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A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group
Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group Ayumi Tanaka a, Mohamed O. Radwan a,b, Akiyuki Hamasaki a, Asumi Ejima a, Emiko Obata a, Ryoko Koga a, Hiroshi Tateishi a, Yoshinari Okamoto a, Mikako Fujita c, Mitsuyoshi Nakao d, Kazuo Umezawa e, Fuyuhiko Tamanoi f, Masami Otsuka a,⇑ a
Department of Bioorganic Medicinal Chemistry, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan Department of Chemistry of Natural Compounds, National Research Center, Dokki 12622, Cairo, Egypt Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan d Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan e Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195, Japan f Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, California NanoSystems Institute, University of California Los Angeles, Los Angeles, CA 90095, USA b c
a r t i c l e
i n f o
Article history: Received 6 May 2017 Revised 18 June 2017 Accepted 19 June 2017 Available online 20 June 2017 Keywords: Farnesyltransferase Zinc protein Ras protein Farnesyl group
a b s t r a c t Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity. Ó 2017 Elsevier Ltd. All rights reserved.
Modification of proteins by prenyl lipids, e.g., farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid lipid, is often crucial for protein function in a cell.1,2 The first step of prenylation is catalyzed by a zinc-containing farnesyltransferase or geranylgeranyltransferase, which conjugates a farnesyl/geranylgeranyl group of farnesyl/geranylgeranyl pyrophosphate to a cysteine residue of the C-terminal CAAX motif (X: aliphatic amino acid; A: any amino acid) of the substrate protein. Subsequently, the terminal AAX tripeptide is removed by endoprotease digestion and the resulting carboxyl of the prenylated cysteine is methylated by methyltransferase. The attached prenyl group, with its hydrophobic character, facilitates the translocation of the prenylated protein to the membrane. Protein prenylation is associated with various diseases, such as cancer, progeria, infectious diseases, glaucoma and neurological diseases.1,3 Thus, many inhibitors of farnesyltransferase/geranylgeranyltransferase have been developed, including some under Abbreviations: DTT, dithiothreitol.
⇑ Corresponding author.
E-mail address: [email protected] (M. Otsuka). http://dx.doi.org/10.1016/j.bmcl.2017.06.047 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
clinical investigation.1–4 Most of the inhibitors are CAAX analogues, farnesyl/geranylgeranyl pyrophosphate analogues and screened library compounds. However, the zinc site of these transferases has yet to be a major inhibitory target. We developed previously a dithiol compound SN-1 (Fig. 1) and showed that SN-1 inhibits the function of zinc proteins5–7 via a zinc-binding mechanism.8 In particular, we successfully endowed SN-1 with farnesyltransferase specificity by introducing a protein-recognizing naphthyl group, i.e., naphthyl-SN-1 (Fig. 1).9 This compound inhibited farnesyltransferase, which is known to farnesylate the Ras oncoprotein to cause cancer.10–12 We herein report novel SN-1 derivatives with a farnesyl/dodecyl group that bind into the farnesyl pyrophosphate-binding pocket of farnesyltransferase. Analogous to the case of naphthyl-SN-1, we placed a farnesyl group at the 4-position of pyridine, i.e., farnesyl-SN-1 (Fig. 1). The structure of farnesyl-SN-1 seemed rational, as indicated by the docking study using the Molecular Operating Environment (MOE) 2014.09. As a template for docking, we selected the X-ray crystal structure (PDB ID: 1SA4) of human farnesyltransferase complexed with farnesyl pyrophosphate and an inhibitor Tipifarnib, which binds to the catalytic domain with high affinity.3,13
A. Tanaka et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866
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Fig. 1. Structures of SN-17 and its derivatives with naphthyl,9 farnesyl and dodecyl groups.
Fig. 2. Binding mode of farnesyl-SN-1 into farnesyltransferase catalytic domain as predicted by the MOE 2014.09 using human farnesyltransferase complexed with the inhibitor Tipifarnib and farnesyl pyrophosphate (PDB ID: 1SA4).13 Structural refinement, protonation, removal of water molecules and energy minimization were carried out using MOE LigX function. Farnesyl-SN-1 was created, energy minimized, and then docked by the default docking protocol implemented in MOE.8 (A) 3D depiction. FarnesylSN-1 (green), Tipifarnib (yellow) and farnesyl pyrophosphate (FPP, blue) are shown. Farnesyl-SN-1 binds to Zn2+, Arg202 and Tyr361 residues. (B) 2D depiction. Proximal amino acids and settled interactions (dashed lines and arrows) are shown.
As shown in Fig. 2, the docking result revealed that both the SN-1 moiety and the farnesyl group of farnesyl-SN-1 fit the catalytic domain of farnesyltransferase well with a binding score of 8.1 kcal/mol. One thiol group of farnesyl-SN-1 binds to zinc and Tyr361, whereas the other thiol interacts with Arg202. Interestingly,
the farnesyl group of farnesyl-SN-1 is accommodated in the farnesyl pyrophosphate pocket. In addition to farnesyl-SN-1, we also designed dodecyl-SN-1 with a saturated straight-chain dodecyl group (Fig. 1). We also synthesized the corresponding disulfides, farnesyl-SN-1 disulfide
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Scheme 1. Synthesis of farnesyl-SN-1 disulfide and dodecyl-SN-1 disulfide.
and dodecyl-SN-1 disulfide. The synthesis was based on our previous synthesis of biotin-linked SN-1.8 As shown in Scheme 1, 4-aminopyridine carrying Boc-protected thiazolidin rings (1) was
reacted with triphosgene followed by addition of farnesol or dodecyl alcohol to give the coupled product 2a or 2b.14 Removal of the Boc group of 2a or 2b furnished 3a or 3b.15 Treatment of
A. Tanaka et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866 Table 1 In vitro inhibitory activity of SN-1 derivatives against farnesyltransferase.a Compound
IC50 (lM)
Farnesyl-SN-1 Dodecyl-SN-1 Naphtyl-SN-1
0.90 2.0 1.9b
a According to a method using human farnesyltransferase, Dansyl-GCVLS and farnesyl pyrophosphate.19 b Reported previously.9
3a or 3b with reducing agent NaCNBH3 furnished farnesyl-SN-1 4a and dodecyl-SN-1 4b that spontaneously gave farnesyl-SN-1 disulfide 5a16 or dodecyl-SN-1 disulfide 5b17 by air oxidation.18 Farnesyl-SN-1 and dodecyl-SN-1 were generated in situ by reduction of their disulfide 5a and 5b with DTT, and the biological
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activity was evaluated. The in vitro inhibitory activity against farnesyltransferase was evaluated by a method that observes intramolecular fluorescence enhancement using a dansyl marked peptide containing CAAX as a substrate.19,20 The results are shown in Table 1. The IC50 against farnesyltransferase of farnesyl-SN-1 and dodecyl-SN-1 were 0.90 and 2.0 lM, suggesting improvement of the inhibitory activity because the previously reported IC50 of naphthyl-SN-1 was 1.90 lM. The stronger activity of farnesyl-SN1 compared with that of dodecyl-SN-1 shows the effect of the p-electron interaction between the double bonds of the farnesyl group and protein aromatic residues, as shown in Fig. 2 We then examined the inhibitory activity against the proliferation of the pancreatic cell line AsPC-121,22 expressing the K-Ras mutant (active form), which causes cancer.10 Expecting the reduction of the disulfide bond by intracellular reductants such as glutathione, the cells were treated with the prodrug farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide, and the number of living cells
(A)
(B)
(C)
Fig. 3. Inhibitory activity of farnesyl-SN-1 and dodecyl-SN-1 against proliferation of AsPC-1 and BxPC-3 cells. Cells were stained by trypan blue, and unstained cell numbers were counted. (A) Living cell numbers of AsPC-1 after treatment with the indicated concentration of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide. (B) Living cell numbers of AsPC-1 after treatment with 10 lM of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide in the presence of DTT. (C) Living cell numbers of BxPC-3 after treatment with the indicated concentration of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide.
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was counted at 1 and 2 d post-treatment.23 The results are shown in Fig. 3A. Farnesyl-SN-1 at 0.5, 5 and 10 lM inhibited cellular proliferation. On the other hand, the inhibitory activity of dodecyl-SN-1 was concentration-dependent. The stronger activity of farnesyl-SN-1 when compared with that of dodecyl-SN-1 is in accordance with the in vitro activity against farnesyltransferase (Table 1). In the activity of farnesyl-SN-1 (0.5, 5 and 10 lM) and that of dodecyl-SN-1 (10 lM), activity 2 d post-treatment was more critical than that observed after 1 d. This is presumably due to the slow reduction of the disulfide in the cells. Thus, the same experiment using 10 lM of the disulfide compounds treated with DTT was performed. As expected, both farnesyl-SN-1 and dodecyl-SN-1 were effective inhibitors 1 d post-treatment (Fig. 3B). Next, the inhibitory effect against proliferation of BxPC-3,24 which is also a pancreatic cell line but does not have the mutation in the Ras protein,10 was examined.23 As shown in Fig. 3C, 10 lM of farnesyl-SN-1 was effective, but activity of 0.5 and 5 lM of the compound was weaker when compared with the results of the AsPC-1 cells. In contrast, no clear difference was not seen between AsPC-1 and BxPC-2 when these cell lines were treated with dodecyl-SN-1. These results suggests that 0.5 and 5 lM of farnesyl-SN-1 inhibit farnesylation of the Ras protein in a cell, thereby suppressing proliferation of AsPC-1 cells. The activity of 10 lM of farnesyl-SN-1 and dodesyl-SN-1 would be different from the inhibitory effect of Ras farnesylation. Taken together, we have developed a new inhibitor against protein prenylation that consists of a zinc-binding part and a prenyl group. The SN-1 moiety is revealed to have binding specificity toward zinc ions among various divalent cations (Table S1). Introduction of a protein-recognition part on SN-1 confers binding specificity toward zinc proteins. Further modification should improve the activity of our compounds to gain IC50 values acceptable to initiate clinical studies. Acknowledgements This work was supported by a Grant-in-Aid for Exploratory Research (B) (23390028) (to M. O.) and a Grant-in-Aid for Scientific Research (C) (26460148) (to Y. O.).
A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.06. 047. References 1. Wang M, Casey PJ. Protein prenylation: unique fats make their mark on biology. Nat Rev Mol Cell Biol. 2016;17:110–122. 2. Palsuledesai CC, Distefano MD. Protein prenylation: enzymes, therapeutics, and biotechnology applications. ACS Chem Biol. 2015;10:51–62. 3. Shen M, Pan P, Li Y, Li D, Yu H, Hou T. Farnesyltransferase and geranylgeranyltransferase I: structures, mechanism, inhibitors and molecular modeling. Drug Discovery Today. 2015;20:267–276. 4. Tamanoi F, Gau CL, Jiang C, Edamatsu H, Kato-Stankiewicz J. Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells. Cell Mol Life Sci. 2001;58:1636–1649. 5. Ejima T, Hirota M, Mizukami T, Otsuka M, Fujita M. An anti-HIV-1 compound that increases steady-state expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G. Int J Mol Med. 2011;28:613–616. 6. Otsuka M, Fujita M, Sugiura Y, et al. Synthetic inhibitors of regulatory proteins involved in the signaling pathway of the replication of human immunodeficiency virus 1. Bioorg Med Chem. 1997;5:205–215.
7. Fujita M, Otsuka M, Sugiura Y. Metal-chelating inhibitors of a zinc finger protein HIV-EP1. Remarkable potentiation of inhibitory activity by introduction of SH groups. J Med Chem. 1996;39:503–507. 8. Radwan MO, Sonoda S, Ejima T, et al. Zinc-mediated binding of a lowmolecular-weight stabilizer of the host anti-viral factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G. Bioorg Med Chem. 2016;24:4398–4405. 9. Hamasaki A, Naka H, Tamanoi F, Umezawa K, Otsuka M. A novel metalchelating inhibitor of protein farnesyltransferase. Bioorg Med Chem Lett. 2003;13:1523–1526. 10. Aoki K, Yoshida T, Sugimura T, Terada M. Liposome-mediated in vivo gene transfer of antisense K-ras construct inhibits pancreatic tumor dissemination in the murine peritoneal cavity. Cancer Res. 1995;55:3810–3816. 11. Manne V, Roberts D, Tobin A, et al. Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci USA. 1990;87:7541–7545. 12. Goodman LE, Judd SR, Farnsworth CC, et al. Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins. Proc Natl Acad Sci USA. 1990;87:9665–9669. 13. Reid TS, Beese LS. Crystal structures of the anticancer clinical candidates R115777 (Tipifarnib) and BMS-214662 complexed with protein farnesyltransferase suggest a mechanism of FTI selectivity. Biochemistry. 2004;43:6877–6884. 14. A solution of 1 (156 mg, 0.333 mmol) and pyridine (222 lL, 2.76 mmol) in CH2Cl2 (4 mL) was cooled at 0 °C. A solution of triphosgene (112 mg, 0.377 mmol) in CH2Cl2 (4 mL) was added at 0 °C, and a solution of 1dodecanol (74.6 lL, 0.333 mmol) in CH2Cl2 (4 mL) was subsequently added at 0 °C. The reaction mixture was stirred at ambient temperature for 2.5 h, and the solution was concentrated in vacuo. Aq-NaHCO3 (50 mL) was then added, and extracted with CH2Cl2 (20 mL) 5 times. The extract was dried over MgSO4, the solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 10:1) to afford 2b (222 mg, 98%). The same procedure using trans,trans-farnesol instead of 1-dodecanol gave 2a (77%). 15. To a solution of 2b (166 mg, 0.244 mmol) in toluene (2 mL), p-toluenesulfonic acid (81.1 mg, 0.471 mmol) was added. The reaction mixture was stirred at 40 °C for 1.5 h, and the solution was concentrated in vacuo. Water (50 mL) was then added, and extracted with CH2Cl2 (20 mL) 5 times. The extract was dried over MgSO4, and the solvent was removed in vacuo to afford 3b (81.8 mg, 70%). The same procedure using 2a instead of 2b gave 3a (50%). 16. 1H NMR (CDCl3) d: 1.45–1.98 (m, 12H), 2.01–2.17 (m, 8H), 2.73–3.65 (m, 12H), 4.09 (bs, 2H), 4.67 (s, 2H), 5.07–5.16 (m, 2H), 5.38–5.50 (m, 1H), 7.51–7.64 (m, 2H), 9.39 (bs, 1H). 13C NMR (CDCl3) d: 16.0, 16.6, 17.6, 21.5, 22.3, 25.6, 26.2, 26.3, 26.6, 38.3, 39.6, 49.9, 62.6, 102.7, 106.2, 118.2, 123.5, 124.3, 131.2, 135.4, 141.8, 142.6, 146.7, 151.1, 153.4. HRMS(FAB) m/z calcd for C27H43N4O2S2 [M +H]+ 519.2827 Found: 519.2823. 17. 1H NMR (CD3OD) d: 0.89 (t, 3H, J = 6.4 Hz), 1.28 (m, 20H), 1.69 (t, 2H, J = 6.8 Hz), 2.93 (t, 4H), 3.08-3.12 (m, 4H) 3.21 (t, 4H), 4.16–4.20 (m, 2H), 7.48–7.51(m, 2H). 13C NMR (CD3OD) d: 14.4, 23.6, 29.9, 30.3, 30.4, 30.6, 30.7, 33.0, 34.9, 38.5, 51.6, 52.0, 52.2, 52.3, 66.7, 111.8, 150.0, 155.0. HRMS(FAB) m/z calcd for C24H43N4O2S2 [M+H]+ 483.2827 Found: 483.2791. 18. To a solution of 3b (117 mg, 0.243 mmol) in dried CH2Cl2, NaCNBH3 (154 mg, 2.45 mmol) and 0.1 M HCl methanol solution were added to adjust pH 3. The reaction mixture was stirred at ambient temperature for 2 h, and neutralized with 1 M aq-NaOH. The solution was concentrated in vacuo, and extracted with CH2Cl2. The solvent was then removed in vacuo to afford dodecyl-SN-1 disulfide 5b (49.0 mg, 42%). The same procedure using 3a instead of 3b gave farnesyl-SN-1 disulfide 5a (42%). 19. Pompliano DL, Gomez RP, Anthony NJ. Intramolecular fluorescence enhancement: a continuous assay of Ras farnesyl:protein transferase. J Am Chem Soc. 1992;114:7945–7946. 20. Human farnesyltransferase (Jena Biosciences, Jena, Germany) (0.03 lM), dansyl-GCVLS (Sigma-Aldrich, St Louis, MO, USA) (1.0 lM), and farnesyl pyrophosphate (Sigma-Aldrich) (10 lM) in assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, and 0.2% n-octyl-b-D-glucopyranoside) were incubated with or without Farnesyl-SN-1/Dodecyl-SN-1 (0.05, 0.50, 5.00 or 50.0 lM) at 37 °C for 15 min. Then, fluorescence at 505 nm with excitation at 340 nm was measured by infinite M1000 (Tecan, Grodig, Austria), and IC50 was calculated. 21. Tan MH, Shimano T, Chu TM. Differential localization of human pancreas cancer-associated antigen and carcinoembryonic antigen in homologous pancreatic tumoral xenograft. J Natl Cancer Inst. 1981;67:563–569. 22. Chen WH, Horoszewicz JS, Leong SS, et al. Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites. In Vitro. 1982;18:24–34. 23. AsPC-1 or BxPC-3 cells (1.0 104 cells) were spreaded in 24-well plate, and incubated at 37 °C for 1 d. Farnesyl-SN-1/Dodecyl-SN-1 was then added, and incubated for 2 days. At 0, 1 and 2 days post-addition, cells were detached from plate, and living cells were counted after staining by 0.4% trypan blue solution (ThermoFisher Scientific, Waltham, MA, USA). 24. Tan MH, Nowak NJ, Loor R, et al. Characterization of a new primary human pancreatic tumor line. Cancer Invest. 1986;4:15–23.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group Ayumi Tanaka a, Mohamed O. Radwan a,b, Akiyuki Hamasaki a, Asumi Ejima a, Emiko Obata a, Ryoko Koga a, Hiroshi Tateishi a, Yoshinari Okamoto a, Mikako Fujita c, Mitsuyoshi Nakao d, Kazuo Umezawa e, Fuyuhiko Tamanoi f, Masami Otsuka a,⇑ a
Department of Bioorganic Medicinal Chemistry, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan Department of Chemistry of Natural Compounds, National Research Center, Dokki 12622, Cairo, Egypt Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan d Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan e Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195, Japan f Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, California NanoSystems Institute, University of California Los Angeles, Los Angeles, CA 90095, USA b c
a r t i c l e
i n f o
Article history: Received 6 May 2017 Revised 18 June 2017 Accepted 19 June 2017 Available online 20 June 2017 Keywords: Farnesyltransferase Zinc protein Ras protein Farnesyl group
a b s t r a c t Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity. Ó 2017 Elsevier Ltd. All rights reserved.
Modification of proteins by prenyl lipids, e.g., farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid lipid, is often crucial for protein function in a cell.1,2 The first step of prenylation is catalyzed by a zinc-containing farnesyltransferase or geranylgeranyltransferase, which conjugates a farnesyl/geranylgeranyl group of farnesyl/geranylgeranyl pyrophosphate to a cysteine residue of the C-terminal CAAX motif (X: aliphatic amino acid; A: any amino acid) of the substrate protein. Subsequently, the terminal AAX tripeptide is removed by endoprotease digestion and the resulting carboxyl of the prenylated cysteine is methylated by methyltransferase. The attached prenyl group, with its hydrophobic character, facilitates the translocation of the prenylated protein to the membrane. Protein prenylation is associated with various diseases, such as cancer, progeria, infectious diseases, glaucoma and neurological diseases.1,3 Thus, many inhibitors of farnesyltransferase/geranylgeranyltransferase have been developed, including some under Abbreviations: DTT, dithiothreitol.
⇑ Corresponding author.
E-mail address: [email protected] (M. Otsuka). http://dx.doi.org/10.1016/j.bmcl.2017.06.047 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
clinical investigation.1–4 Most of the inhibitors are CAAX analogues, farnesyl/geranylgeranyl pyrophosphate analogues and screened library compounds. However, the zinc site of these transferases has yet to be a major inhibitory target. We developed previously a dithiol compound SN-1 (Fig. 1) and showed that SN-1 inhibits the function of zinc proteins5–7 via a zinc-binding mechanism.8 In particular, we successfully endowed SN-1 with farnesyltransferase specificity by introducing a protein-recognizing naphthyl group, i.e., naphthyl-SN-1 (Fig. 1).9 This compound inhibited farnesyltransferase, which is known to farnesylate the Ras oncoprotein to cause cancer.10–12 We herein report novel SN-1 derivatives with a farnesyl/dodecyl group that bind into the farnesyl pyrophosphate-binding pocket of farnesyltransferase. Analogous to the case of naphthyl-SN-1, we placed a farnesyl group at the 4-position of pyridine, i.e., farnesyl-SN-1 (Fig. 1). The structure of farnesyl-SN-1 seemed rational, as indicated by the docking study using the Molecular Operating Environment (MOE) 2014.09. As a template for docking, we selected the X-ray crystal structure (PDB ID: 1SA4) of human farnesyltransferase complexed with farnesyl pyrophosphate and an inhibitor Tipifarnib, which binds to the catalytic domain with high affinity.3,13
A. Tanaka et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866
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Fig. 1. Structures of SN-17 and its derivatives with naphthyl,9 farnesyl and dodecyl groups.
Fig. 2. Binding mode of farnesyl-SN-1 into farnesyltransferase catalytic domain as predicted by the MOE 2014.09 using human farnesyltransferase complexed with the inhibitor Tipifarnib and farnesyl pyrophosphate (PDB ID: 1SA4).13 Structural refinement, protonation, removal of water molecules and energy minimization were carried out using MOE LigX function. Farnesyl-SN-1 was created, energy minimized, and then docked by the default docking protocol implemented in MOE.8 (A) 3D depiction. FarnesylSN-1 (green), Tipifarnib (yellow) and farnesyl pyrophosphate (FPP, blue) are shown. Farnesyl-SN-1 binds to Zn2+, Arg202 and Tyr361 residues. (B) 2D depiction. Proximal amino acids and settled interactions (dashed lines and arrows) are shown.
As shown in Fig. 2, the docking result revealed that both the SN-1 moiety and the farnesyl group of farnesyl-SN-1 fit the catalytic domain of farnesyltransferase well with a binding score of 8.1 kcal/mol. One thiol group of farnesyl-SN-1 binds to zinc and Tyr361, whereas the other thiol interacts with Arg202. Interestingly,
the farnesyl group of farnesyl-SN-1 is accommodated in the farnesyl pyrophosphate pocket. In addition to farnesyl-SN-1, we also designed dodecyl-SN-1 with a saturated straight-chain dodecyl group (Fig. 1). We also synthesized the corresponding disulfides, farnesyl-SN-1 disulfide
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A. Tanaka et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866
Scheme 1. Synthesis of farnesyl-SN-1 disulfide and dodecyl-SN-1 disulfide.
and dodecyl-SN-1 disulfide. The synthesis was based on our previous synthesis of biotin-linked SN-1.8 As shown in Scheme 1, 4-aminopyridine carrying Boc-protected thiazolidin rings (1) was
reacted with triphosgene followed by addition of farnesol or dodecyl alcohol to give the coupled product 2a or 2b.14 Removal of the Boc group of 2a or 2b furnished 3a or 3b.15 Treatment of
A. Tanaka et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3862–3866 Table 1 In vitro inhibitory activity of SN-1 derivatives against farnesyltransferase.a Compound
IC50 (lM)
Farnesyl-SN-1 Dodecyl-SN-1 Naphtyl-SN-1
0.90 2.0 1.9b
a According to a method using human farnesyltransferase, Dansyl-GCVLS and farnesyl pyrophosphate.19 b Reported previously.9
3a or 3b with reducing agent NaCNBH3 furnished farnesyl-SN-1 4a and dodecyl-SN-1 4b that spontaneously gave farnesyl-SN-1 disulfide 5a16 or dodecyl-SN-1 disulfide 5b17 by air oxidation.18 Farnesyl-SN-1 and dodecyl-SN-1 were generated in situ by reduction of their disulfide 5a and 5b with DTT, and the biological
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activity was evaluated. The in vitro inhibitory activity against farnesyltransferase was evaluated by a method that observes intramolecular fluorescence enhancement using a dansyl marked peptide containing CAAX as a substrate.19,20 The results are shown in Table 1. The IC50 against farnesyltransferase of farnesyl-SN-1 and dodecyl-SN-1 were 0.90 and 2.0 lM, suggesting improvement of the inhibitory activity because the previously reported IC50 of naphthyl-SN-1 was 1.90 lM. The stronger activity of farnesyl-SN1 compared with that of dodecyl-SN-1 shows the effect of the p-electron interaction between the double bonds of the farnesyl group and protein aromatic residues, as shown in Fig. 2 We then examined the inhibitory activity against the proliferation of the pancreatic cell line AsPC-121,22 expressing the K-Ras mutant (active form), which causes cancer.10 Expecting the reduction of the disulfide bond by intracellular reductants such as glutathione, the cells were treated with the prodrug farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide, and the number of living cells
(A)
(B)
(C)
Fig. 3. Inhibitory activity of farnesyl-SN-1 and dodecyl-SN-1 against proliferation of AsPC-1 and BxPC-3 cells. Cells were stained by trypan blue, and unstained cell numbers were counted. (A) Living cell numbers of AsPC-1 after treatment with the indicated concentration of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide. (B) Living cell numbers of AsPC-1 after treatment with 10 lM of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide in the presence of DTT. (C) Living cell numbers of BxPC-3 after treatment with the indicated concentration of farnesyl-SN-1 disulfide or dodecyl-SN-1 disulfide.
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was counted at 1 and 2 d post-treatment.23 The results are shown in Fig. 3A. Farnesyl-SN-1 at 0.5, 5 and 10 lM inhibited cellular proliferation. On the other hand, the inhibitory activity of dodecyl-SN-1 was concentration-dependent. The stronger activity of farnesyl-SN-1 when compared with that of dodecyl-SN-1 is in accordance with the in vitro activity against farnesyltransferase (Table 1). In the activity of farnesyl-SN-1 (0.5, 5 and 10 lM) and that of dodecyl-SN-1 (10 lM), activity 2 d post-treatment was more critical than that observed after 1 d. This is presumably due to the slow reduction of the disulfide in the cells. Thus, the same experiment using 10 lM of the disulfide compounds treated with DTT was performed. As expected, both farnesyl-SN-1 and dodecyl-SN-1 were effective inhibitors 1 d post-treatment (Fig. 3B). Next, the inhibitory effect against proliferation of BxPC-3,24 which is also a pancreatic cell line but does not have the mutation in the Ras protein,10 was examined.23 As shown in Fig. 3C, 10 lM of farnesyl-SN-1 was effective, but activity of 0.5 and 5 lM of the compound was weaker when compared with the results of the AsPC-1 cells. In contrast, no clear difference was not seen between AsPC-1 and BxPC-2 when these cell lines were treated with dodecyl-SN-1. These results suggests that 0.5 and 5 lM of farnesyl-SN-1 inhibit farnesylation of the Ras protein in a cell, thereby suppressing proliferation of AsPC-1 cells. The activity of 10 lM of farnesyl-SN-1 and dodesyl-SN-1 would be different from the inhibitory effect of Ras farnesylation. Taken together, we have developed a new inhibitor against protein prenylation that consists of a zinc-binding part and a prenyl group. The SN-1 moiety is revealed to have binding specificity toward zinc ions among various divalent cations (Table S1). Introduction of a protein-recognition part on SN-1 confers binding specificity toward zinc proteins. Further modification should improve the activity of our compounds to gain IC50 values acceptable to initiate clinical studies. Acknowledgements This work was supported by a Grant-in-Aid for Exploratory Research (B) (23390028) (to M. O.) and a Grant-in-Aid for Scientific Research (C) (26460148) (to Y. O.).
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Goodman LE, Judd SR, Farnsworth CC, et al. Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins. Proc Natl Acad Sci USA. 1990;87:9665–9669. 13. Reid TS, Beese LS. Crystal structures of the anticancer clinical candidates R115777 (Tipifarnib) and BMS-214662 complexed with protein farnesyltransferase suggest a mechanism of FTI selectivity. Biochemistry. 2004;43:6877–6884. 14. A solution of 1 (156 mg, 0.333 mmol) and pyridine (222 lL, 2.76 mmol) in CH2Cl2 (4 mL) was cooled at 0 °C. A solution of triphosgene (112 mg, 0.377 mmol) in CH2Cl2 (4 mL) was added at 0 °C, and a solution of 1dodecanol (74.6 lL, 0.333 mmol) in CH2Cl2 (4 mL) was subsequently added at 0 °C. The reaction mixture was stirred at ambient temperature for 2.5 h, and the solution was concentrated in vacuo. Aq-NaHCO3 (50 mL) was then added, and extracted with CH2Cl2 (20 mL) 5 times. The extract was dried over MgSO4, the solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 10:1) to afford 2b (222 mg, 98%). The same procedure using trans,trans-farnesol instead of 1-dodecanol gave 2a (77%). 15. To a solution of 2b (166 mg, 0.244 mmol) in toluene (2 mL), p-toluenesulfonic acid (81.1 mg, 0.471 mmol) was added. The reaction mixture was stirred at 40 °C for 1.5 h, and the solution was concentrated in vacuo. Water (50 mL) was then added, and extracted with CH2Cl2 (20 mL) 5 times. The extract was dried over MgSO4, and the solvent was removed in vacuo to afford 3b (81.8 mg, 70%). The same procedure using 2a instead of 2b gave 3a (50%). 16. 1H NMR (CDCl3) d: 1.45–1.98 (m, 12H), 2.01–2.17 (m, 8H), 2.73–3.65 (m, 12H), 4.09 (bs, 2H), 4.67 (s, 2H), 5.07–5.16 (m, 2H), 5.38–5.50 (m, 1H), 7.51–7.64 (m, 2H), 9.39 (bs, 1H). 13C NMR (CDCl3) d: 16.0, 16.6, 17.6, 21.5, 22.3, 25.6, 26.2, 26.3, 26.6, 38.3, 39.6, 49.9, 62.6, 102.7, 106.2, 118.2, 123.5, 124.3, 131.2, 135.4, 141.8, 142.6, 146.7, 151.1, 153.4. HRMS(FAB) m/z calcd for C27H43N4O2S2 [M +H]+ 519.2827 Found: 519.2823. 17. 1H NMR (CD3OD) d: 0.89 (t, 3H, J = 6.4 Hz), 1.28 (m, 20H), 1.69 (t, 2H, J = 6.8 Hz), 2.93 (t, 4H), 3.08-3.12 (m, 4H) 3.21 (t, 4H), 4.16–4.20 (m, 2H), 7.48–7.51(m, 2H). 13C NMR (CD3OD) d: 14.4, 23.6, 29.9, 30.3, 30.4, 30.6, 30.7, 33.0, 34.9, 38.5, 51.6, 52.0, 52.2, 52.3, 66.7, 111.8, 150.0, 155.0. HRMS(FAB) m/z calcd for C24H43N4O2S2 [M+H]+ 483.2827 Found: 483.2791. 18. To a solution of 3b (117 mg, 0.243 mmol) in dried CH2Cl2, NaCNBH3 (154 mg, 2.45 mmol) and 0.1 M HCl methanol solution were added to adjust pH 3. The reaction mixture was stirred at ambient temperature for 2 h, and neutralized with 1 M aq-NaOH. The solution was concentrated in vacuo, and extracted with CH2Cl2. The solvent was then removed in vacuo to afford dodecyl-SN-1 disulfide 5b (49.0 mg, 42%). The same procedure using 3a instead of 3b gave farnesyl-SN-1 disulfide 5a (42%). 19. Pompliano DL, Gomez RP, Anthony NJ. Intramolecular fluorescence enhancement: a continuous assay of Ras farnesyl:protein transferase. J Am Chem Soc. 1992;114:7945–7946. 20. Human farnesyltransferase (Jena Biosciences, Jena, Germany) (0.03 lM), dansyl-GCVLS (Sigma-Aldrich, St Louis, MO, USA) (1.0 lM), and farnesyl pyrophosphate (Sigma-Aldrich) (10 lM) in assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, and 0.2% n-octyl-b-D-glucopyranoside) were incubated with or without Farnesyl-SN-1/Dodecyl-SN-1 (0.05, 0.50, 5.00 or 50.0 lM) at 37 °C for 15 min. Then, fluorescence at 505 nm with excitation at 340 nm was measured by infinite M1000 (Tecan, Grodig, Austria), and IC50 was calculated. 21. Tan MH, Shimano T, Chu TM. Differential localization of human pancreas cancer-associated antigen and carcinoembryonic antigen in homologous pancreatic tumoral xenograft. J Natl Cancer Inst. 1981;67:563–569. 22. Chen WH, Horoszewicz JS, Leong SS, et al. Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites. In Vitro. 1982;18:24–34. 23. AsPC-1 or BxPC-3 cells (1.0 104 cells) were spreaded in 24-well plate, and incubated at 37 °C for 1 d. Farnesyl-SN-1/Dodecyl-SN-1 was then added, and incubated for 2 days. At 0, 1 and 2 days post-addition, cells were detached from plate, and living cells were counted after staining by 0.4% trypan blue solution (ThermoFisher Scientific, Waltham, MA, USA). 24. Tan MH, Nowak NJ, Loor R, et al. Characterization of a new primary human pancreatic tumor line. Cancer Invest. 1986;4:15–23.