Detection and structural characterization of ras oncoprotein-inhibitors complexes by electrospray mass spectrometry

Ganguly, Ashit K.; Pramanik, Birendra N.; Huang, Eric C.; Liberles, Stephen D.; Heimark, Larry; Liu, Y.H.; Tsarbopoulos, Anthony; Doll, Ronald J.; Taveras, Arthur G.; Remiszewski, Stacy; Snow, Mark E.; Wang, Y.S.; Vibulbhan, Bancha; Cesarz, D.; Brown, J. E.; Rosario, J. del; James, Linda; Kirschmeier, Paul T.; Girijavallabhan, V.

doi:10.1016/s0968-0896(97)00021-7

Cited by 30 publications

(17 citation statements)

References 14 publications

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“…The best-characterized Ras-binding compounds reported so far are SCH54292 (16) and its more water-soluble derivative (7). In silico calculation based on NMR mapping suggests that these compounds bind to a region between switch II and helix 4 of Ras GDP , although other binding areas cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

Maurer¹,

Garrenton²,

Oh³

et al. 2012

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

560

575

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The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.small G protein | guanine nucleotide exchange | nuclear magnetic resonance | crystal structure | small-molecule inhibitors R as is a small GTP-binding protein that functions as a nucleotide-dependent switch for central growth signaling pathways (1, 2). In response to extracellular signals, Ras is converted from a GDP-bound (Ras GDP ) to a GTP-bound (Ras GTP ) state, as catalyzed by guanine nucleotide exchange factors (GEFs), notably the SOS1 protein. Active Ras GTP mediates its diverse growth-stimulating functions through its direct interactions with effectors including Raf, PI3K, and Ral guanine nucleotide dissociation stimulator. The intrinsic GTPase activity of Ras then hydrolyzes GTP to GDP to terminate Ras signaling. The Ras GTPase activity can be further accelerated by its interactions with GTPase-activating proteins (GAPs), including the neurofibromin 1 tumor suppressor (2).Ras, a human oncogene identified and characterized over 30 y ago, is mutated in more than 20% of human cancers. Among the three Ras isoforms (K, N, and H), KRas is most frequently mutated (2). Mutant Ras has a reduced GTPase activity, which prolongs its activated conformation, thereby promoting Rasdependent signaling and cancer cell survival or growth (1, 2).Mutations of Ras in cancer are associated with poor prognosis (2). Inactivation of oncogenic Ras in mice results in tumor shrinkage. Thus, Ras is widely considered an oncology target of exceptional importance. However, development of small-molecule inhibitors against Ras has thus far proven unsuccessful. Given the picomolar affinity between guanine nucleotides and Ras and the high cytosolic concentration of guanine nucleotides, it is very challenging to develop a conventional inhibitor competitive against nucleotide binding (1, 2). Outside of the nucleotide-binding pocket, the Ras protein does not contain obvious cavities for small-molecule binding. A number of small molecules have been reported to bind to Ras (3-7), but their mechanisms of action and the structural basis to achieve Ras inhibition remain elusive.Fra...

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Section: Discussionmentioning

confidence: 99%

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

Maurer¹,

Garrenton²,

Oh³

et al. 2012

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

560

575

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“…Mass spectrometry identified the compounds in ternary complexes with Ras and GDP,and NMR analysis placed them in an area close to the nucleotide binding site. [76] To improve their water solubility and potency, derivatives were designed which can inhibit RasGRF-1-catalyzed nucleotide exchange in vitro (half-maximum inhibitory concentration (IC 50 ) = 35-80 mm). [77] Reduced cell growth in Ras-dependent cell lines as well as decreased downstream signaling were observed at high micromolar concentrations,but the exact mode of action still remains elusive and off-target effects have to be considered in certain cases.F or another compound of the SCH compound family (SCH51344 (9), Figure 5a)which was reported to modulate the Ras signaling pathway through an ovel mechanism, [78] the human mutT homologue hMTH1 was recently identified as the actual target.…”

Section: Ras Gtpasesmentioning

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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“…Since the early 1990s, when several molecular aggregates were successfully observed, 3 -10 ESI has been applied to detect various complexes. The ESI method has become increasingly important for the detection of diverse species of molecular complexes such as protein-inhibitor, 11 antibiotic binding, 12 heme-globin, 13 protein-DNA 14 and ligand-receptor. 15 However, in order to facilitate ionization, ESI is generally performed in the presence of a volatile organic solvent and acid, under conditions that are different from those in the original solution without an organic solvent.…”

Section: Introductionmentioning

confidence: 99%

Determination of micelle mass by electrospray ionization mass spectrometry

2005

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By electrospray ionization (ESI) mass spectrometry, micelle solutions of sodium cholate were investigated in detail in the presence and absence of ethanol. The average aggregation number could be evaluated from the spectra acquired under conditions where soft collisions adequate to measure the micelle solution were induced, and the value agreed well with that obtained previously by other methods. From the dependence on ethanol content, it was also found that the average aggregation number in aqueous solution without organic solvent could be reliably estimated. The ESI method proved to be a useful tool for determining the micelle mass in the original aqueous phase.

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Detection and structural characterization of ras oncoprotein-inhibitors complexes by electrospray mass spectrometry

Cited by 30 publications

References 14 publications

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

Direct Modulation of Small GTPase Activity and Function

Determination of micelle mass by electrospray ionization mass spectrometry

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