Bisphenol A Binds to Ras Proteins and Competes with Guanine

Article pubs.acs.org/jmc

Bisphenol A Binds to Ras Proteins and Competes with Guanine Nucleotide Exchange: Implications for GTPase-Selective Antagonists Miriam Schöpel,†,§ Katharina F. G. Jockers,†,§ Peter M. Düppe,‡ Jasmin Autzen,‡ Veena N. Potheraveedu,† Semra Ince,† King Tuo Yip,† Rolf Heumann,† Christian Herrmann,† Jürgen Scherkenbeck,*,‡ and Raphael Stoll*,† †

Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, Universitätsstraße 150, D-44780 Bochum, Germany Faculty of Chemistry, University of Wuppertal, Gaußstraße 20, D-42119 Wuppertal, Germany

‡

S Supporting Information *

ABSTRACT: We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4′biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4′-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPaseselective antagonists with higher affinity to benefit of the treatment of cancer because KRas inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors.

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INTRODUCTION K-Ras4B and Ras homologue enriched in the brain (Rheb) are small GTPases that belong to the Ras superfamily of guanine nucleotide-binding proteins and are related to Rap and Ral.1 To achieve their full physiological potential, GTPases toggle between an inactive, GDP-bound state and an active, GTPbound state.2 Therefore, small GTPases function as molecular switches in living cells and are key players in intracellular signaling.3 Ras has been identified as an oncogene and is found to carry mutations in more than 20% of human cancers.2,4−6 Several isoforms of Ras exist, such as K-, N-, and H-Ras. Mutations of K-Ras, for example, are frequently found in pancreatic, colon, and lung carcinomas.7 Ras and in particular K-Ras have attracted widespread attention in cancer drug development initiatives.8 The function of Rheb has been studied in a variety of organisms, especially in Drosophila and mammalian cells.9 These reports underscore the role of Rheb as a molecular switch in many cellular processes such as cell volume growth, cell cycle progression, neuronal axon regeneration, autophagy, nutritional deprivation, oxygen stress, and cellular energy status.10−12 The effects of Rheb are mediated via the mammalian target of rapamycin (mTOR), which exists in two different multiprotein complexes: the rapamycin-sensitive mTORC1, which is responsible for the modulation of protein translation, and TORC2, which mediates the spatial control of cell growth by regulating the actin cytoskeleton.13,14 In mammals, mTOR activity plays an important role in © 2013 American Chemical Society

proliferation and inhibition of apoptosis, including that of tumor cells. Thus, mTOR has gained much interest as a therapeutic target in cancer.11,15 Insulin as well as other growth factors stimulate the GTP loading of Rheb via the inhibition of the tuberous sclerosis complex (TSC) 1 and 2, a tumor suppressor protein complex that acts as a Rheb GTPase activating protein (GAP).15,16 Ras-like G-proteins are not only negatively regulated by GAPs but also positively by guanine nucleotide exchange factors (GEFs).2 GEFs interact directly with G-proteins, thereby lowering the affinity of the G-protein for its bound nucleotide. As a consequence, this nucleotide is released and replaced by excess bulk GTP under physiological conditions.2 The son of sevenless (Sos) protein serves as a GEF as it catalyzes the rate-limiting step of replenishing the level of activated, GTP-bound K-Ras4B in the cell. Note that a bona fide GEF for Rheb has not been identified yet.17 The inhibition of such protein−protein interactions by means of small molecules is regarded as a crucial strategy in cancer therapy; however, despite some progress in recent years, the inhibition of protein−protein interactions by small molecules still represents a formidable challenge in many cases. The reason behind this is the fact that these interactions are usually mediated by large surface regions and not by small areas; consequently, each residue contributes only minimally to the overall binding free energy. Nevertheless, during the past years several small molecules and zinc-chelating compounds Received: August 20, 2013 Published: November 22, 2013 9664

dx.doi.org/10.1021/jm401291q | J. Med. Chem. 2013, 56, 9664−9672

Journal of Medicinal Chemistry

Article

Chart 1. Selected Structures from Table S1, Supporting Information, Discussed in Detail

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RESULTS AND DISCUSSION We have applied multidimensional heteronuclear NMR spectroscopy and chemical shift perturbation analysis in order to characterize the interactions between low molecular weight compounds with K-Ras and Rheb, respectively (Chart 1, Table S1, Supporting Information, and Figures 1a,b and 3a,b). The identified binding sites were then used as experimental restraints in a molecular docking procedure (Figures 2, 4, and S7, Supporting Information).31,32 Structures obtained from these docking calculations with the HADDOCK software package were refined by OPLS_2005 force field minimizations with water as solvent (Figure S5, Supporting Information). In the refined docking model, the complex between Rheb and 4,4′-biphenol is stabilized by three hydrogen bridges between Ile69 and Ile78 to one 4,4′-biphenol hydroxyl group and another hydrogen bond between the second 4,4′-biphenol hydroxyl group and Lys109 in the refined docking model, which explain the observed chemical shift perturbation. The hydrogen bonds fix the 4,4′-biphenol (5) horizontally at the top of a deep lipophilic binding pocket with the two aromatic rings conformationally twisted by approximately 30°. On the basis of the observed significant NMR chemical shift perturbations, the lipophilic binding site includes the flexible switch II amino acids Tyr67, Ile69, Phe70-Ile78, and Tyr81 as well as the residues Leu103, Met106, Val107, and Lys109 located in α-helix 3. The bottom of this pocket is mainly formed by residues Tyr67 and Tyr81. As the switch II region of Rheb has been shown to exhibit an increased flexibility on the pico- to nanosecond time scale, conformational selection might play a role in ligand binding to Rheb.36 However, the KD value extracted from multidimensional NMR spectroscopy of 4,4′-biphenol and Rheb is only approximately 1500 ± 200 μM (Figure S6, Supporting Information). Separating the two phenol moieties of 4,4′-biphenol (5) by introducing an sp2 carbon, like in benzophenone, or by a quaternary sp3 carbon with limited conformational freedom, as found in bisphenol A (10), introduces a kink in the structure directing the aromatic rings deeply into the binding pocket. The rather low KD value extracted from multidimensional NMR spectra of bisphenol A (10) and Rheb is approximately 1800 ± 500 μM (Figure S3, Supporting Information). In the refined docking model, bisphenol A (10) is fixed in the lipophilic pocket by three hydrogen bridges, similar to 4,4′biphenol (5). In Rheb, Ser68 and Gln72 form hydrogen bonds with one hydroxyl group of bisphenol A (10) and Tyr67, and one of the residues forming the bottom of the pocket is

have been identified and characterized as Sos antagonists of KRas8,18−21 Inspired by the results of Fesik and co-workers who performed an NMR screening of a large 11.000 member library, we designed a small fragment library based mainly on the privileged structure concept and the “rule of five” [Chart 1 and Table S1, Supporting Information, which lists all compounds tested in this study and also provides information on the structure−activity relationship (SAR)].19,22−24 Furthermore, SARs of known Ras inhibitors were also taken into account.8 This focused library, containing not more than 100 compounds, was screened by multidimensional NMR spectroscopy to identify structures that bind to Rheb, for which small molecule inhibitors still remained unknown. The best Rheb-fragments were additionally examined for their binding and selectivity for K-Ras (Table S1, Supporting Information). The NMR screening of Rheb revealed that almost all hydrophilic compounds exhibited no significant chemical shift perturbations, while in the group of the more lipophilic, polar structures several compounds could be identified as ligands. Remarkably, most binding molecules contained structural elements that had either a linear form, resembling a biphenyl scaffold or a bend, induced by an sp2- or sp3-hybridized bridge between the phenyl rings (Chart 1). In the case of Rheb, the most significant chemical shifts were found for bisphenol A (10), 4,4′-biphenol (5), and 4,4′-dihydroxybenzophenone (9). Here, we also show for the first time that bisphenol A (10) can directly bind to both K-Ras (Figures 1a,b, 2, S1, and S2, Supporting Information) and Rheb (Figures 3a,b, 4, S3, S4, and S6−S8, Supporting Information), suggesting an entirely new mode of action for this ubiquitous compound. According to the Breast Cancer Fund, bisphenol A (10) is one of the most frequent chemicals humans are exposed to as it is a building block of polycarbonate plastics and hence is present in many household products, such as plastic food containers and eating utensils. It has been suggested that bisphenol A (10) might (partly) cause cardiovascular diseases, breast and prostate cancers, and neuronal disorders, to name but a few.25−28 Bisphenol A (10) is regarded as an endocrine disrupting chemical as it is capable of disturbing the normal activity of (estrogen) nuclear hormone receptors.29,30 However, neither the molecular basis of this process has been addressed nor the complex impact of bisphenol A (10) on living cells is fully understood to date. 9665



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Figure 1. (a) NMR chemical shift perturbation for K-Ras bound to GDP upon titration with bisphenol A (10). The left panel shows the region of the 1H−15N HSQC spectra of K-Ras/GDP titrated with bisphenol A (10) for Leu56 and Asp57 (molar ratio of K-Ras/bisphenol A (10) ranging from 1:0 (shown in black) to 1:6 (shown in magenta)). The right panel shows a corresponding region of the 1H−15N HSQC spectra for Gly60, Thr74, and Gly75 of K-Ras/GDP titrated with bisphenol A (10) (molar ratio of K-Ras/bisphenol A ranging from 1:0 (shown in black) to 1:6 (shown in magenta)). (b) Weighted chemical shift differences of Ras/GDP titrated with bisphenol A (10). Weighted chemical shift differences plotted versus the amino acid sequence are shown at the top. On the basis of the previously published assignments and this NMR titration experiment, the KD of bisphenol A (10)/K-Ras is 600 ± 200 μM as shown at the bottom.39 The KD values in this study have been determined by using a binding isotherm as described previously.20,35,36,46

involved in another hydrogen bond with the second hydroxyl group. Additional contributions to the overall binding energy arise from a π-cation interaction between the side-chain amino group of Lys109 and the phenol group of bisphenol A (10)

located deep inside the pocket. While the binding situation is similar for 4,4′-dihydroxybenzophenone (9), the conformationally highly flexible 4,4′-methylenediphenol (11) induces only minor chemical shift changes in Rheb. Significantly reduced 9666



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Figure 2. Weighted chemical shift differences of K-Ras/GDP titrated with bisphenol A (10). On the left, a HADDOCK model of K-Ras/GDP in complex with bisphenol A (10) is shown. On the right, the observed weighted chemical shift differences are projected onto the molecular surface of K-Ras. No significant weighted chemical shift differences could be observed on the back side of K-Ras (data not shown). For the color code, please refer to Figure 1b.

loaded K-Ras, thereby rendering this low molecular weight compound a potential inhibitor of K-Ras activation (Figure 5). It has been shown previously that K-Ras and Rheb are involved in the mechanisms of apoptosis or cellular growth.11,36−38 Therefore, we determined dose−response curves for bisphenol A (10) and 4,4′-biphenol (5) on cellular degeneration and signaling. By means of direct microscopic counting, we found an increase in cellular degeneration at concentrations of bisphenol A (10) exceeding 100 μM (Figure S9, Supporting Information) indicating a toxic effect beyond this concentration. We then tested the effect of various concentrations of 4,4′-biphenol (5) on cellular degeneration after 4 h of treatment using the MTT assay. Degenerating cells were observed already at 25 μM reaching a maximum at 100 μM (Figure 6) and decreasing thereafter until 200 μM was reached. Beyond 200 μM, cellular degeneration increased again. Because of the weak but selective binding of 4,4′-biphenol (5) to Rheb but not to K-Ras, we tested the effect on intracellular signaling. Interestingly, at concentrations of 4,4′biphenol (5) inducing maximal cell death (100 μM), a pronounced decrease in S6 ribosomal protein activating phosphorylation (phospho-S6) was found (Figure 6). Thus, 4,4′-biphenol (5)-induced cell death was correlated with a blockade of phospho-S6, which is involved in the regulation of protein synthesis. However, there was no detectable effect of bisphenol A (10) on the canonical Rheb signaling pathway (data not shown). Although bisphenol A (10) binds to both KRas and Rheb, the data suggest that the cell death-inducing effects by bisphenol A (10) are not due to the regulation of the canonical signaling activity of Rheb. Taken together, we have designed a small fragment library in this study based mainly on the privileged structure concept and Lipinski’s “rule of five”.23 Furthermore, structure−activity relationships of known Ras inhibitors were also taken into account. This focused library, containing not more than 100 compounds, was screened by multidimensional NMR spectroscopy to identify structures that bind to Rheb, for which small molecule inhibitors still remained unknown. The best Rheb ligands were additionally examined for their binding and selectivity for K-Ras. The NMR screening of Rheb revealed that almost all hydrophilic compounds exhibited no significant NMR chemical shift perturbations, while in the group of the more lipophilic, polar structures several compounds could be identified as ligands. Remarkably, most binding molecules

chemical shift perturbations were also found for the diphenylether (12). Bisphenol A (10) also binds to K-Ras, however, at a different site, which includes N-terminal residues of β-strand 1, as well as switch I and II residues. On the basis of the previously published assignments and these NMR titration experiments, the KD of bisphenol A (10)/K-Ras is 600 ± 200 μM and is therefore significantly higher in comparison to that of Rheb (Figure 1a,b).39 The binding site in K-Ras identified in this study for bisphenol A (10) is identical to the one described recently for structurally different compounds.19,20 Much to our surprise, 4,4′-biphenol (5) did not show any binding to K-Ras and thus turns out to be a selective ligand for Rheb. On the basis of our structural analyses, it is evident that the bisphenol A (10) binding site on K-Ras is considerably smaller than the corresponding binding site on Rheb. Stretched, linear molecules such as 4,4′-biphenol (5) cannot be accommodated in this pocket. As expected, a simulation using the Autodock software also confirmed only a very weak binding of 4,4′biphenol (5). In accordance with recent studies of K-Ras ligands, our refined docking models show that two hydroxyl- or aminosubstituted (hetero-) aromatic rings separated by a sp2hybridized or a sp3-carbon with limited conformational freedom represent characteristic motives for K-Ras binding.8,18−20 Furthermore, for optimal binding the aromatic rings must be able to adopt almost a right angle (Figure S5, Supporting Information). Interestingly, very similar structure−activity relationships have been reported for the potent inhibitory effect of bisphenol A (10) on voltage-activated Ca2+ channels.33 Bisphenol A (10) significantly reduces the Sos-mediated nucleotide exchange reaction of both H- and K-Ras by a factor of 1.6 (Figure 5). For H-Ras, even a 2.5-fold reduction of Sosmediated exchange was found (data not shown). This corroborates our in silico molecular models that predict a steric clash between bisphenol A (10) and Sos, thereby preventing complex formation (Figure 5). This is in accordance with recent studies in which a comparable stoichiometric excess of small molecular compounds were used.19−21,34,35 In addition, the affinity of bisphenol A (10) is in the micromolar range and therefore comparable to the affinity between Ras bound to GDP and its GEF, such as Sos.21,34,35 Obviously, bisphenol A (10) might well compete with Sos for the binding of GDP9667



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Figure 3. (a) NMR chemical shift perturbation of Rheb/GDP titrated with bisphenol A (10). The left panel shows the region of the 1H−15N HSQC spectra for Ile78 of Rheb/GDP titrated with bisphenol A (10) (molar ratio of Rheb/bisphenol A (10) ranging from 1:0 (shown in black) to 1:15 (shown in magenta)). The right panel shows the region of the 1H−15N HSQC spectra for Phe70 of Rheb/GDP titrated with bisphenol A (10) (molar ratio of Rheb/BPA ranging from 1:0 (shown in black) to 1:15 (shown in magenta)). (b) Weighted chemical shift differences of Rheb/GDP titrated with bisphenol A (10). The weighted chemical shift differences of Rheb/GDP titrated with bisphenol A (10) plotted versus the ligand concentration is shown in the top panel. On the basis of this NMR titration experiment, the KD of bisphenol A (10)/Rheb is approximately 1830 ± 470 μM as shown in the bottom panel. The assignments of Rheb/GDP have been previously published.46

contained structural elements that had either a linear form, resembling a biphenyl scaffold or a bend, induced by an sp2- or sp3-hybridized bridge between the phenyl rings. In the case of Rheb, the most significant chemical shifts were found for bisphenol A (10) and 4,4′-biphenol (5). On the basis of the

multidimensional NMR data, we were able to map the binding site of these ligands and to generate structural models of both Rheb and K-Ras in complex with these ligands showing for the first time that bisphenol A (10) and 4,4′-biphenol (5) derivatives have indeed the capacity to interact directly with 9668



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Figure 4. Weighted chemical shift perturbation of Rheb bound to GDP upon titration with bisphenol A (10). On the left, a HADDOCK model of Rheb/GDP bound to bisphenol A (10) is shown. On the right, the observed weighted chemical shift differences are projected onto the molecular surface of Rheb. For the color code, refer to Figure 3b.

Figure 6. Measurement of cell death in response to various concentrations of 4,4′-biphenol (5). HeLa cells were treated with increasing concentrations of 4,4′-biphenol (5) for 4 h as indicated. Results are reported as the mean value ± SEM (standard error mean) from triplicates. Statistically significant differences are indicated as * p < 0.1 and ***p ≤ 0.05 using Student’s t test. The insert shows that 4,4′-biphenol (5) causes a decrease of phospho-S6 protein levels at 100 μM.

remarkably different behavior of bisphenol A (10) and 4,4′biphenol (5) might help to design future experiments to decipher the signaling cascade of Rheb. As mentioned before, a GEF has not been characterized for Rheb on a molecular level.17 However, it could be shown previously that Rheb can enhance apoptosis via ASK-1 indicating that the signaling cascade of Rheb could differ from other small GTPases.36 Interestingly, we show here that 4,4′-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth.

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CONCLUSIONS Here, we show for the first time that bisphenol A (10) can directly bind to both K-Ras and Rheb with an affinity comparable to that of known low molecular compounds that interact with K-Ras. We could also establish that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. This suggests an entirely new mode of action of bisphenol A (10), a ubiquitous compound in our civilization. According to the Breast Cancer Fund, bisphenol A (10) is one of the most frequent chemicals humans are exposed to as it is a building block of polycarbonate

Figure 5. Sos-mediated nucleotide exchange assay. Effect of bisphenol A (10) on Sos-mediated K-Ras activation (top panel). (Bottom panel) Model of K-Ras[green]/Sos[blue] (PDB code 1BKD) superimposed with bisphenol A (10)[red].

K-Ras and/or Rheb, respectively. In addition, we have characterized these interactions at atomic resolution and established that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. The 9669



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NMR Spectral Analysis and Molecular Docking Studies. Assignment and data handling were performed using NMRView42 and CcpNmr Analysis.43 Docking was performed using HADDOCK 2.131 and CNS 1.3.44 The topology and parameter files for the low molecular weight compounds were generated using the PRODRG server (www.http://davapc1.bioch.dundee.ac.uk/prodrg/). Docking interfaces were defined by ambiguous interaction restraints (AIR) using the coordinate sets 1XTQ, 2L0X (ensemble of structures), and 4DSO. For K-Ras, Leu6, Val9, Leu56, Thr74, and Gly75 were selected as active residues. In the case of Rheb, residues Ile69-Gly80 and Met106-Val107 were set to be active in the docking protocol. A total of 1000 structures were generated using a rigid body docking procedure in HADDOCK. The 200 best scoring structures thereof were subjected to semiflexible simulated annealing, followed by a refinement in explicit water. Finally, structures with lowest HADDOCK scores were selected for further analysis. Structure visualization and superposition based on RMSD values for Cα, C, and N atoms were performed by PyMol.47 Sos-Mediated Nucleotide Exchange Assay. The Sos-mediated nucleotide exchange assay was performed as previously published.35 Briefly, Ras bound to fluorescent mant-GDP was incubated with a 100fold molar excess of GDP in the presence and in the absence of Sos. Inhibitory compounds were added at various concentrations. The fluorescence was excited at 360 nm and detected at 450 nm, and its time course was recorded with a Perkin-Elmer LS50B instrument. Determination of Cell Numbers. Total number of cells were counted by DAPI (4′,6-diamidino-2-phenylindole) (Sigma) staining, and the fraction of degenerating cells was determined by counting cells with nuclear fragmentation. 1.5 × 106 cells per well were plated in 6 well plates and incubated at 37 °C/10% CO2 for 24 h. Cells were treated with various dilutions of bisphenol A (10) dissolved in DMSO and incubated for 24 h at 37 °C/10% CO2. As a control, cells were also treated with a maximal concentration of DMSO (0.1%), yielding 9.7 ± 2% of cell death considered here as background (Figure S9, Supporting Information). Results are reported as the mean value ± SEM (standard error of the mean) from triplicates. The significance of difference was assessed by one-way Student’s t test with p ≤ 0.05 considered as statistically significant. MTT Assay. The MTT assay was used to determine the viability of HeLa cells in the presence of 4,4′-biphenol (5). Cells (6 ×104) were plated in each 96 wells and incubated for 20 h at 37 °C/10% CO2. Cells were then treated with various dilutions of 4,4′-biphenol (5) dissolved in DMSO. Cells were also treated with the respective concentrations of DMSO without 4,4′-biphenol (5), and background effects of DMSO on cell viability were subtracted. Plates were incubated with 4,4′-biphenol (5) for 4 h at 37 °C/10% CO2. A 5 mg/ mL stock of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was diluted 1:10 into prewarmed medium. Culture media were then replaced with diluted MTT solution. Plates were then incubated for another 3 h at 37 °C/10% CO2. After incubation, supernatants were removed, and 100 μL of 100% DMSO was added. Plates were then placed on an orbital shaker for 10 min, and the absorbance was recorded at 570 and 620 nm. The percentage of degenerating cells in presence of 4,4′-biphenol (5) was expressed as absorbance relative to that of the control. Results are reported as the mean value ± SEM from triplicates. The significance of difference was assessed by Student’s t test with p ≤ 0.05 considered as statistically significant. S6-Kinase Phosphorylation Assay. Cells (1.5 × 106) were seeded per well in 6-well plates and incubated for 24 h at 37 °C/10% CO2. Cells were serum starved for 24 h and then restimulated with serum alone or serum supplemented with bisphenol A (10) or 4,4′biphenol (5) (Figure 6). Cells were then incubated for 4 h at 37 °C/ 10% CO2. Untreated cells were taken as a control. Cells were lysed, and the protein content was measured using the Bradford assay45 to ensure equal loading. Western blot analysis was also performed essentially as described previously.40 Antibodies used in this study comprised antiphospho S6-protein (Cell Signaling Technology) and anti-tubulin (Sigma). Proteins were visualized by using enhanced chemiluminescence reagents (Thermo Scientific).

plastics and hence is present in many household products, such as plastic food containers and eating utensils. It has been suggested that bisphenol A (10) might cause cardiovascular diseases, breast and prostate cancers, and neuronal disorders, to name but a few. In contrast to most of the small GTPase family members, a GEF has not been characterized for Rheb on a molecular level. However, mutations in the Rheb GTPase activating protein tuberous sclerosis protein 1 (TSC1, harmatin) result in enhanced Rheb activity generating ectopic neural progenitor cells. Using a MTT-based and an S6-Kinase phosphorylation assay, we describe here a selective binding of 4,4′-biphenol (5) to Rheb thus delivering a novel target which directly inhibits the TSC1 downstream signaling without affecting Ras signaling. On the one hand, our results do suggest a new mode of action for bisphenol A (10), which, in turn, advocates a reduced exposure to this compound in our environment. On the other hand, our data might also lay the foundation for future design of GTPase-selective antagonists with higher affinity to benefit the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors.19,20 Finally, we believe that our work represents an important contribution not only to the biological activity of bisphenol A (10) and phenol derivatives but also opens an avenue to the design of GTPase-selective antagonists for new therapies in the treatment of cancer and brain disease.

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EXPERIMENTAL SECTION

Recombinant Proteins and Their Purification. Isotopically enriched full-length proteins were expressed and purified as previously published.36,39 Western blot analysis was also performed essentially as described previously.40 UV/visible spectra were recorded with an Analytik Jena SPECORD 200 spectrometer. Low Molecular Weight Compounds and Solvents. Compounds tested in this study were purchased with ≥95% purity, as judged by GC and/or LC, from Alfa Aesar, ABCR, ACROS Organics, AppliChem, Dr. Ehrensdorfer GmbH, Merck, Fluka, MAYBRIDGE, Sigma-Aldrich, and TCI Europe. Deuterated solvents for NMR measurements were obtained from Deutero GmbH. Multidimensional NMR Spectroscopy. All spectra were recorded at 298 K on a Bruker DRX600 spectrometer equipped with pulsed field gradients and a triple resonance probe head. The NMR spectra with Watergate solvent suppression were recorded at 600.13 MHz proton frequency and at 298 K. The 1D 1H NMR spectra were recorded with a time domain of 32 k data points and a spectral width of 12019.23 Hz. The sweep width of the two-dimensional homonuclear spectra was 12019.23 Hz in the direct 1H and 2736.70 Hz in the indirect 15N dimension. The free-induction decay was acquired for 340.9 ms, and the dwell time was set to 41.6 μs. The binding studies were performed in accordance with literature procedures.36 All spectra were processed with NMRPipe41 and analyzed with NMRView42 or CcpNmr Analysis.43 Protein Binding Studies. NMR titrations using putative ligands of Rheb and K-Ras4B consisted of monitoring changes in chemical shifts and line widths of the backbone amide resonances of uniformly 15 N enriched Rheb or K-Ras4B samples as a function of ligand concentration. This resulted in a series of 1H−15N HSQC NMR spectra, following the procedure of SAR by NMR.22 Quantitative analysis of ligand-induced chemical shift perturbation was performed by applying Pythagoras’ equation to the weighted chemical shifts as previously published.36,39 Weighted chemical shift perturbations regarded to be significant are shown in color in Figures 1a and b, 3a and b, as well as S6, Supporting Information. The assignments of KRas amide resonances were obtained from (BMRB 18529).39 KD values were derived as previously described.19,22,35,36,46 9670



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ASSOCIATED CONTENT

S Supporting Information *

List of compounds used in this study; 600 MHz 1H−15N HSQC spectra of K-Ras/GDP titrated with bisphenol A; electrostatic surface potential K-Ras/GDP in complex with bisphenol A (10); 600 MHz 1H−15N HSQC spectra of Rheb/ GDP titrated with bisphenol A (10); electrostatic surface potential Rheb/GDP in complex with bisphenol A (10); superposition of K-Ras/GDP in complex with bisphenol A (10); 600 MHz 1H−15N HSQC spectra of Rheb/GDP titrated with 4,4′-biphenol (5); weighted chemical shift perturbation of Rheb bound to GDP upon titration with 4,4′-biphenol (5); electrostatic surface potential Rheb/GDP in complex with 4,4′biphenol (5); and measurement of degenerating cells in response to various concentrations of bisphenol A (10). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(J.S.) Phone: +49 202 439 2654. Fax: +49 202 439 3464. Email: [email protected]. *(R.S.) Phone: +49 234 32 25466. Fax: +49 234 32 05466. Email: [email protected]. Author Contributions §

M.S. and K.F.G.J. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Deutsche Krebshilfe (109776 and 109777) and the DFG (SFB 642) for generous financial support.

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ABBREVIATIONS USED GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; SAR, structure−activity-relationship; Sos, son of sevenless

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REFERENCES

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Bisphenol A Binds to Ras Proteins and Competes with Guanine

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