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Keywords:

  • acyl protein thioesterases;
  • boronic acids;
  • inhibitors;
  • microarrays;
  • oncogenic Ras proteins

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

The Ras proteins are membrane-bound signal-transducing GTPases that are prominent in the control of cell growth, differentiation, and proliferation. Mutations in Ras play a causative role in about 30 % of all human cancers.1 For biological activity, H- and N-Ras—which are particularly prominent in, for example, melanoma, leukemia, and cancers of the bladder, liver, and kidney2—need to be S-farnesylated and S-palmitoylated at their C termini. Dynamic S-palmitoylation and S-depalmitoylation regulate reversible membrane attachment of H- and N-Ras and their correct subcellular localizations and differing signaling outputs.3

Inhibition of the Ras-depalmitoylating acyl protein thioesterase enzymes has recently become a new approach to modulating the Ras de-/reacylation cycle. We have provided evidence that human acyl protein thioesterases 1 and 2 (APT1/2) are major Ras-depalmitoylating enzyme in cells and that inhibition of Ras depalmitoylation results in impaired Ras localization and downstream signaling, as well as in phenotypic reversion of fibroblasts transformed with oncogenic H-RasG12V.4 These findings suggest that interference with Ras depalmitoylation might hold promise for the discovery of drug candidates that target tumors with mutations in H- and N-Ras and highlight the importance of the identification of new Ras depalmitoylation inhibitors. For these reasons, we4b and others5 have previously developed inhibitors against APT1 and APT2.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

Compound screening and validation

In order to supply proteins for the inhibitor screenings, we cloned the human apt1 and apt2 genes into bacterial expression vectors for subsequent heterologous protein production in E. coli; this allowed us to purify both proteins to homogeneity in high yields. We produced both proteins as glutathione-S-transferase (GST) fusion proteins for screening studies and without tags for activity measurements as described below.

In order to obtain APT1 and APT2 binders that might inspire new chemotypes of APT inhibitors, we screened a large library of small molecules from the RIKEN Natural Products Depository (NPDepo)6 by chemical array (Figure 1).

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Figure 1. Principle of the microarray-based compound screening. Glass slides functionalized with photoaffinity linkers were exposed to UV light and the small molecules were bound to the reactive linkers. For detection of bound GST-APT proteins, an anti-GST antibody and a secondary fluorescent antibody were used.

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This method is based on a trifluoromethylaryldiazirine system as a functional group for photo-crosslinking and allows for the immobilization of structurally diverse small molecules in a functional-group-independent manner, so the pharmacophoric elements should not be influenced.7 In total, 15 675 compounds from NPDepo were screened on both GST-APT1 and GST-APT2 with use of a GST-specific antibody for detection of bound protein. To validate the screening method, the known APT1 inhibitors 138 (Figure 2) were also immobilized on the chemical array as controls. The screen identified several APT1- and APT2-binders with bis-borinic acid and bis-borinate ester structures (Figure 2).9 The bound GST-APT proteins were visualized by using secondary antibodies. As further validation—and to exclude GST-binding effects—hit compounds were immobilized on SPR chips and the adsorption of proteins was monitored in a time-dependent fashion by surface plasmon resonance (SPR).10 These experiments confirmed the potent binding of the bis-borinic acid derivatives 47 to the human acyl protein thioesterases APT1 and APT2 (Figure 2).

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Figure 2. Microarray screening of the NPDepo library and hit validation by surface plasmon resonance (SPR). A) GST-APT1/2 binders (compounds 13). B) Borinic acid derivatives identified as GST-APT1/2 binders on glass array and SPR chip. C), D) Covalent APT1/2 inhibitors and borinic acid derivatives immobilized on glass array. GST-APT1/2 binding is indicated by red fluorescence of GST-specific antibody. E), F) SPR chip. Red color indicates binding of APT1/2 to immobilized small molecules on gold chip. G), H) SPR signals (averaged over four spots) for compounds immobilized on gold chip.

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Enzyme kinetics and inhibitor validation

Subsequent experiments monitoring the influence of the identified hits on enzyme activity showed that compound 4 not only binds to the proteins on microarray and SPR chip but also inhibits their in vitro enzymatic activities, as shown in a Lineweaver–Burk analysis of a fluorescence-based APT1/2 activity assay (Figure 3).

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Figure 3. Lineweaver–Burk plots for boron compound 4. A) APT1; ▪: 1, ▴: 3, and ▪: 5 μM. B) APT2, showing the competitive inhibition of both APTs by compound 4>; ▪: 0.75, ▴: 2, and ▪: 3 μM.

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The analysis revealed competitive inhibition of both APT1 and APT2 by the inhibitors: the same mechanism already observed for other boron compounds that had been shown to inhibit β-hydrolases such as porcine pancreatic lipase, presumably through complex formation with the active site serine residues. The Ki values for 4 were thus determined to be (0.99±0.13) μM [IC50=(3.3±0.2) μM] for APT1 and (0.73±0.20) μM [IC50=(0.98±0.06) μM] for APT2. In search of more potent boron inhibitors of APT1, we screened various related aliphatic and aromatic boronic acids by a fluorescence-based activity assay. A significant proportion of the investigated boron compounds—particularly the relatively low molecular weight compounds 8, 9, 11, and 15 (Table 1)—displayed IC50 values in the low micromolar range for human APT1 and even stronger effects, at nanomolar levels, on human APT2. This not only constitutes the first group of APT inhibitors that is about ten times more specific for one of the two isoenzymes, but it also preferentially targets the second human acyl protein thioesterase APT2, which was shown to depalmitoylate semisynthetic Ras more efficiently than APT1.4c Phenylboronic acids (Table 1) bearing electron-withdrawing groups (e.g., 8, 9, or 10) and therefore showing increasing electrophilicity of the nucleophile-trapping boron atom showed the most potent inhibition of APT1 and APT2. The more electron-rich unsubstituted phenylboronic acid 22 was a weaker inhibitor, and bulky phenylboronic acid 24 did not inhibit at all.

Table 1. IC50 values for different phenylboronic acids that inhibit human APT1 and APT2.
NoStructureIC50M] hAPT1IC50M] hAPT2
8
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0.51±0.031.97±0.10
9
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1.1±0.20.138±0.013
10
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1.4±0.10.418±0.013
11
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2.3±0.10.529±0.133
12
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2.6±0.11.6±0.02
13
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2.7±0.14.1±0.03
14
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3.2±0.11.8±0.06
1511
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3.2±0.20.238±0.025
16
Thumbnail image of
3.7±0.42.95±0.15
17
Thumbnail image of
3.9±12.87±0.63
18
Thumbnail image of
4.6±0.44.18±0.77
19
Thumbnail image of
6.3±0.52.16±0.17
20
Thumbnail image of
10.2±11.98±0.09
21
Thumbnail image of
13.5±1.411.3±4.0
22
Thumbnail image of
14.7±1.625.1±1.41
23
Thumbnail image of
16.9±1.53.55±0.40
7
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33.6±4.641.5±7.1
24
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>50>50

To characterize these inhibitors further in an environment closer to the natural APT substrate Ras, an in vitro depalmitoylation assay was performed with borinic acid ester 4 as inhibitor, APT1 as deacylating enzyme, and palmitoylated N-Ras as substrate. Briefly, after incubation of the inhibitor with N-Ras and APT1, the release of fatty acid was monitored with ADIFAB, which in the presence of free fatty acid changes its fluorescent spectrum, thereby allowing the calculation of liberated palmitic acid.12 Indeed, N-Ras depalmitoylation by APT1 was inhibited completely at 50 μM, indicating potent in vitro inhibition of APT1 (Figure 4 A).

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Figure 4. Inhibition of in vitro depalmitoylation by compound 4, together with toxicity determination A) Compound 4 inhibited deacylation of semisynthetic S-palmitoylated S-farnesyl-N-Ras by human APT1. The amounts of palmitic acid after incubation of N-Ras with APT1 (50 nM) and ADIFAB (200 nM) in the absence (•) or in the presence of 50 μM compound 4 (▴) were detected by measuring the fluorescence spectra (400–650 nm). B) Cytotoxic effect of compound 4. MDCK cells were treated with ▪: 100, ▪: 50, ▪: 12.5, and ▪: 6.25 μM 4 for 24 h. The cytotoxicity was evaluated by LDH assay.

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Cytotoxicity evaluation

To prepare in vivo studies, the cytotoxic effect of compound 4 on MDCK cells was investigated. The cytotoxicity was evaluated by LDH assay13 and compound 4 turned out not to be cytotoxic on MDCK cells (Figure 4 B).

Inhibition of oncogenic Ras activity in cell culture

We further examined whether compound 4 inhibits Ras signaling in cultured cells. MDCK/F3 cells are a constitutively active H/N-Ras subclone of MDCK cells, where ERK phosphorylation is upregulated (as opposed to MDCK cells; Figure S1 A in the Supporting Information).14

The phosphorylation of ERK in MDCK/F3 cells was completely inhibited by 10 μM of U0126, a MEK inhibitor, and partially inhibited by 50 μM of the β-lactone APT1/2 inhibitor palmostatin B.15 Inhibitory effects of both U0126 and palmostatin B were observed at 6 h of treatment with the compounds (Figure S1 B). When MDCK-F3 cells were treated with compound 4 for 6 h, dose-dependent inhibition of ERK phosphorylation comparable to inhibition by palmostatin B was observed (Figure 5 A), without cell viability being affected.

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Figure 5. Compound 4 inhibits oncogenic Ras activity. A) Compound 4 inhibited N-Ras-induced phosphorylation of ERK. MDCK/F3 cells were treated with the indicated concentrations of palmostatin B or compound 4 for 6 h. The cell lysates were prepared and subjected to Western blotting with anti-phospho-ERK and anti-ERK antibodies.16 The intensity of each band was measured with ImageJ (http://rsbweb.nih.gov/ij/). B) Partial phenotypic reversion of MDCK-F3 cells in the presence of compound 4. MDCK-F3 cells were treated with 50 μM of compound 4 for 6 h. The morphologies of the MDCK-F3 cells were observed by phase contrast microscopy.

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Moreover, we also examined the effect of compound 4 on morphological changes. In comparison with MDCK cells, oncogenically transformed MDCK-F3 cells are spindle-shaped and display a reduced number of cell–cell contacts. Compound 4 caused a partial phenotypic reversion in MDCK-F3 cells (Figure 5 B), analogous to the partial phenotypic reversion observed upon treatment with palmostatin B.4a

Taken together, compound 4 inhibited the function of Ras, possibly by inhibition of the enzymatic activities of APT1 and APT2, which resulted in suppression of ERK phosphorylation and induction of a phenotypic reversion of MDCK-F3 cells.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

We have identified boronic and borinic acid derivatives as a new class of potent and nontoxic inhibitors of the human acyl protein thioesterases APT1 and APT2. Different inhibitors for APT1/2 have been reported in the past, but although very potent, they had disadvantages such as fast off-rates from the proteins or not being isoenzyme-specific.4b Current APT inhibitor research therefore mainly focuses on overcoming these issues, and recent inhibitors have been reported to target the APTs isoenzyme-specifically.5

The boronic and borinic acid inhibitors we present here constitute another important step in this direction. They have advantages such as a known binding mechanism to the proteins and the fact that boron-based therapeutic agents are already established and boronic acids are known to bind to active-site serines and have already been reported to inhibit β-hydrolases such as porcine pancreatic lipase,17 the protease subtilisin,18 and the prototypical hormone-processing protease Kex2.19 The proteasome inhibitor Bortezomib also possesses a boron atom that binds at the catalytic site of the 26S proteasome with high affinity and specificity.20 Further boronic-acid-based therapeutics have emerged as β-lactamase inhibitors, contributing to the need for new antibiotics.21 Boron-containing phosphodiesterase 4 (PDE4) inhibitors have also been developed and might play an important role as drugs against inflammatory diseases in the future.22 Moreover, the boron-containing dipeptidyl peptidase 4 inhibitor Januvia is in use against type 2 diabetes.23 and an anti-hepatitis C virus (HCV) drug that is also a boronic acid derivative has been reported.24 More compounds are under development for, for example, a cure for acne.25 All these emerging drugs show that boron-based therapeutics might play an increasing role in the future. However, boron-based compounds have not yet been tested for inhibitory activity against acyl protein thioesterases. We have now identified such compounds by extensive library screening by chemical array and found that they inhibit both APT1 and APT2 in a competitive mode. Individual inhibitors (compounds 9, 10, 11, 15) also display appreciable isoenzymatic specificity for APT2; this is significant because we recently demonstrated that semisynthetic Ras is depalmitoylated by human APT2 more efficiently than by its isoenzyme APT1.4c At this moment, it is purely speculation whether the hydrophobicities of the compounds or steric factors are the cause of their favoring APT2 over APT1. Furthermore, our investigated compounds are borinic acids. Borinic acids have barely emerged in the literature so far26 but constitute an important part of our boron-based inhibitor set. Diphenylborinic acids in particular have been shown to act as potent inhibitors of serine proteases.27 We used the most potent borinic acid—a diphenylborinic acid (compound 4)—and expanded our biochemical data with cell culture experiments and were able to demonstrate that compound 4 can impair N-Ras-induced ERK phosphorylation and can also reverse the phenotype of MDCK/F3 cells. It is very likely that this effect is due to inhibition of the acyl protein thioesterases. In addition, we were able to show that the compound was not cytotoxic to MDCK cells. It is hard to make judgements on the biostabilities of borinic acids in general because they have only been poorly described so far. However, we successfully applied compound 4 in cell culture and did not see any hints of its degradation, so it can be assumed that at least this borinic acid must be quite stable in biological systems. It is plausible that all boron-based inhibitors bind reversibly and covalently to the active sites of the APTs because they are believed to act as nucleophilic traps for the activated serine residues in the catalytic triads of these proteins. This idea is supported by the Lineweaver–Burk data, which clearly show competitive binding of compound 4. The binding off-rate of the boron-based inhibitors from the proteins can be regarded as very slow (most probably in the range of hours to days);28 this is an advantage over the known palmostatin inhibitor group, which is more potent but displays quite fast off-rates and does not have isoenzymatic specificity.

Experimental Section

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

Molecular cloning: Human apt1 and apt2 cDNA (GenomeCube clones IRAUp969F0953D and IRAUp969B0847D) were used as a template for PCR amplification of both genes, each starting at the second methionine residue in the corresponding sequence. The apt1 and apt2 full-length genes were gateway cloned into pGEX 4T1 expression vectors (GE Healthcare) and primers were modified to encode a PreScission protease cleavage site immediately upstream of the appropriate start codon, creating a cloning artifact of Gly-Pro.

Protein expression and purification: E. coli BL21 Codon +RIL cells were transformed by use of the above constructs. Bacterial cells were grown at 25 °C, and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.1 mM) at 20 °C overnight. Cells were harvested and lysed by using a high-pressure cell disruptor, cell debris was then removed by centrifugation at 100 000 g, and the supernatant was loaded onto a GSH affinity column. The column was washed with 15 column volumes of buffer. PreScission protease (GE Healthcare) was then loaded onto the column and slowly circulated over it overnight. Afterwards, all cleaved protein was washed from the column. The GSH affinity chromatographic separations were done under the following conditions: TrisHCl (pH 8.5, 20 mM), NaCl (150 mM), glycerol (5 %), β-mercaptoethanol (5 mM). The proteins were then subjected to gel filtration with use of the following buffer: TrisHCl (pH 8.0, 20 mM), NaCl (30 mM), TCEPHCl (1 mM). Finally, both proteins were concentrated by using MWCO spin filters (10 000 kDa) to concentrations of 20 mg mL−1 and flash-frozen.

Synthesis of bisborinic acid derivatives 47 and 1214: Bisborinic acid derivatives 47 and 1214 were synthesized by a minor modification of the original procedure.9 Triisopropoxyborane was treated with one equivalent of commercially available phenyllithium at −78 °C in anhydrous diethyl ether to afford the diisopropoxyphenylborane, which was used after purification by distillation (b.p. 83–85 °C/7 mm Hg).

Bisborinate ester 4: Colorless solid; 1H NMR (500 MHz, CDCl3): δ=2.55 (s, 12 H), 2.87 (t, J=6.3 Hz, 4 H), 4.27 (t, J=6.3 Hz, 4 H), 4.49 (s, 4 H), 7.18 (br t, J=7.4 Hz, 2 H), 7.26 (br dd, J=7.4, 7.4 Hz, 4 H), 7.27 (br d, J=7.4 Hz, 4 H), 7.73 (br d, J=7.4 Hz, 4 H), 7.75 ppm (dd, J=7.4, 1.1 Hz, 4 H); 13C NMR (125 MHz, CDCl3): δ=47.2, 60.50, 60.54, 72.3, 126.3, 127.0, 127.2, 132.5, 132.6, 136.3, 146.0, 146.8 ppm; 11B NMR (160 MHz, CDCl3): δ=6.4 ppm; HRMS (ESI): m/z calcd for C34H43B2N2O3: 549.3460 [M+H]+; found: 549.3469.

Bisborinic acid 5: Colorless oil; 1H NMR (500 MHz, CD3COCD3): δ=4.63 (s, 4 H), 7.41 (dd, J=7.4, 7.4 Hz, 4 H), 7.42 (dd, J=7.4, 7.4 Hz, 2 H), 7.47 (tt, J=7.4, 1.1 Hz, 2 H), 7.52 (br d, J=7.4 Hz, 2 H), 7.70 (d, J=7.4 Hz, 2 H), 7.79 (dd, J=7.4, 1.1 Hz, 4 H), 7.81 (br s, 2 H), 9.07 ppm (br s, 2 H); 13C NMR (125 MHz, CD3COCD3): δ= 72.8, 128.4, 128.4, 130.7, 131.3, 134.8, 134.9, 135.6, 138.6, 138.7, 138.9. ppm; 11B NMR (160 MHz, CD3COCD3): δ=43.7 ppm; HRMS (ESI): m/z calcd for C26H24B2O3Na: 429.1809 [M+Na]+; found: 429.1812.

Bisborinic acid 6: Colorless oil; 1H NMR (500 MHz, CD3COCD3): δ=4.67 (s, 4 H), 7.42 (dd, J=7.4, 7.4 Hz, 4 H), 7.47 (d, J=7.4 Hz, 4 H), 7.48 (dd, J=7.4, 1.1 Hz, 2 H), 7.79 (dd, J=7.4, 1.1 Hz, 4 H), 7.80 (d, J=7.4, Hz, 4 H), 9.04 ppm (br s, 2 H); 13C NMR (125 MHz, CD3COCD3): δ=72.7, 127.5, 128.4, 131.3, 135.6, 135.8, 137.8, 138.7, 142.1 ppm; 11B NMR (160 MHz, CD3COCD3): δ=43.0 ppm; HRMS (ESI): m/z calcd for C26H28B2NO3: 424.2255 [M+NH4]+; found: 424.2260; HRMS (ESI): m/z calcd for C26H24B2O3Na: 429.1809 [M+Na]+; found: 429.1808.

Bisborinic acid 7: Colorless solid; 1H NMR (500 MHz, CD3OD): δ=4.11 (s, 4 H), 7.01 (br d, J=6.9 Hz, 2 H), 7.22 (dd, J=7.4, 7.4 Hz, 4 H), 7.23–7.30 (m, 6 H), 7.34 (br t, J=7.4, 7.4 Hz, 2 H), 7.44 (d, J=7.4 Hz, 4 H); 13C NMR (125 MHz, CD3OD): δ=74.1, 127.1, 127.9, 128.4, 128.9, 131.3, 131.6, 135.9, 138.7, 139.1, 141.9 ppm; 11B NMR (160 MHz, CD3OD): δ=42.8 ppm; HRMS (ESI): m/z calcd for C26H24B2O3Na: 429.1809 [M+Na]+; found: 429.1807.

Bisborinate ester 14: Colorless solid; 1H NMR (500 MHz, CDCl3): δ=2.49 (s, 12 H), 2.82 (br t, J=6.3 Hz, 4 H), 4.24 (br t, J=6.3 Hz, 4 H), 4.54 (s, 4 H), 7.16 (tt, J=7.4, 1.1 Hz, 2 H), 7.21 (ddd, J=7.4, 1.7, 1.1 Hz, 2 H), 7.23 (dd, J=7.4, 7.4 Hz, 2 H), 7.25 (ddd, J=7.4, 7.4, 1.7 Hz, 4 H), 7.66 (ddd, J=7.4, 1.7, 1.7 Hz, 2 H), 7.69 (br s, 2 H), 7.74 ppm (dd, J=7.4, 1.1 Hz, 4 H); 13C NMR (125 MHz, CDCl3): δ=47.2, 60.47, 60.54, 72.6, 126.0, 126.2, 127.2, 131.8, 132.0, 132.6, 137.0, 146.8, 146.9 ppm; 11B NMR (160 MHz, CDCl3): δ=7.2 ppm; HRMS (ESI): m/z calcd for C26H24B2O3Na: 429.1809 [M−2 (CH3)2NCH2CH2OH+2 H2O+Na]+; found: 429.1813.

In vitro depalmitoylation ADIFAB assay: ADIFAB (AcryloDated Intestinal Fatty Acid Binding) protein was purchased from FFA Sciences LLC (San Diego, CA) and dissolved in storage buffer [Tris (50 mM), EDTA (1 mM), sodium azide (0.05 %)] to a concentration of 100 μM. This stock solution was then diluted in assay buffer [Tris (20 mM), NaCl (150 mM), KCl (5 mM), Na2HPO4 (1 mM), pH 7.4] to a concentration of 13 μM. Fluorescent spectra were recorded with a JASCO FP-6500 fluorimeter and measured between 400 and 650 nm with an excitation wavelength of 386 nm. All samples were measured at room temperature in quartz cuvettes (1 cm path length, 200 μL minimal volume) with a final volume of 400 μL. To obtain comparable data, all measurement parameters and concentrations were kept constant. ADIFAB (200 nM final concentration) was added to the cuvette and the obtained fluorescence intensity ratio was taken as R0. After the addition of N-Ras (2.5 μM final concentration) the reaction mixture was measured and taken as the value for the calculation of released palmitic acid. The inhibitor (final concentration of 50 μM) was pre-incubated with APT1 (final concentration of 50 nM) for 5 min. The reaction was followed for 15 min in 70 s intervals. The release of palmitic acid was subsequently calculated by use of the described equation for ratio mode measurement with Rmax=11.5 and Q=19.5 and the Kd of palmitic acid of 0.32 μM.29 Semisynthetic palmitoylated and farnesylated N-Ras was prepared as described in the literature.30 After preparation on solid support the purified lipidated peptide was ligated to an E. coli expressed truncated N-Ras protein Δ1–181 with a C-terminal cysteine.

Enzymatic protein activity assay: The enzyme activities were determined by measuring the release of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) by the APT hydrolysis of DiFMU octanoate (DiFMUO) in a Tecan Infinite M200 microplate reader in a 96-well format. The final enzyme concentration was 0.1 nM and the final substrate concentration was 7.5 μM. In the assay, inhibitor solutions (30 μL, at varying concentrations) in assay buffer were mixed with APT solution (0.2 nM, 50 μL) in assay buffer. The mixture was incubated for 20 min. at 37 °C. Subsequently, a solution of DiFMUO (38 μM, 20 μL) in assay buffer was added to the reaction mixture and after a 2 min lag time the formation of fluorescent DiFMU was recorded (λex=358 nm, λem=455 nm) over 40 min at 60 s intervals at 37 °C. During the incubation and between measurements the reaction mixture was shaken. The reaction rate was determined by linear regression analyses (r2≥0.98) of the fluorescence emission increase over time. The background reaction rate with no enzyme present was subtracted and the reaction rates were normalized to the reaction rate with no inhibitor present (100 %).

Microarray screening and SPR validation: The Natural Products Depository (NPDepo) slides were prepared and analyzed as reported before.31 The microarray screening was performed in the following buffer: HEPES (20 mM), NaCl (150 mM), pH 7.53–7.55. For protein and antibody incubation, the array slide was covered by a gap cover glass from Matsunami Glass Ind., Ltd (microscope cover glass, 24×60 mm) and incubated with GST-protein solution (1 μM, 50 μL) in buffer containing skim milk (1 %) at 30 °C for 1 h. After washing, array slides were incubated with anti-GST antibody (rabbit IgG fraction, Invitrogen, 30 μg mL−1) in buffer containing skim milk (1 %) at 30 °C for 1 h. This incubation was followed by another wash step and incubation with a second antibody (Millipore, goat anti-rabbit IgG, Cy5 conjugate, 50 μg mL−1) at 30 °C for 1 h. After the final wash step, slides were scanned at 635 nm with a GenePix 4200AL microarray scanner (Molecular Devices). The photo-crosslinked small-molecule array was placed in an SPR imaging instrument (TOYOBO). SPR signals were obtained in the running buffer [HEPES (20 mM), NaCl (150 mM), pH 7.53–7.55]. The buffer and the protein samples in the same buffer were applied to the array surface at 100 μL min−1. All SPR experiments were performed at 30 °C. The SPR image and signal data were collected with an SPR analysis program (TOYOBO), and the SPR difference image was created by use of Scion Image (Scion, MD).

Western blotting: MDCK or MDCK-F3 cells were seeded at a density of 1.0×105 cells per well in a 6-well dish. The next day, test compounds were added to the cells. After 6 h incubation, the cells were collected and lysed in lysis buffer [HEPES (pH 7.0, 50 mM), NaCl (150 mM), ethylenediaminetetraacetic acid (EDTA, 1 mM), ethyleneglycol tetraacetic acid (EGTA, 2.5 mM), NP-40 (1 %), protease inhibitor cocktail (1 %, Roche), and PhosStopPhosphatase inhibitor cocktail (1 %, Roche)] for 30 min on ice. An equal amount of proteins was loaded onto a 10 % SDS-PAGE gel. The phospho-ERK1/2 and ERK1/2 were detected with anti-phospho-ERK and anti-ERK antibodies, respectively.16

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

This study was supported by the International Max Planck Research School (IMPRS) and the Fonds der Chemischen Industrie with fellowships to T.Z. and SFB 642 from the Deutsche Forschungsgemeinschaft to M.B. We thank the RIKEN-Max Planck Joint Research Center for support, Tamio Saito for supplying compounds from the NPDepo, Kaori Honda, Motoko Uchida and Patricia Stege for technical support, and Raed Al-Zoubi for supplying boronic acids.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental Section
  7. Acknowledgements
  8. Supporting Information

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