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Cited by 150Protein Kinase Regulation and GTPase SignalingComputational Drug Discovery MethodsProtein Tyrosine Phosphatases

Discovery and characterization of small molecules that target the GTPase Ral

Open access
Yan, Chao, Liu, Degang, Li, Liwei, Wempe, Michael F., Guin, Sunny, Khanna, May, Meier, Jeremy, Hoffman, Brenton +12 more·2014·Nature
DOI 10.1038/nature13713PMC PMC4351747
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Abstract

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and 1H–15N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

AI snapshot

Written from the paper’s full text.

TL;DR

Structure-based virtual screening identified small molecules that bind the GDP-bound form of Ral GTPases and inhibit Ral signaling, cell growth, and xenograft tumor growth. RBC8 and its derivative BQU57 act selectively through RalA/RalB and show efficacy in Ral-dependent lung cancer models.

Key findings

  • Docking 500,000 ChemDiv compounds to an allosteric site on RalA-GDP yielded 88 candidates; RBC6, RBC8, and RBC10 inhibited RalA activation in cells, Ral-dependent MEF spreading, and anchorage-independent growth, while RBC5, RBC7, and RBC42 were inactive controls.
  • BQU57 bound RalB-GDP with KD = 7.7 ± 0.6 μM by ITC and KD = 4.7 ± 1.5 μM by SPR; RBC8 and BQU57 showed selectivity for Ral over Ras and RhoA and did not bind RalB-GNP.
  • In soft agar, RBC8 IC50 was 3.5 μM in H2122 and 3.4 μM in H358; BQU57 IC50 was 2.0 μM in H2122 and 1.3 μM in H358. Ral-dependent lines H2122 and H358 were sensitive, whereas H460 and Calu6 were not.
  • In H2122 xenografts, RBC8 at 50 mg/kg/d for 21 days inhibited tumor growth similarly to dual RalA/RalB knockdown; BQU57 showed dose-dependent inhibition at 10, 20, and 50 mg/kg/d, and both compounds inhibited RalA/RalB but not Ras or RhoA activity in tumors.

Methods

  • Structure-based virtual screening: compared RalA-GDP, RalA-GNP-exo84, and RalA-GNP-sec5 structures to identify an allosteric site; docked 500,000 ChemDiv compounds into RalA-GDP and selected 88 for testing.
  • Cell-based assays: RalA activity ELISA in J82 cells expressing FLAG-RalA; MEF spreading in wild-type versus Cav1−/− cells; soft agar colony formation in H2122, H358, H460, and Calu6 cells, including siRNA knockdown and constitutively active RalAG23V/RalBG23V rescue.
  • Biophysical and in vivo methods: 15N-HSQC NMR, ITC, and SPR for binding; LC-MS/MS for cellular uptake and pharmacokinetics; nude mouse xenografts with H2122 or H358 cells; RalBP1, Ras, and RhoA pull-down assays for activity.

Limitations

  • The paper states that additional medicinal chemistry optimization is required and describes the compounds as a first generation of tools.
  • The paper states that no RalB-GDP crystal structure is available, so a homology model was used and docking could not be performed on RalB-GDP; RalB-GDP binding was instead assessed by NMR.
  • The paper notes that mouse randomization was done but no blinding was performed; stable overexpression of FLAG-RalAG23V or FLAG-RalBG23V in H2122 cells was unsuccessful, so those rescue experiments used transient transfection.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/nature13713

Cites

34 of 34 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 23 cited sources checked · 6 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Genetically engineered mouse models revealed substantial redundancy for Ral proteins in tumorigenesis.

    “genetically engineered mouse models revealed substantial redundancy for Ral proteins in tumorigenesis12.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [12] Peschard, P. et al. Genetic deletion of RALA and RALB small GTPases reveals redundant functions in development and tumorigenesis. Curr. Biol. 22, 2063–2068 (2012) source
  • Premisecitation

    Active Ral activates cellular processes through effectors including RalBP1, Sec5/Exo85, filamin, and phospholipase D1.

    “Active Ral activates cellular processes through effectors including Ral Binding Protein 1 (RalBP1, RLIP76 or RIP17), Sec5/Exo85, filamin, and phospholipase D18–10.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [7] Awasthi, S., Sharma, R., Singhal, S. S., Zimniak, P. & Awasthi, Y. C. RLIP76, a novel transporter catalyzing ATP-dependent efflux of xenobiotics. Drug Metab. Dispos. 30, 1300–1310 (2002) source
    • cites [8] Oxford, G. et al. RalA and RalB: antagonistic relatives in cancer cell migration. Cancer Res. 65, 7111–7120 (2005) source
    • cites [9] Lim, K. H. et al. Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell 7, 533–545 (2005) source

      The passages on RalA effector binding identify RalBP1, filamin, PLD1, and the exocyst subunits Sec5 and Exo84. The claim says “Sec5/Exo85,” but our copy names Exo84, not Exo85. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The proximity of this site to the C3bot binding site supports the notion that small molecule occupancy at this site could inhibit function.

    “Its proximity to the previously described C3bot binding site17 supports the notion that small molecule occupancy at this site could inhibit function.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Pautsch, A., Vogelsgesang, M., Trankle, J., Herrmann, C. & Aktories, K. Crystal structure of the C3bot–RalA complex reveals a novel type of action of a bacterial exoenzyme. EMBO J. 24, 3670–3680 (2005) source
  • Findingown data

    Overexpression of constitutively active RalAG23V or RalBG23V, which do not bind these compounds, mitigated inhibition of H2122 and H358 cell growth by these compounds.

    “Lastly, overexpression of a constitutively active (GTP-form22) RalAG23V or RalBG23V, which do not bind these compounds (Extended Data Fig. 3d, 4b), mitigated inhibition of H2122 and H358 cell growth by these compounds (Fig. 3g–j, Extended Data Fig. 6f).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [22] Hinoi, T. et al. Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator. J. Biol. Chem. 271, 19710–19716 (1996) source
  • Premisecitation

    Ral promotes anchorage-independence.

    “Ral promotes anchorage-independence1,20, therefore we measured cell growth in soft agar.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [1] Lim, K. H. et al. Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr. Biol. 16, 2385–2394 (2006) source

      The passage labeled Abstract reports that suppressing RalA impaired anchorage-independent growth in nine of ten pancreatic cancer cell lines. It also reports that suppressing RalB did not impair soft-agar growth, so the unqualified claim about Ral is broader than this paper supports. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [20] Balasubramanian, N. et al. RalA–exocyst complex regulates integrin-dependent membrane raft exocytosis and growth signaling. Curr. Biol. 20, 75–79 (2010) source
  • Classified as probably not a substantial claim. 11
  • May combine two claims. 23
  • The quote only partly states this claim. 3

  • Classified as probably not a substantial claim.

    Premisecitation

    Recently, three studies used similar fragment-based small molecule screening to identify compounds that bind sites on the K-Ras surface and block its SOS-mediated activation.

    “Recently, three studies used similar fragment-based small molecule screening to identify compounds that bind sites on the K-Ras surface and block its SOS-mediated activation24–26 suggesting the approach has some promise.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [24] Sun, Q. et al. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew. Chem. Int. Edn Engl. 51, 6140–6143 (2012) source
    • cites [25] Maurer, T. et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc. Natl Acad. Sci. USA 109, 5299–5304 (2012) source
    • cites [26] Shima, F. et al. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc. Natl Acad. Sci. USA 110, 8182–8187 (2013) source

      The Discussion distinguishes this study from compounds that bind K-Ras and inhibit SOS-mediated nucleotide exchange: it says those were reported by Maurer et al., whereas this study's compounds primarily interfere with Ras–effector interactions. The Discovery section describes an in silico docking screen targeting an M-Ras structure, not fragment-based screening for inhibitors of SOS-mediated activation. (LLM, conf=0.91)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    RalA and RalB are important drivers of tumor growth and metastasis.

    “The Ras-like GTPases RalA and B are important drivers of tumor growth and metastasis1.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [1] Lim, K. H. et al. Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr. Biol. 16, 2385–2394 (2006) source

      The Discussion reports that RalA is critical for tumorigenic growth and that both RalA and RalB contribute to metastatic growth in the pancreatic cancer models studied. It also says RalB was dispensable for tumorigenic growth, so the claim overstates their shared role in tumor growth. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Ral is required for lipid raft exocytosis and spreading of murine embryonic fibroblasts on fibronectin-coated coverslips.

    “Ral is required for lipid raft exocytosis and spreading of murine embryonic fibroblasts (MEFs) on fibronectin-coated coverslips20.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Balasubramanian, N. et al. RalA–exocyst complex regulates integrin-dependent membrane raft exocytosis and growth signaling. Curr. Biol. 20, 75–79 (2010) source

      The Results and Discussion sections on Ral inhibition and RalA/RalB knockdown report that inhibiting RalA delayed lipid-raft return and spreading of WT murine embryonic fibroblasts replated on fibronectin-coated surfaces. The copy identifies those surfaces as fibronectin-coated but does not establish that they were coverslips; it also says spreading was delayed, not completely blocked. No page number is available in the indexed passages. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The IC50 for RBC8 is 3.5 μM in H2122 and 3.4 μM in H358; for BQU57 2.0 μM in H2122 and 1.3 μM in H358.

    “The IC50 for RBC8 is 3.5 μM in H2122 and 3.4 μM in H358; for BQU57 2.0 μM in H2122 and 1.3 μM in H358.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    RBC8 and BQU57 were detected in tumor tissue in vivo 3h after dosing.

    “As shown in Extended Data Fig. 7b–c, the compounds were detected in tumor tissue in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RBC6, RBC8 and RBC10 dock into the target site of RalA-GDP.

    “Molecular docking of RBC6 (c), RBC8 (d), and RBC10 (e) into the target site of RalA-GDP.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Compounds RBC5, RBC7, and RBC42 had no effect and served as negative controls.

    “while compounds RBC5, RBC7, and RBC42 (structures not shown) had no effect and thus served as negative controls.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    RBC8 at 50 mg/kg/day initiated 24 hours after inoculation inhibited xenograft tumor growth of human lung cancer cell line H358, with tumor volume statistically different from controls.

    “RBC8 treatment (50 mg/kg/day) initiated 24 h after inoculation inhibited xenograft tumor growth of human lung cancer cell line H358.”
    • Also stated in: § Extended Data · plain
      “Tumor volume in the treatment group was statistically different from controls as determined by the Student’s t-test (*p<0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RBC6 and RBC8 are related structures sharing the same bicyclic core, with specific substitutions giving rise to similar but somewhat different binding orientations in the allosteric binding cavity.

    “RBC6 and RBC8 are related structures sharing the same bicyclic core (Fig. 1e, f), with specific substitutions giving rise to similar but somewhat different binding orientations in the allosteric binding cavity (Extended Data Fig. 2c–e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Although initial library screening was based on RalA structure, selected compounds also bound RalB.

    “Although our initial library screening was based on RalA structure, the selected compounds also bound RalB, which is not surprising given the similarity of the amino acid sequences and predicted structures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both RalA and RalB were significantly inhibited by RBC8 and BQU57 but not by RBC5 in H2122 xenograft tumors.

    “Both RalA and RalB were significantly inhibited by RBC8 (Extended Data Fig. 8a–d) and BQU57 (Fig. 4e) but not by the inactive compound RBC5 (Extended Data Fig. 8e, f).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Attempts to stably overexpress FLAG-RalAG23V or FLAG-RalBG23V in H2122 cells were unsuccessful.

    “Attempts to stably overexpress FLAG-RalAG23V or FLAG-RalBG23V in H2122 cells were unsuccessful”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Colony formation in soft agar showed that the Ral-dependent lines H2122 and H358, but not H460 or Calu6, were sensitive to RBC8 and BQU57 treatment.

    “Colony formation in soft agar showed that the Ral-dependent lines H2122 and H358, but not H460 or Calu6, were sensitive to RBC8 and BQU57 treatment (Fig. 3a, b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Volumes calculated for this binding site were 175 Å3 for RalA-GDP, 155 Å3 for RalA-GNP-exo84, and 116 Å3 for RalA-GNP-sec5.

    “Volumes calculated for this binding site were 175 Å3 for RalA-GDP (Fig. 1b), 155 Å3 for RalA-GNP-exo84 (Fig. 1c), and 116 Å3 for RalA-GNP-sec5 (Fig. 1d).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    H358 xenograft yielded similar results to H2122 for RBC8.

    “A second lung cancer line, H358 yielded similar results (Extended Data Fig. 7e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Depletion of RalA by siRNA inhibits spreading of WT MEFs, whereas caveolin deficient (Cav1−/−) MEFs are resistant to RalA depletion.

    “In these cells, depletion of RalA by siRNA inhibits spreading of WT MEFs, whereas caveolin deficient (Cav1−/−) MEFs are resistant to RalA depletion.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    H2122 and H358, but not H460 or Calu6, were sensitive to Ral knockdown.

    “H2122/H358 (c) but not H460/Calu6 (d) were sensitive to Ral knockdown.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RBC8 and BQU57 but not RBC5 inhibited both RalA and RalB activation in H2122 and H358 cell lines.

    “Additionally, a Ral pull-down assay using RalBP1 agarose beads8 showed that RBC8 and BQU57 but not RBC5 inhibited both RalA and RalB activation in both the H2122 and H358 cell lines (Extended Data Fig. 5e).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Oxford, G. et al. RalA and RalB: antagonistic relatives in cancer cell migration. Cancer Res. 65, 7111–7120 (2005) source

  • May combine two claims.

    Findingown data

    RBC8 induced chemical shift changes in RalB-GDP but not RalB-GNP, indicating that RBC8 shows selectivity for the GDP bound form of Ral.

    “RBC8 induced chemical shift changes in RalB-GDP but not RalB-GNP, indicating that RBC8 shows selectivity for the GDP bound form of Ral (Extended Data Fig. 3c, d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RBC6, RBC8 and RBC10 inhibited cell spreading only in WT MEFs, not Cav1−/− MEFs.

    “When RBC6, RBC8 and RBC10 were tested for effects on cell spreading in WT and Cav1−/− MEFs, inhibition was observed only in the WT MEFs (Fig. 1i, Extended Data Fig. 2b).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The surface model of RalB-GNP shows absence of the allosteric binding site.

    “Surface model showing absence of the allosteric binding site.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The majority of BQU57-induced chemical shift changes localized to the allosteric site, consistent with assignment of BQU57 binding to this site based on modeling.

    “The majority of the chemical shift changes localized to the allosteric site, consistent with assignment of BQU57 binding to this site based on modeling.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    RBC6, RBC8 and RBC10 inhibited Ral-mediated cell spreading in murine fibroblasts.

    “When RBC6, RBC8 and RBC10 were tested for effects on cell spreading in WT and Cav1−/− MEFs, inhibition was observed only in the WT MEFs (Fig. 1i, Extended Data Fig. 2b).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    No blinding was done in the xenograft experiments.

    “Mice were randomized into 6 per group immediately before use (no blinding was done).”
    • Also stated in: § Ral activity in tumor xenografts · plain
      “mice were randomized into 6 per group (no blinding was done)”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Efforts shifted to target signaling components downstream of Ras such as Raf-MEK-ERK and PI3K-AKT-mTOR.

    “efforts shifted to target signaling components downstream of Ras such as Raf-MEK-ERK mitogen-activated protein kinases4 and the phosphoinositide 3-kinase-AKT-mTOR pathway5.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [4] Roberts, P. J. & Der, C. J. Targeting the Raf–MEK–ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26, 3291–3310 (2007) source
    • cites [5] Yap, T. A. et al. Targeting the PI3K–AKT–mTOR pathway: progress, pitfalls, and promises. Curr. Opin. Pharmacol. 8, 393–412 (2008) source

  • May combine two claims.

    Interpretationreasoning

    These Ral inhibitors represent a first generation of valuable tools for elucidating Ral signaling and developing novel agents for cancer therapy.

    “Though additional medicinal chemistry optimization is required, these Ral inhibitors represent a first generation of valuable tools for elucidating Ral signaling and developing novel agents for cancer therapy.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Similar considerations delayed development of protein kinase inhibitors, and some of the best kinase inhibitors are allosteric inhibitors that lock the conformation of kinases such as MEK in a closed state.

    “Similar considerations delayed development of protein kinase inhibitors, and indeed some of the best kinase inhibitors proved to not be competitive with ATP, but are allosteric inhibitors that lock the conformation of kinases such as MEK in a closed state23.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Fang, Z., Grutter, C. & Rauh, D. Strategies for the selective regulation of kinases with allosteric modulators: exploiting exclusive structural features. ACS Chem. Biol. 8, 58–70 (2013) source

  • May combine two claims.

    Interpretationown data

    RBC8 and BQU57 act specifically through the GDP-bound form of Ral proteins.

    “Together, these data provide evidence that RBC8 and BQU57 act specifically through the GDP-bound form of Ral proteins.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    More than one-third of human tumors harbor activating RAS mutations.

    “More than one-third of human tumors harbor activating RAS mutations2”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Schubbert, S., Shannon, K. & Bollag, G. Hyperactive Ras in developmental disorders and cancer. Nature Rev. Cancer 7, 295–308 (2007) source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    H358 cells stably overexpressing FLAG, FLAG-RalAG23V or FLAG-RalBG23V were generated.

    “For the overexpression experiments, H358 cells stably overexpressing FLAG, FLAG-RalAG23V or FLAG-RalBG23V were generated”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisenone given

    The highly conserved guanine nucleotide binding site has high affinity for GDP/GTP present at millimolar concentrations in cells, and would out-compete ligands for this site.

    “One reason for the failures to date to obtain clinically useful inhibitors for Ras and other related GTPases is the highly conserved guanine nucleotide binding site that has high affinity for the guanine nucleotides GDP/GTP present at millimolar concentrations in cells, and would out-compete ligands for this site.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mice used in xenograft experiments were randomized into groups of 6 immediately before use.

    “Mice were randomized into 6 per group immediately before use (no blinding was done).”
    • Also stated in: § Ral activity in tumor xenografts · plain
      “mice were randomized into 6 per group (no blinding was done)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All cell lines were fingerprinted by STR profiling and tested for mycoplasma contamination.

    “All cell line were fingerprinted by STR profiling and tested for mycoplasma contamination.”
    Quote in the paperNot a substantial claim
33 of 33

  • The paper relies on this without support.

    A third pathway downstream of Ras leading to activation of RalA and RalB has not been targeted to date.

    “A third pathway downstream of Ras that leads to activation of the Ras-like small GTPases, RalA and RalB6, has not been targeted to date.”

  • The paper relies on this without support.

    In the ELISA, FLAG-RalA binding to RalBP1 reflects RalA GTP-loading and capacity for effector activation.

    “In this assay, the RalA binding reflects its GTP-loading and capacity for effector activation.”

  • The paper relies on this without support.

    The RalB Q72L mutant used for NMR behaves like wild-type RalB in GDP/GNP binding and compound interactions.

    “RalB (Q72L mutant) in a pET16b (Novagen) plasmid was a kind gift from Dr. Darerca Owen (Cambridge University).”

  • The paper relies on this without support.

    The allosteric binding site in RalA-GDP is structurally conserved in RalB-GDP sufficiently for homology modeling and mapping of NMR chemical shift changes.

    “Because no RalB-GDP crystal structure is available, instead a homology model was generated based on the similarity to RalA-GDP, and the residues that displayed chemical shift changes in response to the compounds were mapped onto this model (Fig. 2e).”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • RBC8 is a selective small-molecule inhibitor of Ral that binds to GDP-bound Ral and prevents its activation by blocking conversion to the GTP-bound active state.

    “RBC8-a selective small-molecule inhibitor of Ral that binds to GDP-bound Ral and prevents its activation by blocking conversion to the GTP-bound active state 34 .”

    From Dietary cholesterol activates a Ral-dependent pathway driving LDLR turnover (2026) · cites this paper as [34]

Cites

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Related

Authors
Yan, C., Liu, D., Li, L., Wempe, M. F., Guin, S., Khanna, M., Meier, J., Hoffman, B., Owens, C., Wysoczynski, C. L., Nitz, M. D., Knabe, W. E., Ahmed, M., Brautigan, D. L., Paschal, B. M., Schwartz, M. A., Jones, D. N. M., Ross, D., Meroueh, S. O., & Theodorescu, D.
Affiliations
  • University of Colorado Anschutz (US)
  • Indiana University Indianapolis (US)
  • University of Virginia (US)
  • Yale University (US)
  • Indiana University School of Medicine (US)
  • University of Colorado Cancer Center (US)
Corresponding author
Theodorescu, Dan
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2014-09-14
Volume
515
Issue
7527
Pages
443-447
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature13713
PMC
PMC4351747
OpenAlex
W2076865122
Semantic Scholar
4d374733558595cd1d9f434c6874e22f2b6e1d3c
Cited by
150
References
34
Citations by year
2026: 10 · 2025: 9 · 2024: 6 · 2023: 8 · 2022: 10 · 2021: 13 · 2020: 18 · 2019: 12 · 2018: 12 · 2017: 7 · 2016: 21 · 2015: 22 · 2014: 2
FWCI
7.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Purdue University Indianapolis (Indiana University) (submittedVersion)
  • PMC
Funders
  • National Institutes of Health (UL1TR001082)
  • National Heart, Lung, and Blood Institute (R01HL075092)
  • National Cancer Institute (CA 075115, CA 091846, P01CA104106, R01CA075115, P50CA091846, CA 104106, R29CA075115, P30CA046934, P30CA044579)
  • National Institute of General Medical Sciences (T32GM007635, GM-47214, R01GM047214)
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
Keywords
Ral GTPase, small molecule inhibitors, RalA, RalB, Ras signaling, RALBP1, structure-based drug design, virtual screening, anchorage-independent growth, cancer therapy
MeSH terms
Animals, Computer Simulation, Drug Screening Assays, Antitumor, Female, Humans, Models, Molecular, Neoplasms, Protein Binding, Substrate Specificity, Signal Transduction, ATP-Binding Cassette Transporters, ras Proteins, GTPase-Activating Proteins, ral GTP-Binding Proteins, Xenograft Model Antitumor Assays, Cell Line, Tumor, Cell Proliferation, Mice, Small Molecule Libraries, Molecular Targeted Therapy

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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