Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Canthinone alkaloids are novel protein tyrosine phosphatase 1B inhibitors Tatsunori Sasaki, Wei Li ⇑, Koji Higai, Kazuo Koike Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
a r t i c l e
i n f o
Article history: Received 5 January 2015 Revised 3 March 2015 Accepted 7 March 2015 Available online xxxx Keywords: Protein tyrosine phosphatase 1B Canthinone Picrasidine L Diabetes
a b s t r a c t Considerable attention has been paid to protein tyrosine phosphatase 1B (PTP1B) inhibitors as a potential therapy for diabetes. Screening of a natural compound library resulted in six canthinone alkaloids, namely, picrasidine L (1), 3,4-dimethyl-canthin-5,6-dione (2), 4-ethyl-3-methyl-canthin-5,6-dione (3), eurycomine E (4), 5-methoxy-canthin-6-one (5), and 5-acethoxy-canthin-6-one (6), as novel PTP1B inhibitors. Among these, 1 is the competitive PTP1B inhibitor with the best inhibitory selectivity between PTP1B and other PTPs and was shown to promote activity in the insulin signaling pathway in cell-based assays. Molecular docking simulations and structure–activity relationship analysis of 1 will add to its potential as a lead compound in future anti-insulin-resistant drug developments. Ó 2015 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase. It is expressed ubiquitously in the classical insulin-targeted tissues and plays critical roles in negatively regulating both insulin and leptin signaling cascades.1,2 Pharmacological studies have shown that PTP1B knockout mice exhibited increased insulin sensitivity and obesity resistance.3 These findings have led to an intense interest in developing PTP1B inhibitors as potential therapies for diabetes and obesity.4,5 The use of natural products to discover novel PTP1B inhibitors has received much more attention because of their advantage in cells with a high structural diversity.6 In the course of our on-going studies on the discovery of novel PTP1B inhibitors from medicinal plants, we have reported on various types of natural compounds, including flavonoids, triterpenoids and lignoids, as new PTP1B inhibitors.7–10 Herein, we report further progress in the discovery of six canthinone alkaloids as novel protein tyrosine phosphatase 1B inhibitors. Our compound library consists of seventy-six alkaloids (1–76), which were natural compounds from Picrasma quassioides, Picrasma javanica, Ailanthus altissima, Simarouba amara, Eurycoma longifolia, Simaba cuspidata, and Quassia amara, and their structural related chemical synthetic analogs. The compounds were screened for their inhibitory activities against PTP1B at a final concentration of 100 lM (data shown in Supporting information).11 A known PTP1B inhibitor, RK-682, was used as the positive control (IC50 4.49 lM) in this bioassay. As a result, six canthinone alkaloids,
namely, picrasidine L (1), 3,4-dimethyl-canthin-5,6-dione (2), 4ethyl-3-methyl-canthin-5,6-dione (3), eurycomine E (4), 5-methoxy-canthin-6-one (5), and 5-acethoxy-canthin-6-one (6), were found to completely inhibit the activity of PTP1B (>90% inhibition) (Fig. 1). Further investigation indicated that the PTP1B inhibition of these compounds was concentration-dependent, and their IC50 values were obtained by regression analyses (Table 1). The bioactive compounds (1–6) were further investigated to determine their inhibition mechanisms. This was accomplished by performing kinetic analyses with various concentrations of the compounds and substrate p-nitrophenylphosphate. Lineweaver– Burk plots, as shown in Figure 2, suggested that compound 1 was a competitive PTP1B inhibitor and that compounds 2–6 were
⇑ Corresponding author. Tel.: +81 47 4721161; fax: +81 47 4721404. E-mail address: [email protected] (W. Li).
Figure 1. Six canthinone alkaloids as PTP1B inhibitors.
http://dx.doi.org/10.1016/j.bmcl.2015.03.014 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
2
T. Sasaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1 Inhibition effects of compounds 1–6 and RK-682 against protein tyrosine phosphatase 1B
a b
Compounds
IC50 (lM)a
Inhibition mode
Ki (lM)
1 2 3 4 5 6 RK-682b
19.80 ± 0.62 24.72 ± 0.26 27.83 ± 0.68 19.18 ± 0.76 20.30 ± 0.24 28.89 ± 0.52 4.49 ± 0.26
Competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive
7.07 19.94 20.05 12.94 10.33 18.39
Inhibition rate (%) are the mean ± SD from three separate experiments. Positive control.
non-competitive PTP1B inhibitors. Secondary plots were created, which plotted the slopes from the Lineweaver–Burk plots on the y-axis against the concentration of the compounds on the x-axis, and showed a good linear relationship between these variables. The Ki values of compounds 1–6 were calculated to be 7.07, 19.94, 20.05, 10.03, 18.39, and 12.94 lM, respectively. Because of the high structural similarity of the catalytic center among protein tyrosine phosphatases,12 the inhibitory selectivity of compounds 1–6 were evaluated by comparing their inhibitory activity against PTP1B and four homologous protein tyrosine phosphatases, namely, TCPTP, VHR, SHP-1, and SHP-2. Compounds 1–6 showed PTP1B inhibitory selectivity indexes (SI) from 6.9 to 1.5
(Table 2). Of the compounds investigated, compound 1 possessed the best inhibitory selectivity as its SI values between PTP1B and all four PTPs of compound 1 were more than 3.2. Due to the limited amount of compound 2, five out of six bioactive compounds (1 and 3–6) were further assessed for their cellular activity in the insulin signal transduction pathway in the human hepatocellular carcinoma cell line HepG2. This was accomplished by measuring the phosphorylation level of Akt, a key downstream effector of the insulin signaling cascade.13 In the Akt phosphorylation assay, after the cells were stimulated with insulin, the pAkt levels were analyzed by western blotting at different times (0, 5 min). Dimethyl sulfoxide was used as a negative control, and sodium orthovanadate at 200 lM was used as a positive control.10 As shown in Figure 3, the rapid increase in pAkt levels after insulin stimulation was promoted by the administration of compound 1, showing that the effective cellular activity was in good accordance with its potency in the enzymatic assay. Because compound 1 is a competitive PTP1B inhibitor, its binding mode to the enzyme was investigated by a docking simulation using Accelrys Discovery Studio 4.1.14 The X-ray crystal structure of PTP1B (PDB-ID: 1nwe) was obtained from a protein data bank (http://www.rcsb.org). The preferred coordination mode of 1 with PTP1B is shown in Figure 4. The binding energy for the docking experiment was calculated to be 43.4876 kcal/mol. From the analysis of the binding interactions, the tetracyclic rings in 1 was interacted with Tyr46, Phe182, and Cys215 amino acid residues, which
Figure 2. Inhibition of protein tyrosine phosphatase 1B-catalyzed hydrolysis of para-nitrophenylphosphate by compounds 1–6. For each compound, A represents the Lineweaver–Burk plots, and B is the secondary plot derived from A.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
T. Sasaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 2 Inhibition rate (%) and selective index of compounds 1–6 and RK-682 against protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), vaccinia H1-related phosphatase (VHR), and Src homology domain 2-containing protein tyrosine phosphatases 1 (SHP-1) and 2 (SHP-2) Compound 1c
PTP
Inhibition rate (%)a PTP1B TCPTP VHR SHP-1 SHP-2
Compound 2c b
SI
91.4 ± 1.9 29.0 ± 1.9 20.6 ± 1.3 13.2 ± 0.9 16.3 ± 1.2
3.2 4.4 6.9 5.6
Inhibition rate (%)a
PTP1B TCPTP VHR SHP-1 SHP-2
SI
93.8 ± 0.8 15.0 ± 0.9 61.4 ± 3.3 28.7 ± 0.7 44.4 ± 1.1
Inhibition rate (%)a 91.0 ± 1.9 40.0 ± 1.1 19.7 ± 2.9 20.6 ± 1.4 25.0 ± 2.3
1.9 3.5 3.8 4.0
Compound 5c b
Inhibition rate (%)a
SI
92.1 ± 2.2 49.7 ± 2.0 26.5 ± 4.3 24.4 ± 0.6 23.3 ± 1.6
Compound 4c
PTP
Compound 3c b
6.3 1.5 3.3 2.1
SI
91.0 ± 2.3 48.4 ± 2.7 27.5 ± 0.2 22.8 ± 2.5 18.3 ± 0.7
2.3 4.6 4.4 3.6
Compound 6c b
Inhibition rate (%)a
SIb
Inhibition rate (%)a 90.3 ± 2.0 22.1 ± 3.7 46.0 ± 2.3 30.2 ± 1.9 23.0 ± 1.1
1.9 3.3 4.0 5.0
SIb
4.1 2.0 3.0 3.9
a
Inhibition rate (%) are the mean ± SD from three separate experiments. SI: selective index value (inhibition rate (%) against PTP1B/inhibition rate (%) against other PTPs). c Sample final concentration: Compound 1, 2, and 5: 75 lM, 3 and 4: 100 lM, 6: 90 lM.
3
(66 and 69) decreased the inhibitory activity. This was further supported by the dimerization of 1 at this position (74 and 75) showing a decreased activity. Despite the activity only slightly decreased below 1 by the C-4 (2 and 3) and C-9 substitutions (4), these substitutions greatly affected the inhibition mode, which is made clear by the fact that 1 is competitive but 2, 3 and 4 are non-competitive PTP1B inhibitors. This fact was also supported by the docking study. Namely, in comparison of the calculated binding energy between 1 (43.4876 kcal/mol), 2 ( 1.4641 kcal/mol), and 3 ( 0.1871 kcal/mol), compounds with C-4 substitutions greatly depressed the binding energy. In conclusion, six canthinone alkaloids (1–6) were discovered as novel PTP1B inhibitors. Of these, picrasidine L (1) was found to be the competitive PTP1B inhibitor with the best inhibitory selectivity between PTP1B and other PTPs. It also promoted activity on the insulin signaling pathway in cellular-based assays. The molecular docking simulation and structure–activity relationship analysis of 1 will add to its potential as a lead compound in future anti-insulin-resistant drug developments. Acknowledgements
b
Insulin stimulation
1
DMSO 0
5
0
6 5
0
4 5
0
3 5
0
5 5
0
5
(min)
pAkt
Supplementary data
Akt Density (pAkt/Akt)
The research was partially supported by a Grant-in-Aid for Young Scientists (B) (No. 23790025) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Sasakawa Scientific Research Grant (No. 25–316) from The Japan Science Society.
1.0
1.6
1.0
2.4
1.0
2.1
1.0
1.8
1.0
1.2
1.0
1.3
Figure 3. Cellular activity of DMSO (negative control) and compounds 1, 3–6 (at 50 lM) on Akt phosphorylation in HepG2 cells.
Figure 4. Docked molecular model of compound 1 in the active site of the PTP1B enzyme (PDB ID: 1nwe).
were amino acids of pTyr, WPD, and PTP loop in PTP1B,15–17 via p–p interactions. It was also found that the 6-carbonyl moiety of 1 interacted with Lys120 amino acid residues of the substrate recognition loop via a hydrogen binding interaction (hydrogen bond distance is 1.970 Å), and both hydrogen atom at position 2 and methyl moiety at position 3 interacted with Asp48 amino acid residues via a hydrogen binding interaction (hydrogen bond distance is 2.680 Å). Compounds 1–6 shared a common canthin-6-one structure. In comparison to 1, the aliphatic side chain extension at 3-position
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 014. References and notes 1. Byon, C. H. J.; Kusari, B. A. J. Mol. Cell. Biochem. 1998, 182, 101. 2. Kennedy, B. P. Biomed. Pharmacother. 1999, 53, 466. 3. Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C. C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544. 4. Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373. 5. Yip, S. C.; Saha, S.; Chernoff, J. Trends Biochem. Sci. 2010, 35, 442. 6. Jiang, C.; Liang, L.; Guo, Y. Acta Pharmacol. Sin. 2012, 33, 1217. 7. Li, S.; Li, W.; Wang, Y.; Asada, Y.; Koike, K. Bioorg. Med. Chem. Lett. 2010, 20, 5398. 8. Sasaki, T.; Li, W.; Morimura, H.; Li, S.; Li, Q.; Asada, Y.; Koike, K. Chem. Pharm. Bull. 2011, 59, 1396. 9. Li, W.; Li, S.; Higai, K.; Sasaki, T.; Asada, Y.; Ohshima, S.; Koike, K. Bioorg. Med. Chem. Lett. 2013, 23, 5836. 10. Sasaki, T.; Li, W.; Higai, K.; Quang, T. H.; Kim, Y. H.; Koike, K. Planta Med. 2014, 80, 557. 11. PTP1B and other PTP inhibitory activity assays: The assay was carried out as reported previously.10 12. Tabernero, L.; Aricescu, A. R.; Jones, E. Y.; Szedlacsek, S. E. FEBS J. 2008, 275, 867. 13. Insulin-stimulated Akt phosphorylation assay: the assay was carried out as reported previously.10 14. Molecular docking simulation. To further identify the putative binding site and corresponding binding conformation of the compounds, molecular docking simulation was performed against the PTP1B protein carried out using CDOCKER docking protocol of Discovery studio 4.1 (Accelrys K.K.). CDOCKER (CHARMm-based DOCKER) is a molecular dynamics based docking algorithm. It uses the CHARMm family of force fields and offers full flexibility to ligand including dihedrals, angles and bonds. Docking helps to predict best binding compounds based on various scoring functions. The high-resolution crystal structure of PTP1B complexed with inhibitor was obtained from the Protein Data Bank (PDB code: 1nwe). All cocrystalized ligands and water were removed. The starting 3D conformations of compound 1 were prepared with Dock Ligands Fit. The calculated binding score of all docking positions were evaluated. The best ranked position from each of the binding sites was determined by the highest calculated binding score. 15. Zhang, Z. Y. Annu. Rev. Pharmocol. Toxicol. 2008, 42, 209. 16. Tonks, N. K. FEBS Lett. 2003, 546, 140. 17. Taylor, S. D.; Hill, B. Expert Opin. Investig. Drugs 2004, 13, 199.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Canthinone alkaloids are novel protein tyrosine phosphatase 1B inhibitors Tatsunori Sasaki, Wei Li ⇑, Koji Higai, Kazuo Koike Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
a r t i c l e
i n f o
Article history: Received 5 January 2015 Revised 3 March 2015 Accepted 7 March 2015 Available online xxxx Keywords: Protein tyrosine phosphatase 1B Canthinone Picrasidine L Diabetes
a b s t r a c t Considerable attention has been paid to protein tyrosine phosphatase 1B (PTP1B) inhibitors as a potential therapy for diabetes. Screening of a natural compound library resulted in six canthinone alkaloids, namely, picrasidine L (1), 3,4-dimethyl-canthin-5,6-dione (2), 4-ethyl-3-methyl-canthin-5,6-dione (3), eurycomine E (4), 5-methoxy-canthin-6-one (5), and 5-acethoxy-canthin-6-one (6), as novel PTP1B inhibitors. Among these, 1 is the competitive PTP1B inhibitor with the best inhibitory selectivity between PTP1B and other PTPs and was shown to promote activity in the insulin signaling pathway in cell-based assays. Molecular docking simulations and structure–activity relationship analysis of 1 will add to its potential as a lead compound in future anti-insulin-resistant drug developments. Ó 2015 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase. It is expressed ubiquitously in the classical insulin-targeted tissues and plays critical roles in negatively regulating both insulin and leptin signaling cascades.1,2 Pharmacological studies have shown that PTP1B knockout mice exhibited increased insulin sensitivity and obesity resistance.3 These findings have led to an intense interest in developing PTP1B inhibitors as potential therapies for diabetes and obesity.4,5 The use of natural products to discover novel PTP1B inhibitors has received much more attention because of their advantage in cells with a high structural diversity.6 In the course of our on-going studies on the discovery of novel PTP1B inhibitors from medicinal plants, we have reported on various types of natural compounds, including flavonoids, triterpenoids and lignoids, as new PTP1B inhibitors.7–10 Herein, we report further progress in the discovery of six canthinone alkaloids as novel protein tyrosine phosphatase 1B inhibitors. Our compound library consists of seventy-six alkaloids (1–76), which were natural compounds from Picrasma quassioides, Picrasma javanica, Ailanthus altissima, Simarouba amara, Eurycoma longifolia, Simaba cuspidata, and Quassia amara, and their structural related chemical synthetic analogs. The compounds were screened for their inhibitory activities against PTP1B at a final concentration of 100 lM (data shown in Supporting information).11 A known PTP1B inhibitor, RK-682, was used as the positive control (IC50 4.49 lM) in this bioassay. As a result, six canthinone alkaloids,
namely, picrasidine L (1), 3,4-dimethyl-canthin-5,6-dione (2), 4ethyl-3-methyl-canthin-5,6-dione (3), eurycomine E (4), 5-methoxy-canthin-6-one (5), and 5-acethoxy-canthin-6-one (6), were found to completely inhibit the activity of PTP1B (>90% inhibition) (Fig. 1). Further investigation indicated that the PTP1B inhibition of these compounds was concentration-dependent, and their IC50 values were obtained by regression analyses (Table 1). The bioactive compounds (1–6) were further investigated to determine their inhibition mechanisms. This was accomplished by performing kinetic analyses with various concentrations of the compounds and substrate p-nitrophenylphosphate. Lineweaver– Burk plots, as shown in Figure 2, suggested that compound 1 was a competitive PTP1B inhibitor and that compounds 2–6 were
⇑ Corresponding author. Tel.: +81 47 4721161; fax: +81 47 4721404. E-mail address: [email protected] (W. Li).
Figure 1. Six canthinone alkaloids as PTP1B inhibitors.
http://dx.doi.org/10.1016/j.bmcl.2015.03.014 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
2
T. Sasaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1 Inhibition effects of compounds 1–6 and RK-682 against protein tyrosine phosphatase 1B
a b
Compounds
IC50 (lM)a
Inhibition mode
Ki (lM)
1 2 3 4 5 6 RK-682b
19.80 ± 0.62 24.72 ± 0.26 27.83 ± 0.68 19.18 ± 0.76 20.30 ± 0.24 28.89 ± 0.52 4.49 ± 0.26
Competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive
7.07 19.94 20.05 12.94 10.33 18.39
Inhibition rate (%) are the mean ± SD from three separate experiments. Positive control.
non-competitive PTP1B inhibitors. Secondary plots were created, which plotted the slopes from the Lineweaver–Burk plots on the y-axis against the concentration of the compounds on the x-axis, and showed a good linear relationship between these variables. The Ki values of compounds 1–6 were calculated to be 7.07, 19.94, 20.05, 10.03, 18.39, and 12.94 lM, respectively. Because of the high structural similarity of the catalytic center among protein tyrosine phosphatases,12 the inhibitory selectivity of compounds 1–6 were evaluated by comparing their inhibitory activity against PTP1B and four homologous protein tyrosine phosphatases, namely, TCPTP, VHR, SHP-1, and SHP-2. Compounds 1–6 showed PTP1B inhibitory selectivity indexes (SI) from 6.9 to 1.5
(Table 2). Of the compounds investigated, compound 1 possessed the best inhibitory selectivity as its SI values between PTP1B and all four PTPs of compound 1 were more than 3.2. Due to the limited amount of compound 2, five out of six bioactive compounds (1 and 3–6) were further assessed for their cellular activity in the insulin signal transduction pathway in the human hepatocellular carcinoma cell line HepG2. This was accomplished by measuring the phosphorylation level of Akt, a key downstream effector of the insulin signaling cascade.13 In the Akt phosphorylation assay, after the cells were stimulated with insulin, the pAkt levels were analyzed by western blotting at different times (0, 5 min). Dimethyl sulfoxide was used as a negative control, and sodium orthovanadate at 200 lM was used as a positive control.10 As shown in Figure 3, the rapid increase in pAkt levels after insulin stimulation was promoted by the administration of compound 1, showing that the effective cellular activity was in good accordance with its potency in the enzymatic assay. Because compound 1 is a competitive PTP1B inhibitor, its binding mode to the enzyme was investigated by a docking simulation using Accelrys Discovery Studio 4.1.14 The X-ray crystal structure of PTP1B (PDB-ID: 1nwe) was obtained from a protein data bank (http://www.rcsb.org). The preferred coordination mode of 1 with PTP1B is shown in Figure 4. The binding energy for the docking experiment was calculated to be 43.4876 kcal/mol. From the analysis of the binding interactions, the tetracyclic rings in 1 was interacted with Tyr46, Phe182, and Cys215 amino acid residues, which
Figure 2. Inhibition of protein tyrosine phosphatase 1B-catalyzed hydrolysis of para-nitrophenylphosphate by compounds 1–6. For each compound, A represents the Lineweaver–Burk plots, and B is the secondary plot derived from A.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
T. Sasaki et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 2 Inhibition rate (%) and selective index of compounds 1–6 and RK-682 against protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), vaccinia H1-related phosphatase (VHR), and Src homology domain 2-containing protein tyrosine phosphatases 1 (SHP-1) and 2 (SHP-2) Compound 1c
PTP
Inhibition rate (%)a PTP1B TCPTP VHR SHP-1 SHP-2
Compound 2c b
SI
91.4 ± 1.9 29.0 ± 1.9 20.6 ± 1.3 13.2 ± 0.9 16.3 ± 1.2
3.2 4.4 6.9 5.6
Inhibition rate (%)a
PTP1B TCPTP VHR SHP-1 SHP-2
SI
93.8 ± 0.8 15.0 ± 0.9 61.4 ± 3.3 28.7 ± 0.7 44.4 ± 1.1
Inhibition rate (%)a 91.0 ± 1.9 40.0 ± 1.1 19.7 ± 2.9 20.6 ± 1.4 25.0 ± 2.3
1.9 3.5 3.8 4.0
Compound 5c b
Inhibition rate (%)a
SI
92.1 ± 2.2 49.7 ± 2.0 26.5 ± 4.3 24.4 ± 0.6 23.3 ± 1.6
Compound 4c
PTP
Compound 3c b
6.3 1.5 3.3 2.1
SI
91.0 ± 2.3 48.4 ± 2.7 27.5 ± 0.2 22.8 ± 2.5 18.3 ± 0.7
2.3 4.6 4.4 3.6
Compound 6c b
Inhibition rate (%)a
SIb
Inhibition rate (%)a 90.3 ± 2.0 22.1 ± 3.7 46.0 ± 2.3 30.2 ± 1.9 23.0 ± 1.1
1.9 3.3 4.0 5.0
SIb
4.1 2.0 3.0 3.9
a
Inhibition rate (%) are the mean ± SD from three separate experiments. SI: selective index value (inhibition rate (%) against PTP1B/inhibition rate (%) against other PTPs). c Sample final concentration: Compound 1, 2, and 5: 75 lM, 3 and 4: 100 lM, 6: 90 lM.
3
(66 and 69) decreased the inhibitory activity. This was further supported by the dimerization of 1 at this position (74 and 75) showing a decreased activity. Despite the activity only slightly decreased below 1 by the C-4 (2 and 3) and C-9 substitutions (4), these substitutions greatly affected the inhibition mode, which is made clear by the fact that 1 is competitive but 2, 3 and 4 are non-competitive PTP1B inhibitors. This fact was also supported by the docking study. Namely, in comparison of the calculated binding energy between 1 (43.4876 kcal/mol), 2 ( 1.4641 kcal/mol), and 3 ( 0.1871 kcal/mol), compounds with C-4 substitutions greatly depressed the binding energy. In conclusion, six canthinone alkaloids (1–6) were discovered as novel PTP1B inhibitors. Of these, picrasidine L (1) was found to be the competitive PTP1B inhibitor with the best inhibitory selectivity between PTP1B and other PTPs. It also promoted activity on the insulin signaling pathway in cellular-based assays. The molecular docking simulation and structure–activity relationship analysis of 1 will add to its potential as a lead compound in future anti-insulin-resistant drug developments. Acknowledgements
b
Insulin stimulation
1
DMSO 0
5
0
6 5
0
4 5
0
3 5
0
5 5
0
5
(min)
pAkt
Supplementary data
Akt Density (pAkt/Akt)
The research was partially supported by a Grant-in-Aid for Young Scientists (B) (No. 23790025) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Sasakawa Scientific Research Grant (No. 25–316) from The Japan Science Society.
1.0
1.6
1.0
2.4
1.0
2.1
1.0
1.8
1.0
1.2
1.0
1.3
Figure 3. Cellular activity of DMSO (negative control) and compounds 1, 3–6 (at 50 lM) on Akt phosphorylation in HepG2 cells.
Figure 4. Docked molecular model of compound 1 in the active site of the PTP1B enzyme (PDB ID: 1nwe).
were amino acids of pTyr, WPD, and PTP loop in PTP1B,15–17 via p–p interactions. It was also found that the 6-carbonyl moiety of 1 interacted with Lys120 amino acid residues of the substrate recognition loop via a hydrogen binding interaction (hydrogen bond distance is 1.970 Å), and both hydrogen atom at position 2 and methyl moiety at position 3 interacted with Asp48 amino acid residues via a hydrogen binding interaction (hydrogen bond distance is 2.680 Å). Compounds 1–6 shared a common canthin-6-one structure. In comparison to 1, the aliphatic side chain extension at 3-position
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 014. References and notes 1. Byon, C. H. J.; Kusari, B. A. J. Mol. Cell. Biochem. 1998, 182, 101. 2. Kennedy, B. P. Biomed. Pharmacother. 1999, 53, 466. 3. Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C. C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544. 4. Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373. 5. Yip, S. C.; Saha, S.; Chernoff, J. Trends Biochem. Sci. 2010, 35, 442. 6. Jiang, C.; Liang, L.; Guo, Y. Acta Pharmacol. Sin. 2012, 33, 1217. 7. Li, S.; Li, W.; Wang, Y.; Asada, Y.; Koike, K. Bioorg. Med. Chem. Lett. 2010, 20, 5398. 8. Sasaki, T.; Li, W.; Morimura, H.; Li, S.; Li, Q.; Asada, Y.; Koike, K. Chem. Pharm. Bull. 2011, 59, 1396. 9. Li, W.; Li, S.; Higai, K.; Sasaki, T.; Asada, Y.; Ohshima, S.; Koike, K. Bioorg. Med. Chem. Lett. 2013, 23, 5836. 10. Sasaki, T.; Li, W.; Higai, K.; Quang, T. H.; Kim, Y. H.; Koike, K. Planta Med. 2014, 80, 557. 11. PTP1B and other PTP inhibitory activity assays: The assay was carried out as reported previously.10 12. Tabernero, L.; Aricescu, A. R.; Jones, E. Y.; Szedlacsek, S. E. FEBS J. 2008, 275, 867. 13. Insulin-stimulated Akt phosphorylation assay: the assay was carried out as reported previously.10 14. Molecular docking simulation. To further identify the putative binding site and corresponding binding conformation of the compounds, molecular docking simulation was performed against the PTP1B protein carried out using CDOCKER docking protocol of Discovery studio 4.1 (Accelrys K.K.). CDOCKER (CHARMm-based DOCKER) is a molecular dynamics based docking algorithm. It uses the CHARMm family of force fields and offers full flexibility to ligand including dihedrals, angles and bonds. Docking helps to predict best binding compounds based on various scoring functions. The high-resolution crystal structure of PTP1B complexed with inhibitor was obtained from the Protein Data Bank (PDB code: 1nwe). All cocrystalized ligands and water were removed. The starting 3D conformations of compound 1 were prepared with Dock Ligands Fit. The calculated binding score of all docking positions were evaluated. The best ranked position from each of the binding sites was determined by the highest calculated binding score. 15. Zhang, Z. Y. Annu. Rev. Pharmocol. Toxicol. 2008, 42, 209. 16. Tonks, N. K. FEBS Lett. 2003, 546, 140. 17. Taylor, S. D.; Hill, B. Expert Opin. Investig. Drugs 2004, 13, 199.
Please cite this article in press as: Sasaki, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.014
