Sulindac‐Derived Ras Pathway Inhibitors Target the Ras–Raf Interaction and Downstream Effectors in the Ras Pathway

Waldmann, Herbert; Karaguni, Ioanna‐Maria; Carpintero, Mercedes; Gourzoulidou, Eleni; Herrmann, Christian; Brockmann, Christoph; Oschkinat, Hartmut; Müller, Oliver

doi:10.1002/ange.200353089

Cited by 30 publications

(23 citation statements)

References 7 publications

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“…Although several previous studies have reported potential small-molecule association with Ras, none have characterized the binding site by a high-resolution structure analysis and none reported a defined binding pocket (3)(4)(5)(6)(7). The best-characterized Ras-binding compounds reported so far are SCH54292 (16) and its more water-soluble derivative (7).…”

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.

531

572

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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.

531

572

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“…Standard compounds were purchased from SigmaAldrich. For the synthesis of sulindac analogues, the structure of sulindac as guiding scaffold has been chemically modified by solid phase synthesis (19,28). For peptide synthesis, see refs.…”

Section: Methodsmentioning

confidence: 99%

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

Richter

Munter

Neß

et al. 2010

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

101

148

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Following ectodomain shedding by β-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by γ-secretase result in the release of amyloid-β (Aβ) peptides of variable length. Aβ peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent γ-secretase modulator (GSM), selectively reduces Aβ42 production in favor of shorter Aβ species, such as Aβ38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Aβ42 levels and concomitantly increased Aβ38 levels. However, the precise molecular mechanism by which GSMs modulate Aβ production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Aβ42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Aβ sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Aβ42-lowering activity and binding strength to the Aβ sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Aβ42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

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“…Hydrolysis of GTP to GDP is the key reaction through which RAS signaling is turned off and is the center of action for oncogenic mutations at G12, G13, and Q61 that impair both intrinsic and GAP-catalyzed hydrolysis (28). The challenge of directly targeting the RAS G-domain, thus, amounts to three approaches, all of which have been attempted with little or moderate success: increasing hydrolysis rates of mutant RAS (29,30), inhibiting the exchange of GDP for GTP (31)(32)(33)(34)(35)(36)(37)(38)(39), or interfering with effector binding and activation (40)(41)(42)(43)(44)(45)(46)(47)(48)(49). Recent efforts based on high-throughput screening of fragments to identify compounds that affect downstream signaling, accompanied by crystal structures that allowed structure-based optimization in some cases, have for the first time provided a high-resolution view of how small molecules are accommodated in binding pockets near the active site to interfere with RAS signaling (33)(34)(35)39).…”

Section: The State Of Small Molecules That Directly Bind Rasmentioning

confidence: 99%

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

Marcus

Mattos

2015

Clinical Cancer Research

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The crystal structure of RAS was first solved 25 years ago. In spite of tremendous and sustained efforts, there are still no drugs in the clinic that directly target this major driver of human cancers. Recent success in the discovery of compounds that bind RAS and inhibit signaling has fueled renewed enthusiasm, and in-depth understanding of the structure and function of RAS has opened new avenues for direct targeting. To succeed, we must focus on the molecular details of the RAS structure and understand at a high-resolution level how the oncogenic mutants impair function. Structural networks of intramolecular communication between the RAS active site and membraneinteracting regions on the G-domain are disrupted in oncogenic mutants. Although conserved across the isoforms, these networks are near hot spots of protein-ligand interactions with amino acid composition that varies among RAS proteins. These differences could have an effect on stabilization of conformational states of interest in attenuating signaling through RAS. The development of strategies to target these novel sites will add a fresh direction in the quest to conquer RAS-driven cancers.

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Sulindac‐Derived Ras Pathway Inhibitors Target the Ras–Raf Interaction and Downstream Effectors in the Ras Pathway

Abstract: In the preceding article [1] we introduced a method to synthesize a library of compounds structurally based on the nonsteroidal antiinflammatory drug (NSAID) sulindac (1,[*] Dr.

Cited by 30 publications

References 7 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

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

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