taken from its x-ray structure (19). All mutations were modeled by using SWISS PDB VIEWER (20).…”

Section: Methodsmentioning

confidence: 99%

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex

Kiel

Selzer

Shaul

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

104

128

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Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by ⌽ analysis of the encounter complex and transition state of the RalGDS M26K,D47K,E54K ͞Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

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Section: Methodsmentioning

confidence: 99%

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex

Kiel

Selzer

Shaul

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

104

128

View full text Add to dashboard Cite

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“…S8), suggesting sharing of a common binding pocket with H-Ras T35S ·GppNHp. Superimposition of the NMR structure of the H-Ras T35S -Kobe2601 complex with the crystal structures of various Raseffector complexes (15)(16)(17) revealed that Kobe2601 overlapped with the effector-binding interfaces (Fig. S7 B-E).…”

Section: Molecular Basis For Interaction Of Ras·gtp With the Kobe0065mentioning

confidence: 99%

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

Shima

Yoshikawa

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

279

271

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Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras–type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H- ras G12V –transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras G12V gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP T35S , exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

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“…Interestingly, it was observed that NSCLC cell lines carrying the KRAS G12C or G12V mutation have higher RAL GTPase activities and lower PI3K/ AKT or RAF-MEK-ERK pathway activation compared with cell lines carrying other KRAS mutations (Figure 3) or wildtype KRAS [20] . It is known that KRAS homodimer formation is important for RAL GTPase activation via RALGDS binding to KRAS [75] . Ihle et al used extensive molecular modeling to show that in case of the KRAS G12C mutation, the smaller cysteine residue allows for KRAS homodimer formation, RALGDS binding and subsequent RAL activation [20] .…”

Section: Dependency Of Ral Activity On Ras Mutationmentioning

confidence: 99%

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer

Guin

Theodorescu

2015

Acta Pharmacol Sin

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Activating RAS mutations are common in human tumors. These mutations are often markers for resistance to therapy and subsequent poor prognosis. So far, targeting the RAF-MEK-ERK and PI3K-AKT signaling pathways downstream of RAS is the only promising approach in the treatment of cancer patients harboring RAS mutations. RAL GTPase, another downstream effector of RAS, is also considered as a therapeutic option for the treatment of RAS-mutant cancers. The RAL GTPase family comprises RALA and RALB, which can have either divergent or similar functions in different tumor models. Recent studies on non-small cell lung cancer (NSCLC) have showed that different RAS mutations selectively activate specific effector pathways. This observation requires broader validation in other tumor tissue types, but if true, will provide a new approach to the treatment of RAS-mutant cancer patients by targeting specific downstream RAS effectors according to the type of RAS mutation. It also suggests that RAL GTPase inhibition will be an important treatment strategy for tumors harboring RAS glycine to cysteine (G12C) or glycien to valine (G12V) mutations, which are commonly found in NSCLC and pancreatic cancer. Review RAS GTPase overviewRAS is the most extensively studied GTPase [1] . It is known to regulate various cellular functions, including proliferation, survival, growth, migration, differentiation and cytoskeletal dynamics [2][3][4] (Figure 1). Not coincidentally, increased activities of all of these processes are required by tumor cells to promote tumor growth. Activating oncogenic RAS mutations lead to treatment resistance in various tumor models and poor patient outcomes [3,[5][6][7][8][9] . Three human RAS genes have been identified: HRAS, KRAS, and NRAS [1,2] . These encode the related proteins HRAS, KRAS and NRAS, respectively, of 189 amino acids in length [1,4] . KRAS exists as two isoforms, 4A and 4B, which are generated by alternative exon splicing [10] . These different RAS genes are well known to have differential cellular specificities and intrinsic transforming potentials [3,4,10] .In normal cells, RAS proteins act as molecular switches for critical cellular functions. RAS proteins are activated by upstream receptors such as receptor tyrosine kinases * To whom correspondence should be addressed. E-mail dan.theodorescu@ucdenver.edu Received 2014-08-07 Accepted 2014-10-30 Figure 1. RAS GTPase activation and downstream signaling. The cartoon shows the mechanism of RAS GTPase activation by upstream stimuli and numerous downstream effectors stimulated by activated RAS. RAS regulates critical cellular functions through these effectors. Constitutive RAS activation due to mutations leads to protumorigenic signaling through these effectors.

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Structural basis for the interaction of Ras with RaIGDS

Cited by 238 publications

References 30 publications

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer

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