The Isoprenoid Substrate Specificity of Isoprenylcysteine Carboxylmethyltransferase
2005; Elsevier BV; Volume: 280; Issue: 33 Linguagem: Inglês
10.1074/jbc.m504982200
ISSN1083-351X
AutoresJessica L. Anderson, Brian Henriksen, Richard A. Gibbs, Christine A. Hrycyna,
Tópico(s)Click Chemistry and Applications
ResumoIsoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational α-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-l-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (KI = 17.1 ± 1.7 μm). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast KIC = 35.4 ± 3.4 μm, KIU = 614.4 ± 148 μm; human KIC = 119.3 ± 18.1 μm, KIU = 377.2 ± 42.5 μm). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors. Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational α-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-l-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (KI = 17.1 ± 1.7 μm). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast KIC = 35.4 ± 3.4 μm, KIU = 614.4 ± 148 μm; human KIC = 119.3 ± 18.1 μm, KIU = 377.2 ± 42.5 μm). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors. Ras proteins, notably wild-type and oncogenic K-Ras as well as many other important signal transduction proteins, must undergo post-translational modification to function in eukaryotic cells (1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1719) Google Scholar, 2Young S.G. Ambroziak P. Kim E. Clarke S. 3rd Ed. The Enzymes. 21. Academic Press, San Diego, CA2000: 155-213Google Scholar, 3Gibbs R.A. Zahn T.J. Sebolt-Leopold J.S. Curr. Med. Chem. 2001; 8: 1437-1466Crossref PubMed Scopus (51) Google Scholar). These proteins contain a signature C-terminal CAAX box motif, where C is cysteine, A is generally an aliphatic residue, and X can be one of several different amino acids (Fig. 1A). This consensus sequence is recognized by one of two isoprenyltransferases, FTase 1The abbreviations used are: FTase, protein farnesyltransferase; GGTaseI, protein geranylgeranyltransferase I; Rce1, Ras-converting enzyme 1; Icmt, isoprenylcysteine carboxylmethyltransferase; FPP, farnesylpyrophosphate; AFC, N-acetyl-S-farnesyl-l-cysteine; AGC, N-acetyl-S-geranyl-l-cysteine; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; ESI, electrospray ionization.1The abbreviations used are: FTase, protein farnesyltransferase; GGTaseI, protein geranylgeranyltransferase I; Rce1, Ras-converting enzyme 1; Icmt, isoprenylcysteine carboxylmethyltransferase; FPP, farnesylpyrophosphate; AFC, N-acetyl-S-farnesyl-l-cysteine; AGC, N-acetyl-S-geranyl-l-cysteine; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; ESI, electrospray ionization. (protein-farnesyltransferase) or GGTase I (protein-geranylgeranyltransferase I), which transfers either a 15- or 20-carbon isoprene moiety, respectively, to the cysteine residue. Ras and certain other proteins are farnesylated, but the majority of naturally occuring CAAX proteins are geranylgeranylated. Subsequent to isoprenylation, the three -AAX residues are removed by the endoproteases Rce1 or Ste24, (4Hrycyna C.A. Clarke S. J. Biol. Chem. 1992; 267: 10457-10464Abstract Full Text PDF PubMed Google Scholar, 5Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (304) Google Scholar, 6Ashby M.N. King D.S. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4613-4617Crossref PubMed Scopus (97) Google Scholar), and the resulting cysteine carboxylate is methylated by the S-adenosylmethionine-dependent isoprenylcysteine carboxylmethyltransferase, Icmt (7Hrycyna C.A. Clarke S. Mol. Cell. Biol. 1990; 10: 5071-5076Crossref PubMed Scopus (93) Google Scholar, 8Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar, 9Stephenson R.C. Clarke S. J. Biol. Chem. 1990; 265: 16248-16254Abstract Full Text PDF PubMed Google Scholar, 10Stephenson R.C. Clarke S. J. Biol. Chem. 1992; 267: 13314-13319Abstract Full Text PDF PubMed Google Scholar, 11Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 12Perez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar). The overall effect of this trio of post-translational modification steps is an increase in the hydrophobicity of the modified protein, directing otherwise soluble proteins to their proper intracellular membrane location. Ste14p from Saccharomyces cerevisiae is the founding member of a homologous family of Icmt enzymes present in all eukaryotic organisms (13Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar). Ste14p is a 26-kDa integral membrane protein localized to the endoplasmic reticulum membrane and contains six putative transmembrane spans. A 33-kDa functional human ortholog of the yeast protein, hIcmt (also called pcCMT), which is also localized to the endoplasmic reticulum membrane, was recently identified and characterized (11Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The yeast and human Icmt share 41% identity and 63% similarity, overall, suggesting that their three-dimensional structures and mechanisms of action are similar. In fact, human Icmt expressed in yeast complements a ste14Δ deletion (11Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). We have recently overexpressed both yeast and human Icmt in S. cerevisiae to high levels and characterized their activities in vitro (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, this study). Both of these Icmt variants recognize and modify both farnesylated and geranylgeranylated substrates (1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1719) Google Scholar, 2Young S.G. Ambroziak P. Kim E. Clarke S. 3rd Ed. The Enzymes. 21. Academic Press, San Diego, CA2000: 155-213Google Scholar, 12Perez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar, 14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and are evaluated in the present studies to compare their substrate preferences. To date, the nature of the isoprenylcysteine binding site in all Icmt enzymes remains relatively unexplored. Previous studies have indicated that the isoprene moiety is a crucial recognition element for Icmt, as the minimal compounds N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) are good substrates (1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1719) Google Scholar, 2Young S.G. Ambroziak P. Kim E. Clarke S. 3rd Ed. The Enzymes. 21. Academic Press, San Diego, CA2000: 155-213Google Scholar, 12Perez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar, 14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Shi Y.Q. Rando R.R. J. Biol. Chem. 1992; 267: 9547-9551Abstract Full Text PDF PubMed Google Scholar) and are recognized equivalently by the enzyme (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, aminoacyl modifications were also shown to block protein methylation by crude Icmt (16Marciano D. Ben-Baruch G. Marom M. Egozi Y. Haklai R. Kloog Y. J. Med. Chem. 1995; 38: 1267-1272Crossref PubMed Scopus (110) Google Scholar, 17Ding J. Lu D.J. Perez-Sala D. Ma Y.T. Maddox J.F. Gilbert B.A. Badwey J.A. Rando R.R. J. Biol. Chem. 1994; 269: 16837-16844Abstract Full Text PDF PubMed Google Scholar). In this study, to explore the substrate specificity of Icmt, we focused on the isoprene moiety itself via the synthesis of N-acetyl isoprenylcysteine analogs. Our goals are to use these synthetic compounds as tools for further biochemical exploration of the mechanism of action of the enzyme and starting points for the development of inhibitors. Icmt has emerged as a particularly intriguing target for potential anticancer agents, especially against K-Ras. K-Ras is the most commonly mutated form of Ras found in human malignancies, particularly in solid malignancies (18Adjei A.A. J. Natl. Cancer Inst. 2001; 93: 1062-1074Crossref PubMed Scopus (737) Google Scholar). Targeted inactivation of the Icmt gene in mammalian cells led to a profound mislocalization of K-Ras and a blockage of its ability to promote cellular transformation (19Bergo M.O. Gavino B.J. Hong C. Beigneux A.P. McMahon M. Casey P.J. Young S.G. J. Clin. Investig. 2004; 113: 539-550Crossref PubMed Scopus (143) Google Scholar, 20Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Gomes A.Q. Seabra M.C. Young S.G. J. Biol. Chem. 2001; 276: 5841-5845Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Young S.G. J. Biol. Chem. 2000; 275: 17605-17610Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It has also been demonstrated that blocking the methylation of K-Ras blocks its association with microtubules, which may be crucial for the localization and biological activity of this Ras variant. These findings suggest that the development of inhibitors of the methylation step may prove to be useful for the treatment of human cancers (22Clarke S. Tamanoi F. J. Clin. Investig. 2004; 113: 513-515Crossref PubMed Scopus (23) Google Scholar, 23Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (272) Google Scholar). Recently, potent FTase inhibitors have exhibited promise as anticancer agents in human clinical trials. These inhibitors are thought to have multiple cellular targets, including some Ras proteins. Although these agents can inhibit the growth of H-Ras tumors, they have surprisingly little effect on many K-Ras-transformed tumor types. Importantly, K-Ras can be alternatively geranylgeranylated in the presence of FTase inhibitors whereas H-Ras is not (24Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J.K. J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 25Rowell C.A. Kowalczyk J.J. Lewis M.D. Garcia A.M. J. Biol. Chem. 1997; 272: 14093-14097Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). This alternative modification by GGTase I (24Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J.K. J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 25Rowell C.A. Kowalczyk J.J. Lewis M.D. Garcia A.M. J. Biol. Chem. 1997; 272: 14093-14097Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) apparently allows mutant K-Ras to continue its growth-promoting actions. Recent studies with Icmt–/– fibroblasts have indicated that the methylation of K-Ras proteins by Icmt plays a central role in the cellular localization and transformation ability of this key oncoprotein (19Bergo M.O. Gavino B.J. Hong C. Beigneux A.P. McMahon M. Casey P.J. Young S.G. J. Clin. Investig. 2004; 113: 539-550Crossref PubMed Scopus (143) Google Scholar, 20Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Gomes A.Q. Seabra M.C. Young S.G. J. Biol. Chem. 2001; 276: 5841-5845Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Young S.G. J. Biol. Chem. 2000; 275: 17605-17610Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Combined, these data provide compelling reasons that inhibitors of Icmt have great potential as novel anticancer agents (22Clarke S. Tamanoi F. J. Clin. Investig. 2004; 113: 513-515Crossref PubMed Scopus (23) Google Scholar, 23Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (272) Google Scholar). Many signaling proteins undergo -CAAX processing, and inhibiting Icmt could also target the abnormally high activity of these other signaling proteins in tumor cells, regardless of the specific prenyl group attached to the protein. However, although a recent study confirms that methylation is required for the proper localization of Ras, it also demonstrates that the modification is not necessary for localization of the Rho proteins, another class of CAAX proteins (26Michaelson D. Ali W. Chiu V.K. Bergo M. Silletti J. Wright L. Young S.G. Philips M. Mol. Biol. Cell. 2005; 16: 1606-1616Crossref PubMed Scopus (126) Google Scholar). This differential effect was linked to the fact that Ras is farnesylated, and the Rho proteins are geranylgeranylated, suggesting that Icmt inhibition will have a much more profound inhibitory effect on the activity of farnesylated proteins, such as Ras, than geranylgeranylated proteins. To study both the nature of the substrate binding site as well as to develop potentially useful inhibitors of Icmt, modified analogs of the minimal Icmt substrate AFC, 1 (Fig. 1B), were synthesized (Fig. 2) (27Brown M.J. Milano P.D. Lever D.C. Epstein W.W. Poulter C.D. J. Am. Chem. Soc. 1991; 113: 3176-3177Crossref Scopus (70) Google Scholar) and evaluated as substrates and inhibitors of Icmt using crude membrane preparations from yeast cells overexpressing either Ste14p or human Icmt. These compounds were found to vary widely in their ability to act as substrates, and revealed structural requirements of the key isoprene moiety necessary for recognition by Icmt. Two of the compounds synthesized, the isobutenyl derivative, 3, and the isobutenyl biphenyl derivative, 4, were selected for further study because of their minimal substrate activity, combined with significant inhibitory activity. Both compounds were found to be inhibitors of both yeast and human Icmt in micromolar concentrations. Such inhibitors may be valuable lead compounds for the development of novel anticancer agents (22Clarke S. Tamanoi F. J. Clin. Investig. 2004; 113: 513-515Crossref PubMed Scopus (23) Google Scholar, 23Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (272) Google Scholar). Synthesis of Analogs—The AFC analogs described here, compounds 3 and 4 (Fig. 1B), were synthesized from farnesol analogs that were prepared using close variants of our reported procedures (28Xie H. Shao Y. Becker J.M. Naider F. Gibbs R.A. J. Org. Chem. 2000; 65: 8552-8563Crossref PubMed Scopus (30) Google Scholar, 29Zhou C. Shao Y. Gibbs R.A. Bioorg. Med. Chem. Lett. 2002; 12: 1417-1420Crossref PubMed Scopus (15) Google Scholar, 30Gibbs B.S. Zahn T.J. Mu Y.Q. Sebolt-Leopold J. Gibbs R.A. J. Med. Chem. 1999; 42: 3800-3808Crossref PubMed Scopus (55) Google Scholar, 31Rawat D.S. Gibbs R.A. Org. Lett. 2002; 4: 3027-3030Crossref PubMed Scopus (32) Google Scholar, 32Mu Y.Q. Eubanks L.M. Poulter C.D. Gibbs R.A. Bioorg. Med. Chem. 2002; 10: 1207-1219Crossref PubMed Scopus (25) Google Scholar, 33Reigard S.A. Zahn T.J. Haworth K.B. Hicks K.A. Fierke C.A. Gibbs R.A. Biochemistry. 2005; 44 (in press)Crossref PubMed Scopus (19) Google Scholar). Detailed methods for the synthesis of 3 and 4 are given below and outlined in Fig. 2, A and B. Descriptions of the syntheses of the intermediates for the preparation of 4 are also given, along with proton NMR and MS characterization data. N-Acetyl-S-(3-(3-methylbut-2-enyl)-7,11-dimethyldodeca-2Z,6E,10-trien-1-yl)-l-cysteine (Compound 3)—Chloride 8 (64 mg, 0.24 mmol, 1 equivalent; details for the synthesis of 8 are presented elsewhere) (33Reigard S.A. Zahn T.J. Haworth K.B. Hicks K.A. Fierke C.A. Gibbs R.A. Biochemistry. 2005; 44 (in press)Crossref PubMed Scopus (19) Google Scholar) and N-acetyl-l-cysteine (50 mg, 0.3 mmol, 2 equivalents) were dissolved in 7.0 n NH3/MeOH (10 ml/mmol chloride), stirred at 0 °C for 1 h, and then at 20 °C for 1 h. The resulting mixture was concentrated by rotary evaporation. The crude compound was taken up in MeOH/CH2Cl2 and directly purified by flash column (gradient of 10–30% methanol in CH2Cl2) to afford compound 3 in a 60% yield of 61 mg, based on the alcohol 7. 1H NMR (300 MHz, CDCl3): 1.57 (s, 6H), 1.63 (s, 3H), 1.70 (two s, 6H), 2.0–2.1 (narrow m, 14H), 2.71 (narrow m, 2H), 2.9 (br, 2H), 3.16 (narrow m, 2H), 4.7 (narrow m, 1H), 4.95 (app t, 1H), 5.11 (app t, 2H), 5.24 (t, 1H), 6.45 (d, 1H), 9.3–9.7 (very br, 1H). 13C (75 MHz, CDCl3) 16.42, 18.09, 18.3, 23.36, 26.11, 27.12, 30.49, 34.02, 37.42, 40.11, 122.38, 124.24, 124.74, 131.02, 132.67, 135.74, 144.307, 171.52. MS-ESI (M-H) = 420. Elemental Analysis: calculated for C24H38NO3SK0.70Na0.30: C, 63.38; H, 8.42; found: C, 63.28, H, 8.52. Ethyl 3-(But-3-methyl-2-en-1-yl)-5-(4-phenyl)phenylpent-2Z-enoate (10Stephenson R.C. Clarke S. J. Biol. Chem. 1992; 267: 13314-13319Abstract Full Text PDF PubMed Google Scholar)—Triflate 9 (synthesized as previously described, Ref. 29Zhou C. Shao Y. Gibbs R.A. Bioorg. Med. Chem. Lett. 2002; 12: 1417-1420Crossref PubMed Scopus (15) Google Scholar; 350 mg, 0.78 mmol), CuO (620 mg, 7.8 mmol), Ph3As (23 mg, 0.078 mmol), and bis(benzonitrile)-palladium (II) chloride (16.5 mg, 0.0429 mmol) were placed in an argon-flushed flask and dissolved in N-methylpyrrolidone (6 ml). The mixture was immersed in an oil bath maintained at a temperature of 100–104 °C, (3-methylbut-2-enyl)tributyltin (0.393 ml, 1.17 mmol) was added, and the reaction mixture was stirred for 12 h. It was then cooled, taken up in ethyl acetate (25 ml), and washed with aqueous KF (2 × 20 ml) and H2O(2 × 20 ml). The aqueous layers were back-extracted with ethyl acetate (30 ml), and the combined organic layers were dried (MgSO4), filtered, and concentrated. Purification by flash chromatography (hexane/ethyl acetate 98:2) gave 10, in an 83% yield (230 mg). 1H NMR (300 MHz, CDCl3): 1.3 (t, 3H), 1.8 (t, 6H), 2.5 (t, 2H), 2.9 (t, 2H), 3.6 (d, 2H), 4.3 (q, 2H), 5.3 (t, 1H), 5.8 (t, 1H), 7.2 (t, 2H), 7.3 (t, 1H), 7.35 (d, 2H), 7.45 (d, 2H), and 7.5 (d, 2H). 3-(But-3-methyl-2-en-1-yl)-5-(4-phenyl)phenylpent-2Z-en-1-ol (11Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar)— Compound 10 (230 mg, 0.65 mmol) was dissolved in anhydrous toluene (3 ml) and chilled to –78 °C. A 1.0 m solution of DIBAL-H (1.83 ml, 1.83 mmol) was added dropwise. The solution was stirred for 1 h at –78 °C and was then warmed slightly and quenched with 10% aqueous sodium potassium tartrate. The layers were separated, and the aqueous layer was extracted (3 × 20 ml) with ethyl acetate. The organic layers were combined, washed with brine (10 ml), dried (MgSO4), filtered, and concentrated. Purification by flash chromatography (hexane/ethyl acetate 90:10) gave 11, in a 76% yield (150 mg). 1H NMR (300 MHz, CDCl3): 1.8 (t, 6H), 2.5 (t, 2H), 2.9 (t, 2H), 3.6 (d, 2H), 4.1 (d, 2H), 5.3 (t, 1H), 5.8 (t, 1H), 7.2 (t, 2H), 7.3 (t, 1H), 7.35 (d, 2H), 7.45 (d, 2H), and 7.5 (d, 2H). 1-Chloro-3-(but-3-methyl-2-en-1-yl)-5-(4-phenyl)phenylpent-2Z-ene (12Perez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar)—NCS (N-chlorosuccinimide; 55 mg, 0.39 mmol) was dissolved in CH2Cl2 (distilled from CaH2), and the resulting solution was cooled to –30 °C with a dry ice/acetonitrile bath. Dimethyl sulfide (0.028 ml, 0.39 mmol) was added dropwise by syringe, and the mixture was warmed to 0 °C, maintained at that temperature for 15 min, and cooled to –30 °C. To the resulting milky white suspension was added dropwise a solution of the alcohol 11 (80 mg, 0.26 mmol; dissolved in CH2Cl2). The suspension was warmed to 0 °C and stirred for 3 h. The ice bath was removed, and reaction mixture was warmed to room temperature and stirred for an additional 2 h. The resulting solution was washed with hexane (2 × 20 ml). The hexane layers were then washed with brine (2 × 20 ml) and dried over MgSO4. The compound was further elaborated to compound 4 without any purification. 1H NMR (300 MHz, CDCl3): 1.8 (t, 6H), 2.5 (t, 2H), 2.9 (t, 2H), 3.6 (d, 2H), 4.0 (d, 2H), 5.3 (t, 1H), 5.8 (t, 1H), 7.2 (t, 2H), 7.3 (t, 1H), 7.35 (d, 2H), 7.45 (d, 2H), and 7.5 (d, 2H). N-Acetyl-S-(3-(3-methylbut-2-enyl)-5-(4-phenyl)phenylpent-2Z-en-1-yl)-l-cysteine (Compound 4)—Chloride 12 (70 mg, 0.216 mmol, 1 equivalent) and N-acetyl-l-cysteine (39 mg, 0.238 mmol, 1.1 equivalents) were dissolved in 7.0 n NH3/MeOH (10 ml/mmol chloride), stirred at 0 °C for 1 h and then at 20 °C for 1 h. The resulting mixture was concentrated by rotary evaporation. The crude compound was taken up in MeOH/CH2Cl2 and directly purified by flash column (eluting with a gradient of 10–30% methanol in CH2Cl2) to afford compound 4 in a 50% yield (49 mg), based on the alcohol 11. 1H NMR: (300 MHz, CDCl3) 1.63 (s, 6H), 2.4 (t, 2H), 2.8 - 3.0 (m, 6H), 3.2 (m, 2H) 4.7 (m, 1H), 5.2 (t, 1H), 5.5 (t, 1H), 7.2 (t, 2H), 7.3 (t, 1H), 7.35 (d, 2H), 7.45 (d, 2H), and 7.5 (d, 2H). MS ESI (M-H) = 450. Yeast Strains and Media—Plasmid-bearing strains were created by transformation of the indicated plasmid into SM1188, which does not express Ste14p, using the method of Elble (34Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar) with the following modification: dithiothreitol was added to a final concentration of 50 mm to increase the transformation efficiency. All strains were grown at 30 °C on synthetic complete solid media without uracil (SC-URA). The SM1188 strain was kindly provided by S. Michaelis (Johns Hopkins University School of Medicine). Cloning of His-Ste14p—pCHH10m3N-STE14 (His-Ste14p), which encodes Ste14p with a 10× histidine tag followed by a triply iterated myc epitope repeat at the N terminus under the constitutive control of the phosphoglycerate kinase (PGK) promoter, was constructed as previously described (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cloning of Human Icmt—Wild-type human Icmt (hIcmt) cDNA was a generous gift from S. Michaelis (Johns Hopkins University School of Medicine). The hIcmt gene was amplified by PCR after removal of an internal EagI restriction site, which does not result in a change in the amino acid sequence, and the product cloned into the pCHH10m3N vector. The resulting plasmid was pCHH10m3N-hIcmt (His-hIcmt). The plasmid was sequenced bidirectionally to confirm the DNA sequence. Isolation of Membrane Fraction from Yeast Cells—Membrane fractions from yeast cells were isolated as previously described (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 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 prepared by the same procedure. Immunoblot Analysis—Protein samples in the presence of 1× Laemmli sample buffer were heated to 65 °C for 15 min and resolved by 12% SDS-PAGE. Proteins were transferred to a pure nitrocellulose membrane (0.2 μm; Schleicher & Schuell BioScience GmbH) at 400 mA for 1 h. The filter was blocked with 20% milk in phosphate-buffered saline with Tween (PBST; 137 mm NaCl, 2.7 mm KCl, 4 mm Na2HPO4, 1.8 mm KH2PO4, and 0.05% Tween 20, pH 7.4) and then incubated with the primary antibody (1:10,000 α-myc) dissolved in 5% milk in PBST for 3 h at room temperature. Following three washes with PBST, the filter was incubated with the secondary antibody (1:4,000 goat α-mouse horseradish peroxidase). After several washes with PBST, protein bands were visualized by enhanced chemiluminescence (Super Signal West Pico Chemiluminescent Substrate; Pierce). In Vitro Vapor Diffusion Methyltransferase Assay—Methyltransferase assays were performed as described (7Hrycyna C.A. Clarke S. Mol. Cell. Biol. 1990; 10: 5071-5076Crossref PubMed Scopus (93) Google Scholar, 9Stephenson R.C. Clarke S. J. Biol. Chem. 1990; 265: 16248-16254Abstract Full Text PDF PubMed Google Scholar, 35Hrycyna C.A. Wait S.J. Backlund Jr., P.S. Michaelis S. Methods Enzymol. 1995; 250: 251-266Crossref PubMed Scopus (27) Google Scholar, 36Ota I.M. Clarke S. J. Biol. Chem. 1989; 264: 12879-12884Abstract Full Text PDF PubMed Google Scholar) with minor modifications. The assay mixture contained a total volume of 60 μl and a final Tris-HCl concentration of 100 mm, pH 7.5. All reactions contained 20 μm S-adenosyl-[14C-methyl]methionine and 5 μg of membrane protein from the His-Ste14p-overexpressing strain CH2704 or the His-hIcmt-overexpressing strain CH2766. Substrate curves were generated by varying the amount of compound in each reaction. Inhibition curves were generated by varying the amount of compound in each reaction while in the presence of 33 μm AFC. KI curves were generated by varying the amount of AFC in the presence of a constant concentration of 3 or 4. 3 was dissolved in ethanol. 1 and 4 were dissolved in dimethylsulfoxide. In all cases, the solvent was kept at a final concentration of <1% (v/v). The 60-μl reactions were incubated at 30 °C for 30 min. The reaction was stopped with the addition of 50 μl of 1 m NaOH/1% SDS. 100 μl of this mixture was spotted on folded filter paper (5.5 cm × 1.5 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 2–3 h, the vials were shaken, and counted in a liquid scintillation analyzer. Substrate and Inhibition Assays with Purified His-Ste14p Protein— His-Ste14p was purified as previously described (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), except samples were eluted in buffer containing 200 mm EDTA, pH 8.0 instead of 1 m imidazole. For substrate curves with purified His-Ste14p, liposomes containing substrate were prepared by rapid filtration through a Sephacryl S-100 high resolution (Amersham Biosciences) column, the substrate concentration was quantified, and the liposomes used in in vitro vapor diffusion methyltransferase assays as previously described (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). For inhibition assays, both the substrate, AFC, and the inhibitor, either compound 3 or compound 4, were incubated with lipid. The substrate was at a constant concentration (50 μm), and the inhibitor was in increasing concentrations. The purified protein was reconstituted, and in vitro vapor diffusion methyltransferase assays were performed as previously described (14Anderson J.L. Frase H. Michaelis S. Hrycyna C.A. J. Biol. Chem. 2005; 280: 7336-7345Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). All m
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