Amide-Modified Prenylcysteine based Icmt Inhibitors: Structure Activity Relationships, Kinetic Analysis and Cellular Characterization

Abstract

Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-termimal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng – Prusoff method, the Ki values were determined from the IC₅₀ values. Analog 1a has a K_IC of 1.4 ± 0.2 μM and a K_IU of 4.8 ± 0.5 μM while 1b has a K_IC of 0.5 ± 0.07 μM and a K_IU of 1.9 ± 0.2 μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.

1.0 Introduction

A target of growing interest in the field of cancer chemotherapeutics is the human protein isoprenylcysteine carboxyl methyltransferase (hIcmt).^{1, 2, 3, 4} Icmt is the enzyme responsible for the last common step of the posttranslational processing pathway of CaaX proteins.^{5, 6} The family of CaaX proteins includes many key eukaryotic regulatory proteins, most notably proteins in the Ras family. These proteins terminate with a C-terminal CaaX sequence that is composed of four amino acids in the order of cysteine, followed by two generally aliphatic residues (aa), and a final residue (X) that varies in its identity from protein to protein. The first step of the CaaX protein posttranslational processing pathway is mediated by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I).^7-9 FTase and GGTase-I respectively attach either a 15-carbon or 20-carbon isoprenoid group to the CaaX cysteine. The type of lipid group added is directed, in part, by the nature of the terminal X residue. Lipidation is followed by proteolytic removal of the –aaX residues by the endoprotease Ras converting enzyme-1 (Rce1).¹⁰ Icmt then carries out the final carboxyl methylation step. Disruption of this pathway via enzyme inhibition prevents Ras proteins from localizing properly in the cell, thereby hindering cellular functionality and obstructing cellular growth.^{2, 11, 12} Therefore, the development of inhibitors of the enzymes in this pathway is desirable for the treatment of cancer driven by oncogenic Ras.

Mutant forms of K-Ras are a frequent cause of several human malignancies, including pancreatic cancers. Research on farnesyltransferase inhibitors (FTIs), once promising cancer chemotherapeutic agents, showed that K-Ras tumors are often resistant to FTI treatment due to alternative geranylgeranylation.^13-16 The reason for this difference lies in the fact that K-Ras can be alternatively geranylgeranylated by GGTase I in the presence of FTIs.^17-19 Although, Icmt recognizes both farnesylated and geranylgeranylated proteins,^{20, 21} previous studies suggest that the methylation is more important for the proper localization of farnesylated CaaX proteins than for many geranylgeranylated proteins.^{22, 23}

These data further support that Icmt may prove to be an important target in preventing the function of farnesylated oncogenic Ras proteins, but may not be detrimental to the function of all CaaX proteins. Furthermore, recent studies in cells and mouse models also support Icmt as a promising chemotherapeutic target.^{24, 25} Wang et al. have shown that small molecule Icmt inhibitors can induce autophagic cell death in PC3 prostate cancer cells.²⁴ In mice, inactivation of Icmt has also been shown to inhibit transformation by oncogenic K-Ras and B-Raf.² These key findings further support the development of hIcmt inhibitors as chemotherapeutic agents for Ras-driven cancers.

Our laboratories have recently identified 2-phenoxy-phenylfarnesylcysteine (POP-FC, Figure 1a) and 2-phenoxyphenyl-3-methylbutenyl-farnesylcysteine (POP-3-MB-FC, Figure 1b) as lead Icmt inhibitors.²⁶ These findings prompted us to examine the role of the phenoxy-phenyl motif in achieving Icmt inhibition. The simplest and most widely used amino acid-based Icmt substrate is an amide-modified farnesylated cysteine, N-acetyl-S-farnesylcysteine (AFC).^27-33 The important substrate recognition features of AFC, which mimics the –aaX proteolyzed C-terminus of protein substrates, are the presence of an S-farnesyl moiety, a free α-carboxyl group and an amide modified α-amino group.^{27, 29} Note that the peptide Icmt substrate LARYK-S-farnesyl-C was found to have a 10-fold lower K_M for mammalian Icmt than AFC,³³ suggesting that the upstream sequence plays a role in recognition and methylation efficiency.

Figure 1.

Phenoxyphenyl-farnesylcysteine (a, POP-FC), phenoxy-phenyl-3-methylbutenyl-biphenyl farnesylcysteine (b, POP-3MB-FC) and anthranilic farnesylcysteine (c).

Previous studies from our laboratory have indicated that the human homolog of Icmt may possess an active site that can accommodate substrates or inhibitors with bulky, hydrophobic amide moieties and that certain bulky amide-modified farnesylcysteine (AMFC) analogs can act as low micromolar inhibitors of human Icmt.^{26, 34, 35}In order to further our understanding of the role the phenoxy-phenyl motif plays in human Icmt (hIcmt) inhibition, a library of compounds was designed and synthesized based on the lead POP-FC (1a). Results from this study will help us better understand the role of the phenoxy-phenyl motif in enzyme recognition with the goal of enhancing the potency of these small molecules.

2.0 Chemistry

The analogs described herein were synthesized using previously established methods in our laboratory.^{26, 35} The general synthetic design involved utilizing a known synthesis of farnesylcysteine methyl ester³⁵ and coupling it with the appropriate carboxylic acid using HOBT and HBTU followed by saponification using lithium hydroxide (as shown schematically in Scheme 2).³⁵ In cases where the carboxylic acid was not commercially available, the appropriate aromatic carboxylic acid was synthesized and coupled to farnesylcysteine methylester.

Scheme 2.

General Synthetic Scheme showing Synthesis of Analogs 2a-2h

The structures of the compounds synthesized utilizing this approach are shown in Figure 2. Carboxylic acids needed for the synthesis for compounds 2a-c and 2e were commercially available. For compounds 2d and 2f-h, each carboxylic acid was synthesized as shown in Scheme 1. The general method involved using an alkoxide anion as the nucleophile to displace a benzylic halide to obtain the respective phenoxy-phenyl analog. The aromatic bromide was then converted into a Grignard reagent using magnesium turnings in THF and quenching the formed Grignard with dry ice. Carboxylic acid 5 used to prepare analog 2d was synthesized using an Ullmann-type ether coupling as reported previously.³⁶

Figure 2.

Structures of the phenoxy-phenyl modified analogs synthesized and screened as inhibitors of hIcmt.

Scheme 1. Synthesis of various carboxylic acids.

We also designed and synthesized molecules in which the phenoxy phenyl amide scaffold was maintained but the prenyl chain was modified. To this end, we synthesized analogs 17-21 shown in Figure 3. Their synthesis is depicted in Schemes 3 and 4. Analog 17 is the enantiomer of our lead 1a, while analogs 18 and 19 contain a shorter geranyl (10 carbon) and a longer geranylgeranyl (20 carbon) chain, respectively. Analog 20 (synthesis shown in Scheme 4) was specifically designed to investigate the role of the amide proton in hIcmt inhibition and analog 21, with an undecyl moiety was designed to probe the importance of the prenyl chain in achieving hIcmt inhibition. We have recently reported that a similar change from prenyl to straight chain alkyl is detrimental to hIcmt inhibition when the amide linkage was replaced with a sulfonamide bond.³⁷ We wanted to investigate whether such a change is consistent across different classes of inhibitors. The N-methyl analog 20 was synthesized using a solid-phase methodology using the advanced intermediate 23, which was synthesized as reported previously.³⁸ Compound 23 was loaded on the 2-chlorotrityl-chloride resin, which is essential to this synthesis due to the acid sensitivity of the prenyl chain. After loading, the disulfide-protecting group was removed using DTT and the farnesyl group was attached to the free thiol using farnesyl chloride. The 2-phenoxyphenyl carboxylic acid was then coupled to the secondary amine using standard coupling procedures previously used in our laboratory.²⁶ Compound 20 was obtained upon cleavage from the resin using 0.2% TFA in DCM. Following their syntheses, the compounds were evaluated as substrates and inhibitors of hIcmt using a vapor diffusion assay.^{5, 39, 40}

Figure 3.

Structures of the phenoxy-phenyl prenyl modified analogs synthesized and screened as inhibitors of hIcmt.

Scheme 3. Synthesis of Prenyl-Modified Phenoxy-phenyl Analogs.

Scheme 4. Synthesis of N-Methyl POP-FC.

3.0 Results and Discussion

3.1 In vitro Biochemical Evaluation of Analogs

Analogs 2a-h and 17-21 were first tested as substrates for hIcmt at 25 μM as described in the experimental section. Upon evaluation, none of the analogs showed substrate activity (data not shown). The compounds were subsequently tested as inhibitors at 10 μM in the presence of 25 μM of the substrate AFC and all the analogs were inhibitors of hIcmt to varying degrees. Compounds 2a and 2b were specifically synthesized and tested to evaluate the importance of the positioning of the phenoxy-phenyl motif in hIcmt inhibition. Both of these analogs were poor inhibitors of hIcmt, both inhibiting hIcmt by less than 30% at 10 μM. We experimentally determined the IC₅₀ of compound 2b to be 22.6 ± 1.2 μM. This value reflects an approximately five-fold loss in activity between the ortho and para isomers of the phenoxy-phenyl motif, suggesting that the ortho-phenoxyphenyl motif is important for hIcmt inhibition. Note that the para regio-isomer is a significantly poorer inhibitor compared to both the meta and the ortho isomer.

We have previously reported the synthesis of the anthranilic acid analog (Figure 1c), where the phenoxy-phenyl oxygen is replaced by a nitrogen atom.²⁶ This compound has an IC₅₀ of 7.1 μM against hIcmt. Although less potent than the parent compound 1a, the less electronegative nitrogen does not result in a significant loss of activity. We next wanted to evaluate the effect of changing the size and electronegativity of the central atom connecting the ortho-substituted phenyl ring. To this end, we synthesized analogs 2c and 2d. The decreased electronegativity of the carbon and sulfur atom linkers resulted in a roughly four-fold loss of inhibitory activity as compared to compound 1a, suggesting that the electronegativity of the connecting atom between the two-phenyl rings may be a determining factor for inhibitory activity. More electronegative atoms such as oxygen and nitrogen present in the structure resulted in better inhibitors than those containing less electronegative atoms such as carbon and sulfur.

The biochemical evaluation described above suggested that the ortho substituted phenoxy-phenyl motif was important for hIcmt inhibition. To further investigate the effect of the spatial orientation of the oxygen connector atom and the second phenyl ring on hIcmt inhibition, we synthesized and evaluated analogs 2e-2h. Conformational restriction of the two phenyl rings through a dibenzofuran scaffold (compound 2e) diminished inhibitory activity significantly, as did replacing the central one oxygen linker with a two-atom linker in analogs 2f and 2g. It is worth noting that analog 2g, which retains the position of the oxygen atom relative to the amide bond, displays much greater inhibition as compared to analog 2f. The inhibitory effect of analog 1a could result from the two oxygen atoms’ (the amide carbonyl oxygen and the linker oxygen), involvement in a critical interaction because increasing the distance between those two oxygen atoms reduced the inhibitory potency of the molecule. Analog 2h, where the central oxygen linker is more flexible compared to 1a is a poor inhibitor compared to compound 1a.

To evaluate the effect of the stereochemistry of the amino acid derivative POP (1a) on hIcmt inhibition, we synthesized its enantiomer (17). We have recently demonstrated that the stereochemistry at the alpha carbon is not critical for hIcmt inhibition in a sulfonamide series³⁷ and consistent with this result, analog 17 inhibited hIcmt with an IC₅₀ of 7.8 ± 0.4 μM, nearly equivalent to 1a, which demonstrated an IC₅₀ of 6.2 ± 0.7 μM. Next, to interrogate the importance of the farnesyl chain in analog 1a, we synthesized analogs 18, 19 and 21. The farnesyl group appears to be very important to hIcmt inhibition because the short and longer prenyl analogs 18 and 19 exhibited significant loss of hIcmt inhibition at 10 μM. The undecyl analog, 21, was a particularly poor inhibitor of hIcmt at the test concentration. These data suggest that the presence of a farnesyl chain on the cysteine sulfur is a critical pharmacophore for hIcmt inhibition. These data corroborate our previous findings about the importance of the farnesyl chain for hIcmt inhibition.^{34, 37} Finally, to interrogate the effect of the amide proton on hIcmt inhibition, we synthesized analog 20 using the solid-phase strategy described above. This analog was equivalent to our previous lead, 1a, in terms of percent inhibition of hIcmt activity and has a similar IC₅₀ of 6.7 ± 0.4 μM. The presence of a methyl group on the amide nitrogen appears to have minimal effect on the inhibition potential of the analog, suggesting that the amide region of the molecule may be no more important than serving as a linker between the phenoxy-phenyl and the farnesyl moieties.

Overall, our biochemical data suggest that the ortho-phenoxyphenyl motif is important and replacement with other regioisomers leads to a significant decrease in potency. The presence and the position of the oxygen atom connecting the two phenyl rings are also important features, as any change in the atom itself or its spatial positioning reduces hIcmt inhibition. Stereochemistry about the alpha carbon of cysteine and the amide proton both appear inconsequential to hIcmt inhibition. The most important element in hIcmt inhibition with this class of compounds is the farnesyl chain itself, as any deviation from the 15-carbon isoprene results in a loss of inhibition, which is particularly evident with the undecyl analog 21, a very poor hIcmt inhibitor.

3.2 Kinetic Mode of Inhibition for Compounds 1a and 1b

In order to determine the mode of inhibition of hIcmt by compounds 1a and 1b, the most potent inhibitors in this series of compounds, kinetic assays were performed in which the small molecule substrate (AFC) concentration was varied while the inhibitor concentration remained fixed. The data for each inhibitor concentration were averaged from three trials, and plotted as double reciprocal plots (Figures 4 and 5). These inhibitors displayed mixed-type inhibition in which the competitive component dominated, as demonstrated by an α value greater than 1.⁵³ K_i values for Compounds 1a and 1b were then determined from the IC₅₀ values using the Cheng – Prusoff method as adapted by Cer et al.⁵⁴ For compound 1a, the competitive (K_IC) and uncompetitive (K_IU) values were 1.4 ± 0.2 μM and 4.8 ± 0.5 μM, respectively. For compound 1b, the K_IC and K_IU values were 0.5 ± 0.07 μM and K_IU 1.9 ± 0.2 μM, respectively.

Figure 4. Mode of inhibition and K_I determination of compound 1a.

1a displays mixed-type inhibition. Lineweaver-Burk plots illustrate the mode (mixed) of inhibition of hIcmt by 1a. These are double reciprocal plots from AFC substrate curves with hIcmt, in the presence of the indicated concentration of inhibitor, which were obtained as described in Materials and Methods. The K_IC is 2.6 ± 0.5 μM and the K_IU is 10.5 ± 1.4 μM.

4.0 Cellular Effects of Icmt Inhibitor 1b

4.1 Pop-3MB (1b) alters the subcellular localization of GFP-K-Ras in Jurkat T cells

Proper posttranslational processing is a prerequisite for Ras membrane localization and function. To determine the cellular effects of our most potent inhibitor 1b, the membrane localization of fluorescently tagged K-Ras was visualized in the presence and absence of 1b.^{41, 42} We transfected Jurkat T cells with GFP-K-Ras and treated the cells with delivery vehicle alone, the statin drug simvastatin (which blocks K-Ras prenylation), or 1b. The cells were fixed and GFP-K-Ras localization was visualized using fluorescent microscopy. After 24 hours of treatment, GFP-K-ras localization was categorized into three groups: normal plasma membrane localization, partial mislocalization, and complete mislocalization. This was determined via visual inspection of randomly selected fields containing 100 cells. Representative examples of normal, partial and complete mislocalization are shown in Figure 6b (determined as described in the Experimental section). The results demonstrated that 0.1μM 1b prevented approximately 35% protein membrane localization while treatment with 10 μM compound 1b prevented 75% of membrane localization (Figures 6a and 6b). These data suggest that 1b is taken up into mammalian cells and functions as an Icmt inhibitor, preventing K-Ras membrane localization. Surprisingly, the data indicate that significant cellular mislocalization of GFP-K-Ras is seen at compound levels that are significantly lower than the biochemical IC₅₀ value for 1b. While we are unclear as to the reason for this result, there are at least two possibilities. First, this compound may preferentially accumulate in Jurkat cells. Alternatively, this compound may, in addition to inhibiting Icmt, block GFP-K-Ras through another mechanism, possibly related to that proposed for Salirasib by Kloog and coworkers.^46-48

Figure 6. 1b alters the subcellular localization of GFP K-Ras in Jurkat T cells.

Jurkat T cells were transiently transfected with GFP-K-Ras and treated with DMSO or 1b at indicated concentrations for 24 hrs. (A) Histograms depict the differences in GFP-K-Ras localization after indicated treatments. Differences were defined as partial or complete loss of normal localization, as defined by the images shown in (B). Results are representative of three independent experiments.

4.2 Pop-3MB (1b) inhibits Erk phosphorylation and Ras activation in Jurkat T cells

Low-grade stimulation through the T-cell receptor (TCR) in Jurkat cells results in Ras dependent Erk activation.⁴³ We therefore utilized signaling assays with low grade TCR stimulation to evaluate the effects of 1b on Erk and Ras activation. Cells were treated with delivery vehicle (DMSO), simvastatin, or 1b. After 24-hour vehicle or drug treatment, Jurkat cells were low-grade stimulated through the TCR with anti-CD-3ε to activate the Ras-MAPK-Erk pathway. To test Ras activation, the stimulated cells were lysed and active GTP-bound Ras was precipitated from whole cell lysates using a GST-RBD (Ras Binding Domain) pulldown assay.⁴⁴ As shown in Figure 7b, compound 1b decreased Erk activation approximately three-fold compared to the control. Furthermore, 1b also inhibited Ras activation significantly as seen in Figure 7c. These data suggest that the alterations in TCR-dependent Erk phosphorylation and Ras activation in Jurkat T cells are due to the inhibition of Ras methylation by compound 1b.

Figure 7. Inhibition of anti-CD-3 ε dependent Erk phosphorylation and Ras activation in Jurkat T cells following 1b treatment (25μM).

Jurkat T cells were treated with vehicle, simvastatin (36.7 μM) or 1b for 24 h followed by stimulation with anti-CD-3ε (20μg/mL) (A) The phosphorylation of Erk was detected by immunoblotting the cell lysate with an anti-phospho-Erk antibody (top). The active form of Ras was precipitated from the cell lysate with a GST-RBD fusion protein. Precipitated Ras was detected by immunoblotting using an anti-pan-Ras antibody (bottom). Immunodetection of GAPDH was used as the loading control. Erk phosphorylation (B) and Ras activation (C) were quantified using ImageJ (NIH). The results are representative of three independent experiments.

5.0 Conclusion

We have demonstrated that low-micromolar inhibitors of hIcmt alter the sub-cellular location of GFP-Ras and also have an effect in the downstream pathway. Inhibition of anti-CD-3ε dependent Erk phosphorylation and Ras activation in Jurkat T cells by compound 1b suggests that hIcmt inhibitors hold promise in treatment of K-Ras driven cancers. Our SAR data with the entire series of compounds reported here have provided insight into the potential pharmacophoric requirements for hIcmt inhibition. As there is yet no three-dimensional structural information for hIcmt, given that it is an integral membrane protein, these data generated from our compounds are a small step forward in understanding the inhibition requirements for hIcmt. Stereochemistry at the alpha carbon has no significant bearing on the outcome for inhibition. Our data further suggest that the farnesyl chain is the most important scaffold for substrate-based Icmt inhibitors and any change in length or overall hydrophobicity decreases inhibition. Cysmethynil, a small molecule inhibitor of Icmt⁴⁵ has demonstrated induction of autophagic cell death in PC3 prostate cancer cells,²⁴ and thus has shown promise for cancer therapy. Salirasib, a partial inhibitor of Icmt has also shown promise in phase I clinical trials.^46-48 In contrast, a recent report demonstrated that very potent Icmt inhibitors reduced cell viability only modestly in several cancer cell lines.⁴⁹ These reports, coupled with our results published here suggest that Icmt inhibitors have utility in treating Ras-driven cancers. Further studies with substrate-based hIcmt inhibitors, such as those developed in our laboratory, will help to clarify the therapeutic potential of hIcmt inhibition. Analog 1b is a promising lead and suggests that appropriate prenyl mimics can be developed to achieve potent hIcmt inhibition and efforts are currently underway to exploit these findings.

6.0 Experimental

6.1 Synthesis and Spectral Analysis

General Procedure for the synthesis of analogs 2a-2h

Synthesis of farnesylcysteine methylester

L-cysteine methylester hydrochloride (1.0 eq.) (obtained from Sigma-Aldrich) was charged to a dry round bottom flask and the atmosphere inside the flask was replaced with argon. The reaction mixture was maintained at 0 °C and 5mL of 7 N ammonia in methanol (purchased from Sigma-Aldrich) was added to the L-cysteine methylester hydrochloride. The contents were allowed to stir for 15 min following which, trans-trans-farnesylchloride (0.95 eq.) (purchased from Sigma-Aldrich) was syringed into the flask. The reactants were allowed to react for 3 hours at 0 °C. The reaction was monitored by TLC analysis. The contents of the round bottom flask were evaporated to dryness and purified by column chromatography via isocratic elution using a mixture of methanol and dichloromethane (1:9). This yielded farnesylcysteine methylester in 80% yield. The spectral data matched the reported data.^{35, 50}

Synthesis of the amide-modified farnesylcysteine methylester intermediate

The appropriate carboxylic acid (1.0 eq) was charged to a dry round bottom flask and was dissolved in dimethylformamide (DMF). Diisopropylethylamine (1.1 eq), HOBT (1.1 eq.) and HBTU (1.1 eq.) were added to this and the contents were allowed to react at 0 °C for 30 min. This was followed by addition of a solution of farnesylcysteine methylester in DMF. The contents were allowed to react for two hours. The reaction was monitored by TLC analysis. On completion of the reaction, 10% aqueous citric acid was added to the reaction mixture and the product was extracted using ethylacetate (20 mL) as the solvent. The solvent was removed under vacuum utilizing a rotary evaporator and the residue was loaded on a short (2-3 inches) silica gel plug. The silica gel plug was flushed with 3% methanol in dichloromethane to obtain the crude coupling product.

Synthesis of the amide-modified farnesylcysteine (free acid)

The crude product obtained from the previous step was dissolved in methanol and was transferred to a round-bottom flask. The contents were allowed to cool to 0 °C. Lithium hydroxide (1.1 eq.) was added to the mixture and the contents were allowed to react for two hours. Following the completion of the reaction, the solvent was removed under reduced pressure and the mixture was loaded on a silica gel column. Chromatographic separation was performed under the influence of gravity using a 1:9 mixture of methanol and methylenechloride.

Compound 2a: (R)-2-(4-phenoxybenzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 7.80 (m, 3H), 7.33 (dd, J = 18.3, 10.6 Hz, 2H), 7.06 (m, 4H), 5.41 - 4.74 (m, 4H), 3.13 (m, 4H), 2.10 - 1.87 (m, 8H), 1.67 (d, J = 6.5 Hz, 3H), 1.59 (d, J = 4.5 Hz, 3H), 1.56 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 176.81, 135.09, 134.77, 131.17, 131.06, 129.86, 124.31, 124.03, 123.81, 119.74, 117.51, 39.66, 39.56, 26.69, 26.48, 25.70, 25.66, 25.63, 17.65, 17.59, 16.08, 15.95, 15.79.

Compound 2b: (R)-2-(3-phenoxybenzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 8.03 - 7.68 (m, 4H), 7.67 - 7.46 (m, 3H), 7.37 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 18.4 Hz, 1H), 5.38 (dd, J = 13.7, 7.3 Hz, 3H), 4.98 (d, J = 29.8 Hz, 1H), 3.61 - 2.94 (m, 4H), 2.48 - 2.08 (m, 8H), 1.94 (d, J = 14.1 Hz, 3H), 1.88 (s, 3H), 1.85 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 176.38, 167.17, 157.37, 156.40, 139.73, 135.21, 135.05, 131.12, 129.73, 124.26, 124.22, 123.76, 123.57, 121.85, 121.50, 119.32, 119.07, 117.94, 53.49, 39.61, 39.51, 29.98, 26.64, 26.43, 25.62, 25.59, 17.61, 16.01, 15.91.

Compound 2c: (R)-2-(2-benzylbenzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, MeOH) δ 7.32 (d, J = 6.7 Hz, 1H), 7.18 - 6.89 (m, 8H), 5.05 (t, J = 7.4 Hz, 1H), 4.92 (t, J = 6.3 Hz, 2H), 4.51 (s, 1H), 4.13 - 3.93 (m, 2H), 3.11 - 2.99 (m, 2H), 2.78 (ddd, J = 22.1, 13.6, 5.6 Hz, 2H), 1.98 - 1.72 (m, 8H), 1.50 (d, J = 4.1 Hz, 6H), 1.42 (s, 6H). ¹³C NMR (75 MHz, MeOH) δ 172.60, 160.82, 142.20, 140.57, 140.30, 137.60, 136.13, 132.03, 131.45, 131.01, 130.28, 129.36, 128.46, 127.10, 127.01, 125.43, 125.11, 121.51, 40.83, 40.71, 27.77, 27.42, 25.98, 17.85, 16.35, 16.21.

Compound 2d: (R)-2-(2-(phenylthio)benzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 7.61 (d, J = 32.1 Hz, 1H), 7.37 (s, 3H), 7.28 (d, J = 3.6 Hz, 3H), 7.14 (s, 2H), 7.01 (s, 1H), 5.23 - 4.93 (m, 3H), 4.80 (s, 1H), 3.12 (s, 2H), 2.93 (t, J = 28.2 Hz, 2H), 1.99 (dd, J = 17.7, 6.9 Hz, 8H), 1.67 (s, 3H), 1.59 (s, 5H), 1.55 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 171.57, 168.04, 139.63, 136.60, 135.04, 133.98, 133.09, 131.15, 130.88, 129.32, 128.84, 127.92, 126.13, 124.27, 123.81, 119.51, 77.42, 77.00, 76.57, 58.58, 39.62, 39.53, 33.17, 30.02, 26.65, 26.46, 25.65, 17.64, 16.13, 15.95.

Compound 2e: (R)-2-(dibenzo[b,d]furan-4-carboxamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 8.56 (s, 1H), 8.09 (d, J = 17.2 Hz, 1H), 7.85 - 7.42 (m, 3H), 7.35 (s, 1H), 7.22 (s, 2H), 5.15 (s, 1H), 5.09 - 4.81 (m, 3H), 3.38 - 3.04 (m, 4H), 1.94 (dd, J = 19.0, 7.0 Hz, 8H), 1.64 (s, 3H), 1.56 (s, 3H), 1.47 (d, J = 12.5 Hz, 5H). ¹³C NMR (75 MHz, CDCl₃) δ 176.57, 168.75, 156.31, 152.11, 140.64, 136.04, 132.16, 129.75, 128.50, 125.29, 124.77, 124.21, 123.82, 121.35, 120.57, 112.87, 61.28, 40.60, 40.49, 34.29, 31.15, 27.65, 27.39, 26.64, 18.63, 17.00, 16.89, 12.33.

Compound 2f: (R)-2-(2-(phenoxymethyl)benzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 7.61 (s, 1H), 7.47 (d, J = 6.6 Hz, 2H), 7.30 (d, J = 17.7 Hz, 1H), 7.21 (t, J = 7.0 Hz, 3H), 6.91 (dd, J = 20.5, 7.4 Hz, 3H), 5.34 - 4.92 (m, 5H), 4.61 (s, 1H), 2.97 (s, 3H), 2.62 (s, 1H), 2.10 - 1.79 (m, 8H), 1.68 (s, 3H), 1.60 (s, 3H), 1.55 (s, 3H), 1.51 (s, 3H).

Compound 2g

¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 7.2 Hz, 1H), 8.22 (dd, J = 7.8, 1.6 Hz, 1H), 7.50 (d, J = 6.5 Hz, 2H), 7.46 - 7.29 (m, 4H), 7.13 - 6.98 (m, 2H), 5.26 (d, J = 10.5 Hz, 2H), 5.10 (d, J = 18.0 Hz, 3H), 4.94 (dd, J = 12.1, 6.7 Hz, 1H), 3.08 (ddd, J = 20.4, 13.2, 7.8 Hz, 3H), 2.79 (ddd, J = 20.5, 13.9, 7.8 Hz, 2H), 2.05 - 1.90 (m, 8H), 1.67 (s, 3H), 1.62 - 1.57 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 175.33, 164.65, 156.96, 139.67, 135.43, 135.19, 133.04, 132.37, 131.20, 129.17, 128.49, 127.99, 124.33, 123.68, 121.39, 120.94, 119.57, 112.68, 77.43, 77.00, 76.58, 71.22, 52.44, 52.29, 39.61, 39.53, 32.90, 29.68, 26.63, 26.34, 25.62, 17.62, 16.01, 15.93.

Compound 2h: (R)-2-(2-((benzyloxy)methyl)benzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 8.10 (d, J = 6.8 Hz, 1H), 7.71 (t, J = 8.2 Hz, 1H), 7.36 (dd, J = 11.4, 4.5 Hz, 2H), 7.33 - 7.29 (m, 4H), 7.27 (d, J = 3.7 Hz, 1H), 5.19 - 4.99 (m, 3H), 4.66 (d, J = 11.3 Hz, 1H), 3.36 (s, 4H), 3.07 (dd, J = 12.5, 7.4 Hz, 2H), 2.87 - 2.71 (m, 2H), 2.11 - 1.88 (m, 8H), 1.65 (d, J = 12.1 Hz, 3H), 1.58 (s, 6H), 1.56 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.72, 170.17, 140.64, 138.58, 136.17, 136.09, 132.14, 131.54, 131.21, 130.05, 129.28, 129.22, 128.65, 128.59, 125.21, 124.69, 120.37, 73.07, 71.12, 51.07, 40.57, 40.47, 34.25, 30.78, 27.60, 27.36, 26.58, 18.57, 16.97, 16.87.

Synthesis of Compound 5

To a solution of potassium hydroxide (5.0 eq.) in water were added 2-iodobenzoic acid (1.0 eq.), thiophenol (1.0 eq.) and Copper powder (0.1 eq.). The contents were refluxed for 12 hours, cooled to room temperature and acidified with 4 N hydrochloric acid. Acidification resulted in precipitation of product.

Compound 5: 2-(phenylthio)benzoic acid

¹H NMR (300 MHz, CDCl₃) δ 7.85 (dd, J = 7.8, 1.4 Hz, 1H), 7.37 - 7.27 (m, 2H), 7.24 - 7.12 (m, 3H), 7.02 - 6.95 (m, 1H), 6.87 (dd, J = 10.9, 4.2 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 172.50, 145.62, 136.84, 134.13, 133.11, 133.00, 130.81, 130.30, 128.12, 126.19, 125.24.

General Synthetic methodology for bromides 9, 13 and 15

In general, phenol (1.0 eq.) (or the corresponding benzyl alcohol) was charged to a dry round-bottom flask and anhydrous DMF was added to it. The atmosphere in the flask was replaced with argon. Upon complete dissolution of the phenol (or the benzyl alcohol), the contents were cooled to 0 °C and allowed to stir for 15 min. This was followed by addition of sodium hydride (1.1 eq.) and the compounds were allowed to react for one hour. Following this time period, a solution of the appropriate benzyl bromide (1.2 eq.) was added as a solution in anhydrous DMF over a period of 10 min. The contents were allowed to react for 3 hours. The reaction was quenched with ice and the product was extracted with ether, followed by washing with 10% citric acid (3×20 mL), then washed with 2M NaOH (to remove the unreacted phenol) and brine. The organic fractions were pooled and concentrated under vacuum to yield product. A chromatographic separation was performed using 2:8 ethylacetate and hexanes if it was necessary.

General Procedure for synthesis of carboxylic acids 10, 14 and 16

Magnesium turnings (4.0 eq.) were placed in a dry round bottom flask and flame-dried under a steady stream of argon. Upon cooling of the flask to room temperature, anhydrous THF and one drop of 1,2-dibromoethane were added to the flask. The contents were allowed to react for 5 min followed by addition of the appropriate bromide (as a solution in anhydrous THF) over a period of 5 min. The Grignard reagent was allowed to form for 2.5 hours and was quenched with dry ice. This was followed by addition of 10% hydrochloric acid (10 mL). The contents from the reaction quench were transferred to a separatory funnel, followed by addition of ethylacetate (20 mL). The aqueous phase was extracted with ethylacetate (3 × 20 mL). The combined organic extracts were treated with 5% sodium hydroxide (3 × 5 mL). Concentrated hydrochloric acid was added to the combined aqueous phase until the pH of the solution was 2.5, which resulted in the precipitation of the product. Filtration of the precipitate yielded the desired carboxylic acid in 45 – 78% yields.

Compound 9: 1-bromo-2-(phenoxymethyl)benzene

¹H NMR (300 MHz, CDCl₃) δ 7.71 (d, J = 8.0 Hz, 2H), 7.45 (dt, J = 7.6, 6.4 Hz, 3H), 7.29 (dd, J = 12.1, 4.4 Hz, 1H), 7.14 (t, J = 8.1 Hz, 3H), 5.27 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 159.27, 137.20, 133.40, 130.41, 130.01, 129.69, 128.38, 123.11, 122.05, 115.71, 70.09.

Compound 10: 2-(phenoxymethyl)benzoic acid

¹H NMR (300 MHz, CDCl₃) δ 11.72 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.45 (s, 1H), 7.38 - 7.25 (m, 2H), 7.11 - 6.93 (m, 3H), 5.61 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.87, 159.53, 141.79, 134.68, 132.73, 130.52, 130.48, 128.25, 127.24, 122.00, 115.93, 115.90, 69.02.

Compound 13: 1-(benzyloxy)-2-bromobenzene

¹H NMR (300 MHz, CDCl₃) δ 7.42 (dd, J = 7.8, 1.6 Hz, 1H), 7.33 (d, J = 7.3 Hz, 2H), 7.27 - 7.10 (m, 3H), 7.02 (dd, J = 7.8, 1.1 Hz, 1H), 6.76 - 6.61 (m, 2H), 4.90 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 155.71, 137.26, 134.12, 129.29, 129.20, 128.63, 127.73, 127.71, 122.85, 114.49, 71.29.

Compound 14: 2-(benzyloxy)benzoic acid

¹H NMR (300 MHz, CDCl₃) δ 10.52 (s, 1H), 7.77 (dt, J = 27.4, 13.7 Hz, 2H), 7.36 (d, J = 4.1 Hz, 1H), 7.32 (s, 2H), 7.00 (t, J = 7.5 Hz, 1H), 6.79 (ddd, J = 19.3, 17.3, 8.3 Hz, 3H), 5.15 (s, 2H).

Compound 15: 1-((benzyloxy)methyl)-2-bromobenzene

¹H NMR (300 MHz, CDCl₃) δ 7.62 - 7.49 (m, 2H), 7.49 - 7.27 (m, 6H), 7.23 - 7.08 (m, 1H), 4.69 (s, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 138.97, 138.57, 133.43, 130.06, 129.84, 129.41, 129.37, 128.67, 128.33, 123.68, 73.67, 72.50.

Compound 16

¹H NMR (300 MHz, CDCl₃) δ 7.72 - 7.49 (m, 3H), 7.49 - 7.31 (m, 5H), 7.25 - 7.11 (m, 1H), 4.73 (s, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 171.87, 137.97, 135.77, 133.43, 130.06, 129.84, 129.41, 129.37, 128.67, 127.45, 123.68, 72.58, 71.65.

Compound 17: (S)-2-(2-phenoxybenzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 10.02 (bs, 1H), 8.77 (d, J = 6.7 Hz, 1H), 8.22 (d, J = 7.1 Hz, 1H), 7.35 (t, J = 7.9 Hz, 3H), 7.23 - 7.04 (m, 4H), 6.83 (d, J = 8.2 Hz, 1H), 5.07 (dd, J = 8.1, 3.9 Hz, 3H), 4.95 (d, J = 5.5 Hz, 1H), 3.18 - 2.86 (m, 4H), 1.99 (tt, J = 27.6, 13.8 Hz, 8H), 1.64 (d, J = 14.5 Hz, 3H), 1.58 (d, J = 4.3 Hz, 6H), 1.55 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.65, 165.04, 155.79, 155.01, 139.55, 134.99, 132.96, 132.15, 131.03, 129.85, 124.49, 124.17, 123.67, 123.26, 122.50, 119.66, 119.41, 117.77, 52.82, 39.52, 39.40, 32.71, 29.74, 26.54, 26.27, 25.55, 17.54, 15.85.

Compound 18: (R,E)-3-((3,7-dimethylocta-2,6-dien-1-yl)thio)-2-(2-phenoxybenzamido)propanoic acid

¹H NMR (300 MHz, CDCl₃) δ 10.28 (s, 1H), 8.69 (d, J = 6.6 Hz, 1H), 8.20 (dd, J = 7.8, 1.7 Hz, 1H), 7.36 (dd, J = 10.7, 4.8 Hz, 3H), 7.16 (t, J = 7.4 Hz, 2H), 7.12 – 7.05 (m, 2H), 6.84 (d, J = 8.2 Hz, 1H), 5.10 – 4.98 (m, 2H), 4.91 (dd, J = 11.4, 6.0 Hz, 1H), 3.17 – 2.85 (m, 4H), 2.07 – 1.88 (m, 4H), 1.64 (s, 3H), 1.54 (d, J = 4.7 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 174.74, 165.43, 156.12, 155.24, 139.95, 133.33, 132.39, 131.75, 130.15, 124.84, 123.98, 123.55, 122.66, 119.94, 119.64, 118.08, 52.89, 39.63, 32.63, 29.93, 26.51, 25.78, 17.79, 16.09.

Compound 19: (R)-2-(2-phenoxybenzamido)-3-(((2E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl)thio)propanoic acid

¹H NMR (500 MHz, CDCl₃) δ 9.73 – 9.72 (m, 1H), 8.85 (d, J = 6.8 Hz, 1H), 8.03 (dd, J = 7.8, 1.6 Hz, 1H), 7.46 – 7.32 (m, 3H), 7.18 (td, J = 7.6, 1.0 Hz, 2H), 7.12 – 7.05 (m, 2H), 6.89 – 6.82 (m, 1H), 5.16 – 4.98 (m, 4H), 4.68 (d, J = 4.8 Hz, 1H), 3.16 – 2.89 (m, 5H), 2.12 – 1.86 (m, 12H), 1.63 (d, J = 0.9 Hz, 3H), 1.61 – 1.49 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 177.26, 166.97, 157.50, 157.26, 140.42, 136.37, 136.08, 134.39, 132.73, 132.29, 131.40, 125.96, 125.70, 125.44, 125.36, 124.70, 121.87, 121.20, 119.63, 114.89, 50.10, 41.12, 41.05, 40.93, 34.66, 31.04, 28.07, 27.83, 27.68, 26.19, 18.06, 16.47, 16.43, 16.40.

Compound 20: (R)-2-(N-methyl-2-phenoxybenzamido)-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)propanoic acid

¹H NMR (300 MHz, MeOH) δ 7.33 - 7.11 (m, 4H), 7.10 - 6.93 (m, 3H), 6.92 - 6.81 (m, 2H), 6.80 - 6.56 (m, 1H), 4.94 (d, J = 1.3 Hz, 3H), 4.30 - 4.13 (m, 1H), 3.12 - 2.98 (m, 1H), 2.94 (d, J = 7.6 Hz, 1H), 2.87 (d, J = 5.8 Hz, 1H), 2.81 (s, 2H), 2.72 (dd, J = 13.9, 4.2 Hz, 1H), 2.03 - 1.97 (m, 2H), 1.97 - 1.75 (m, 8H), 1.51 (s, 4H), 1.44 (s, 6H). ¹³C NMR (75 MHz, MeOH) δ 171.94, 157.82, 136.14, 132.05, 131.93, 130.91, 130.87, 125.44, 125.38, 125.13, 124.55, 121.60, 121.09, 120.98, 120.36, 119.12, 62.48, 40.84, 40.70, 30.69, 29.84, 27.78, 27.46, 25.97, 17.84, 16.31, 16.24, 16.20.

Compound 21: ((R)-2-(2-phenoxybenzamido)-3-(undecylthio)propanoic acid)

¹H NMR (300 MHz, MeOH) δ 7.93 (dd, J = 7.8, 1.7 Hz, 1H), 7.37 - 7.18 (m, 3H), 7.18 - 6.93 (m, 4H), 6.88 - 6.65 (m, 1H), 4.48 (t, J = 5.0 Hz, 1H), 2.94 (ddd, J = 33.3, 13.6, 5.0 Hz, 2H), 2.34 (dd, J = 10.9, 3.9 Hz, 2H), 1.39 - 1.23 (m, 2H), 1.12 (d, J = 17.7 Hz, 16H), 0.78 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, MeOH) δ 178.93, 174.66, 166.37, 157.14, 134.02, 133.69, 132.52, 130.86, 125.56, 124.19, 121.04, 119.56, 37.67, 35.60, 33.33, 33.05, 30.97, 30.81, 30.74, 30.71, 30.49, 23.49, 14.39, 5.36.

6.2 Materials and Methods

Materials

N-2-Phenoxybenzoyl-S-3-(3-methyl-but-2-enyl)-5-(4-biphenyl)pent-2-enyl-L-cysteine (POP-3MB, compound 1b) was synthesized in our laboratory²⁶ via established solution-phase methods and characterized to confirm identity and purity. Stock solutions were prepared at 50mM in DMSO and stored at -80 °C. EGFP-C3 vectors containing GFP K-Ras was a generous gift from Mark Philips (New York University, School of Medicine). pGEX vector containing GST-RBD was a kind gift from Lawrence Quilliam (Indiana University School of Medicine). The following antibodies were used: Pan Ras (Oncogene, OP40), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) Rabbit mAb (Cell Signaling Technology, 4377L), GAPDH (Ambion, 4300), CD-3ε (UCHT1) (BD Pharmingen, 555329), ImmunoPure Goat Anti-Rabbit IgG, Peroxidase Conjugated (GARP, Pierce Biotechnology, 31462), ImmunoPure Goat Anti-Mouse IgG, Peroxidase Conjugated (GAMP, Pierce Biotechnology, 31432). Formaldehyde 37% solution was purchased from Mallinckrodt.

Cell culture

Jurkat T cells (E 6.1) were obtained and maintained as described previously.⁴² For all experiments, cells were treated using a vehicle (DMSO) or drug (diluted from a 50mM DMSO stock solution) at indicated concentrations for 24 h.

Fluorescence microscopy

Jurkat T cells (1.5 × 107 cells/500 ul RPMI), transiently transfected with 20 μg of either GFP-K-Ras construct (a kind gift from M. Phillips) using the Cell Porator (Life Technologies, 800 uF, 250 V, low ohms), were treated with delivery group (DMSO), simvastatin alone (20 μM), or various concentrations of POP-3MB (0.1, 1 or 10 μM) for 24 hours in an incubator at 37 °C. The cells were harvested, washed with PBS three times, and plated on polylysine coverslips for 10 min. The coverslips were washed with PBS for 10 min (3 times) and the cells were fixed with 3% formaldehyde for 10 min. The coverslips were washed again for 10 min with PBS (3 times). After mounting the coverslips onto the slides using FluorSaveTM Reagent (Calbiochem), the slides were dried for 45 min at room temperature. Images of cells were viewed using an Olympus BH-2 microscope and captured with a QImaging Microimager II. The captured images were viewed using Northern Eclipse software. Cells were classified as either full (fully mislocalized GFP-K-Ras construct with diffuse cellular staining), partial (partial proper localization), and normal (normal, primarily plasma membrane localization of GFP-K-Ras) by visual inspection.^{41, 42} The subcellular localization of GFP K-Ras was quantified using fluorescence microscopy (performed on an Olympus BH-2RFCA) as previously described.^{24, 51}

Isolation of Membrane Fraction from Yeast Cells

Membrane fractions from yeast cells were isolated as previously described.⁵ Crude membrane protein concentration was determined using Coomassie Plus Protein Assay Reagent (Pierce) according to the manufacturer’s instructions, and compared with a bovine serum albumin standard curve.

Substrate and Inhibition Assays by In Vitro Vapor Diffusion Methyltransferase Assay

Methyltransferase assays were performed as previously described^{5, 39, 40} with minor modifications. The assay mixture contained a total volume of 60 μL and a final Tris-HCl buffer concentration of 100 mM, pH 7.5. All reactions contained 20 μM S-adenosyl-[¹⁴C-methyl]methionine (¹⁴C-SAM) and 5 μg of membrane protein from His-hIcmt-overexpressing strain CH 2766.⁵ Initial substrate screening was performed at a single concentration of 25 μM in duplicate, three times. For inhibition screening, protein and compound (10 μM) were pre-incubated on ice in buffer, followed by the addition of 25 μM AFC substrate and ¹⁴C-SAM. Reactions were incubated at 30 °C for 30 min and stopped by the addition of 50 μL of 1 M NaOH/1% SDS. 100 μL of this mixture was spotted on folded filter paper (6 cm x 2 cm) and lodged in the neck of a scintillation vial containing 10 mL of scintillation fluid, and capped to allow for diffusion of the released [¹⁴C]methanol into the scintillant. The filters were pulled out after 2.5 h, the vials tightly recapped and shaken, and then counted in a liquid scintillation analyzer.

Immunoblotting

Jurkat T cells (3–5 × 105 cells mL -1) were treated with vehicle (DMSO), simvastatin, or POP-3MB (1a). Cells were lysed in 1% Triton X-100 lysis buffer (25 mM Hepes, pH 7.2, 150 mM NaCl, 1% NP40, 5 mM EDTA, 10 μg mL -1 of leupeptin and aprotinin, and 1 mM sodium vanadate), resolved on SDS-PAGE gels, resolved by SDS-PAGE on 12% gels and transferred to polyvinylidene difluoride membranes (Immun-Blot PVDF; Bio-Rad). Membranes were blocked with 5% goat serum and incubated with primary antibody (anti-Pan Ras (Oncogene OP40) for N/K Ras; or anti-GAPDH (Ambion 4300) for GAPDH) at room temperature for 1 h, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. Membranes were developed using enhanced chemiluminescence reagents (ECL; Amersham Pharmacia Biotech) and autoradiography film. Quantitative western blotting was performed using ImageJ system.

Signaling Assay

Jurkat T cells (1 × 107 cells/1 ml) were washed in PBS, incubated with anti-CD-3ε (20μg/ml) for 15 min on ice, and stimulated at 37 °C for 10min. Cells were washed and lysed in lysis buffer containing 1% Triton X-100, 20 mM Tris/HCl, pH 7.6, 150 mM NaCl, 20 μg/ml each of leupeptin and aprotinin, 1 mM pyrophosphate, 1 mM glycerol phosphate, and 1 mM sodium orthovanadate. Samples were separated on 6-15% gradient SDS-polyacrylamide gels and analyzed by immunoblotting with anti-phosphoERK antibodies (at 4 °C overnight with anti-p-Erk (Cell Signaling Technology 4377L)). Membranes were developed using enhanced chemiluminescence reagents (ECL; Amersham Pharmacia Biotech) and autoradiography film. Quantitative western blotting was performed using ImageJ system.

GST-RBD Pull Down Assay

GST-RBD fusion protein was expressed in bacteria as previously described.⁵². After stimulation with anti CD3 antibody, Jurkat T cells (2×10⁷) were lysed in 1% NP40 lysis buffer (1% Nonidet P-40, 25 mM HEPES, 150 mM NaCl, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl₂, 1 mM EDTA, 1 mM sodium vanadate, 10 mM NaF, 20 g/ml aprotinin and leupeptin, pH 7.4) and centrifuged at 464 x g for 3 min at 4 °C. The lysate was incubated with GST-RBD fusion protein conjugated glutathione-Sepharose 4B beads for 30 min at 4 °C. The resin was washed three times with Magnesium Lysis Buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate 10% glycerol, 10 mM MgCl₂), solubilized in 2X PSB (Protein Sample Buffer, Protein Sample Buffer = 100 mM Tris-Hcl, pH 6.8, 20% glycerol, 4%SDS, 0.2% bromophenol blue, 200 mM dithiothreitol) and the presence of activated Ras was detected by immunoblot analysis and quantified using the ImageJ system as mentioned above.

Figure 5. Mode of inhibition and K_I determination of compound 1b.

The phenoxyphenyl analog, 1a, demonstrated mixed-type inhibition with a K_IC of 1.2 ± 0.2 μ M and a K_IU of 11.2 ± 2.4 μM. Lineweaver-Burk plots were obtained by the same method as that described for Figure 4.

Table 1.

Inhibition profiles of the analogs

Structure	Analog	IC50 (μM)a,b
	AFC (Negative Control)	NA
	1a (POP-FC – Pos. Control)	6.2 ± 0.7 a
	1b (POP-3MB- FC) – Pos. Control	2.5± 0.3 a
	2a	63b
	2b	22.6 ± 1.2 a
	2c	38b
	2d	14.7 ± 0.8 a
	2e	58b
	2f	75b
	2g	13.4 ± 0.6 a
	2h	49b
	17	7.8 ± 0.4 a
	18	149b
	19	38b
	20	6.7 ± 0.4 a
	21	210b

^aIC₅₀ values are mean of three experiments, were determined using the vapor diffusion assay and calculated using GRAPH-PAD PRISM 5.0. Concentration of the substrate used (AFC) was 25 μM.

^bEstimated IC₅₀ values obtained using an in-house equation (y = −26.813x + 77.293; where y = percent inhibition at 10 μM and x = log [IC₅₀]) that correlates percent inhibition value at 10μM inhibitor concentration (in presence of 25 μM substrate, AFC, concentration) to experimental IC₅₀ values. This logarithmic correlation has a R² = 0.85.