Supporting Information. Pterosin Sesquiterpenoids from Pteris cretica as Hypolipidemic Agents. via Activating Liver X Receptors

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1 Supporting Information Pterosin Sesquiterpenoids from Pteris cretica as Hypolipidemic Agents via Activating Liver X Receptors Xiangkun Luo, Chanjuan Li, Pan Luo, Xin Lin, Hang Ma, Navindra P. Seeram, Ching Song, Jun Xu, *, and Qiong Gu *, Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou , People s Republic of China Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island, 02881, United States School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing , People s Republic of China 1

2 List of Contents No. Content Page 1 Molecular Dynamics Simulation S4 2 Binding Energy Calculation S5 Figure S1. The IR Spectra of Compound 1 S8 Figure S2. The HRESIMS Spectroscopic Data of Compound 1 S9 Figure S3. The 1 H NMR Spectrum of Compound 1 in Methanol-d 4 S10 Figure S4. The 13 C NMR Spectrum of Compound 1 in Methanol-d 4 S11 Figure S5. The 1 H- 1 H COSY Spectrum of Compound 1 in Methanol-d 4 S12 Figure S6. The HSQC Spectrum of Compound 1 in Methanol-d 4 S13 Figure S7. The HMBC Spectrum of Compound 1 in Methanol-d 4 S14 Figure S8. The NOESY Spectrum of Compound 1 in Methanol-d 4 S15 Figure S9. The IR Spectra of Compound 2 S16 Figure S10. The HRESIMS Spectroscopic Data of Compound 2 S17 Figure S11. The 1 H NMR Spectrum of Compound 2 in Methanol-d 4 S18 Figure S12. The 13 C NMR Spectrum of Compound 2 in Methanol-d 4 S19 Figure S13. The 1 H- 1 H COSY Spectrum of Compound 2 in Methanol-d 4 S20 Figure S14. The HSQC Spectrum of Compound 2 in Methanol-d 4 S21 Figure S15. The HMBC Spectrum of Compound 2 in Methanol-d 4 S22 Figure S16. The NOESY Spectrum of Compound 2 in Methanol-d 4 S23 Figure S17. The IR Spectra of Compound 3 S24 Figure S18. The HRESIMS Spectroscopic Data of Compound 3 S25 Figure S19. The 1 H NMR Spectrum of Compound 3 in Methanol-d 4 S26 Figure S20. The 13 C NMR Spectrum of Compound 3 in Methanol-d 4 S27 Figure S21. The 1 H- 1 H COSY Spectrum of Compound 3 in Methanol-d 4 S28 Figure S22. The HSQC Spectrum of Compound 3 in Methanol-d 4 S29 Figure S23. The HMBC Spectrum of Compound 3 in Methanol-d 4 S30 Figure S24. The NOESY Spectrum of Compound 3 in Methanol-d 4 S31 Figure S25. The IR Spectra of Compound 4 S32 Figure S26. The HREIMS Spectroscopic Data of Compound 4 S33 Figure S27. The 1 H NMR Spectrum of Compound 4 in Methanol-d 4 S34 Figure S28. The 13 C NMR Spectrum of Compound 4 in Methanol-d 4 S35 Figure S29. The 1 H- 1 H COSY Spectrum of Compound 4 in Methanol-d 4 S36 Figure S30. The HSQC Spectrum of Compound 4 in Methanol-d 4 S37 Figure S31. The HMBC Spectrum of Compound 4 in Methanol-d 4 S38 2

3 Figure S32. The NOESY Spectrum of Compound 4 in Methanol-d 4 S39 Figure S33. The IR Spectra of Compound 17 S40 Figure S34. The HREIMS Spectroscopic Data of Compound 17 S41 Figure S35. The 1 H NMR Spectrum of Compound 17 in Pyridine-d 5 S42 Figure S36. The 13 C NMR Spectrum of Compound 17 in Pyridine-d 5 S43 Figure S37. The 1 H- 1 H COSY Spectrum of Compound 17 in Pyridine-d 5 S44 Figure S38. The HSQC Spectrum of Compound 17 in Pyridine-d 5 S45 Figure S39. The HMBC Spectrum of Compound 17 in Pyridine-d 5 S46 Figure S40. The NOESY Spectrum of Compound 17 in Pyridine-d 5 S47 Figure S41. Four Stereoisomers of Compound 17 S48 Figure S42. RMSD Curves of Four Complexes During 20 ns MD Simulations S49 Figure S43. Decomposition of Binding Free Energy on the Key Residue for S49 Complex 4 to LXRs. Table S1. Binding Energy Analysis for the binding of 4 to LXRα and LXRβ S50 3

4 1. Molecular Dynamics Simulation. The initial coordinates of compound 4 were obtained through molecular docking. The initial coordinates of GW3965 was obtained from RCSB Protein Data Bank (PDB code: 3IPQ, 1PQ6). The structures of LXRα and LXRβ obtained from RCSB Protein Data Bank (PDB code: 3IPQ, 1PQ6) were fixed through homology modeling. GPU-based 1,2 MD simulations were performed using the PMEMD module in AMBER The partial atomic charges of the ligands were calculated through the Gaussian 09 4 program by using the Hartree-Fock method with the 6-31G(d) basis set. The Antechamber program was then used for fitting the restricted electrostatic potential (RESP) and assigning the GAFF force field parameters 5. For the protein receptors, the AMBER ff12sb force field was used 6,7. The ligand-receptor complexes were neutralized by adding sodium/chlorine counter ions, and solvated in an octahedral box of TIP3P 8 water molecules with solvent layers 10 Å between the box edges and solute surface. The SHAKE 9,10 algorithm was used to restrict all covalent bonds involving hydrogen atoms with a time step of 2 femtoseconds (fs). The Particle mesh Ewald (PME) method 11 was performed to treat long-range electrostatic interactions. For each ligand-receptor system, three steps of minimization were performed before the heating step. First, all atoms in the receptor-ligand complex were restrained with 50 kcal/(mol Å2), whereas the solvent molecules were not restrained. This step included 2,000 cycles of steepest descent minimization and 2,000 cycles of conjugated gradient minimization. Second, all heavy atoms were restrained with 10 kcal/(mol Å2) during the minimization steps, which included 2,500 cycles of steepest descent minimization and 2,500 cycles of conjugated gradient minimization. The third step included 5,000 cycles of steepest descent minimization and 5,000 cycles of conjugated gradient minimization without restraint. After the energy minimizations, the whole system was first heated from 0 to 300 K in 50 ps using Langevin dynamics at a constant volume and then equilibrated for 400 ps at a constant pressure of 1 atm. A weak constraint of 10 kcal/ (mol Å2) was used to restrain all heavy atoms in the receptor-ligand complexes during the heating steps. Finally, periodic boundary dynamic simulations were conducted on the whole system with an NPT (constant composition, pressure, and temperature) ensemble at a constant pressure of 1 atom and 300 K in the production step. Each receptor-ligand solution complex was simulated for 20 ns. 4

5 The coordinates of each system were saved every 2 ps. The root-mean-square deviations (RMSDs) of the complexes were calculated using PTRAJ module. Trajectories were analyzed using average linkage algorithm to produce 3 clusters using the pairwise RMSD between frames as a metric comparing the atoms named CA. 2. Binding Energy Calculations. The MM/PBSA method 12 in the Amber Tools suite was used to calculate the binding energies for each complex. For each ligand target complex, the trajectory was generated through MD simulation from the initial pose. The free energy of binding, ΔGbinding, was calculated using Eq. (1) from the free energy of the receptorligand complex (Gcpx) with respect to the unbound receptor (Grec) and ligand (Glig): ΔGbinding = Gcpx (Grec + Glig) (1) The MM-PBSA (Molecular Mechanics-Possion-Boltzmann/Surface Area) methodology allows the calculation of the complete binding reaction energy, including the desolvation of the ligand and the unbound protein, on the basis of a thermodynamic cycle. Therefore, Eq. (1) can be approximated as ΔGbinding = ΔEMM TΔS + ΔGsol (2) ΔEMM = ΔEele + ΔEvdw (3) All energies expressed in the above equations were averaged over the course of the molecular dynamics trajectories. In Eq. (3), ΔEMM is the molecular mechanical energy obtained from the electrostatic (ΔEele) and the van der waals (ΔEvdw) interactions within the system. Here, TΔS is the solute entropic contribution at temperature T (kelvin) and the solvation free energy (ΔGsol) represents the electrostatic and nonpolar free energy of solvation, and therefore can be expressed as ele G = G G nonpolar sol sol sol ele where G sol is the polar contribution to solvation and G nonpolar sol is the nonpolar solvation term. The former component was calculated using the PB calculation, whereas the latter term is determined using Eq. (5): G SASA b nonpolar sol where SASA is the solvent-accessible surface area (Å2) and γ and b represent (4) (5) 5

6 experimental solvation parameters. References (1) Salomon-Ferrer, R.; Götz, A. W.; Poole, D.; Le Grand, S.; Walker, R. C. J. Chem. Theory Comput. 2013, 9, (2) Gotz, A. W.; Williamson, M. J.; Xu, D.; Poole, D.; Le Grand, S.; Walker, R. C. J. Chem. Theory Comput. 2012, 8, (3) D.A. Case, T. A. D., T.E. Cheatham, III, C.L. Simmerling, J. Wang, R.E. Duke, R. Luo, R.C. Walker, W. Zhang, K.M. Merz, B. Roberts, S. Hayik, A. Roitberg, G. Seabra, J. Swails, A.W. Goetz, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, R.M. Wolf, J. Liu, X. Wu, S.R. Brozell, T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.-J. Hsieh, G. Cui, D.R. Roe, D.H. Mathews, M.G. Seetin, R. Salomon-Ferrer, C. Sagui, V. Babin, T. Luchko, S. Gusarov, A. Kovalenko, and P.A. Kollman University of California, San Francisco, (4) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Wallingford CT,

7 (5) Mukherjee, G.; Patra, N.; Barua, P.; Jayaram, B. J. Comput. Chem. 2011, 32, (6) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Proteins 2006, 65, (7) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, (8) Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. J. Mol. Graphics Model. 2006, 25, (9) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, (10) Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, (11) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, (12) Hou, T.; Wang, J.; Li, Y.; Wang, W. J. Chem. Inf. Model. 2011, 51,

8 Figure S1. The IR Spectra of Compound 1 8

9 Relative Abundance 1503A #18 RT: 0.10 AV: 1 NL: 4.58E4 T: FTMS - c ESI Full ms [ ] m/z SPECTRUM simulation : m/z Theo. Mass Delta (ppm) RDB equiv. Composition C21 H29 O8 Figure S2. The HRESIMS Spectroscopic Data of Compound 1 9

10 Figure S3. The 1 H NMR Spectrum of Compound 1 in Methanol-d 4 10

11 Figure S4. The 13 C NMR Spectrum of Compound 1 in Methanol-d 4 11

12 Figure S5. The 1 H- 1 H COSY Spectrum of Compound 1 in Methanol-d 4 12

13 Figure S6. The HSQC Spectrum of Compound 1 in Methanol-d 4 13

14 Figure S7. The HMBC Spectrum of Compound 1 in Methanol-d 4 14

15 Figure S8. The NOESY Spectrum of Compound 1 in Methanol-d 4 15

16 Figure S9. The IR Spectra of Compound 2 16

17 Relative Abundance 1503A #14 RT: 0.07 AV: 1 NL: 3.67E6 T: FTMS + c ESI Full ms [ ] m/z SPECTRUM - simulation : m/z Theo. Mass Delta (ppm) RDB equiv. Composition C14 H17 O5 Figure S10. The HRESIMS Spectroscopic Data of Compound 2 17

18 Figure S11. The 1 H NMR Spectrum of Compound 2 in Methanol-d 4 18

19 Figure S12. The 13 C NMR Spectrum of Compound 2 in Methanol-d 4 19

20 Figure S13. The 1 H- 1 H COSY Spectrum of Compound 2 in Methanol-d 4 20

21 Figure S14. The HSQC Spectrum of Compound 2 in Methanol-d 4 21

22 Figure S15. The HMBC Spectrum of Compound 2 in Methanol-d 4 22

23 Figure S16. The NOESY Spectrum of Compound 2 in Methanol-d 4 23

24 Figure S17. The IR Spectra of Compound 3 24

25 Relative Abundance 1503A #13 RT: 0.06 AV: 1 NL: 5.32E6 T: FTMS + c ESI Full ms [ ] m/z SPECTRUM - simulation : m/z Theo. Mass Delta (ppm) RDB equiv. Composition C14 H19 O4 Figure S18. The HRESIMS Spectroscopic Data of Compound 3 25

26 Figure S19. The 1 H NMR Spectrum of Compound 3 in Methanol-d 4 26

27 Figure S20. The 13 C NMR Spectrum of Compound 3 in Methanol-d 4 27

28 Figure S21. The 1 H- 1 H COSY Spectrum of Compound 3 in Methanol-d 4 28

29 Figure S22. The HSQC Spectrum of Compound 3 in Methanol-d 4 29

30 Figure S23. The HMBC Spectrum of Compound 3 in Methanol-d 4 30

31 Figure S24. The NOESY Spectrum of Compound 3 in Methanol-d 4 31

32 Figure S25. The IR Spectra of Compound 4 32

33 Relative Abundance Instrument:MAT 95XP(Thermo) D:\DATA-HR\15\ lpc-18a-c1 3/19/ :30:19 AM LPC-18A lpc-18a-c1 #18 RT: 0.63 AV: 1 NL: 1.39E4 T: + c EI Full ms [ ] m/z Mass Relative Theoretical Delta Delta RDB Composition Intensity Mass [ppm] [mmu] C14H18O4 Figure S26. The HREIMS Spectroscopic Data of Compound 4 33

34 Figure S27. The 1 H NMR Spectrum of Compound 4 in Methanol-d 4 34

35 Figure S28. The 13 C NMR Spectrum of Compound 4 in Methanol-d 4 35

36 Figure S29. The 1 H- 1 H COSY Spectrum of Compound 4 in Methanol-d 4 36

37 Figure S30. The HSQC Spectrum of Compound 4 in Methanol-d 4 37

38 Figure S31. The HMBC Spectrum of Compound 4 in Methanol-d 4 38

39 Figure S32. The NOESY Spectrum of Compound 4 in Methanol-d 4 39

40 Figure S33. The IR Spectra of Compound 17 40

41 Relative Abundance Instrument:MAT 95XP(Thermo) D:\DATA-HR\15\ lpc-c1 4/2/ :43:40 AM LPC-21-A lpc-c1 #13 RT: 0.51 AV: 1 NL: 2.33E3 T: + c EI Full ms [ ] m/z Mass Relative Theoretical Delta Delta RDB Composition Intensity Mass [ppm] [mmu] C20H34O4 Figure S34. The HREIMS Spectroscopic Data of Compound 17 41

42 Figure S35. The 1 H NMR Spectrum of Compound 17 in Pyridine-d 5 42

43 Figure S36. The 13 C NMR Spectrum of Compound 17 in Pyridine-d 5 43

44 Figure S37. The 1 H- 1 H COSY Spectrum of Compound 17 in Pyridine-d 5 44

45 Figure S38. The HSQC Spectrum of Compound 17 in Pyridine-d 5 45

46 Figure S39. The HMBC Spectrum of Compound 17 in Pyridine-d 5 46

47 Figure S40. The NOESY Spectrum of Compound 17 in Pyridine-d 5 47

48 Figure S41. Four Stereoisomers of Compound 17 48

49 Figure S42. RMSD Curves of Four Complexes During 20 ns MD Simulations Figure S43. Decomposition of Binding Free Energy on the Key Residue for Complex 4 to LXRs. The unit for energy contribution per residue is kcal/mol. 49

50 Table S1. Binding Energy Analysis for the Binding of 4 to LXRα and LXRβ Energy terms LXRα/4 (kal/mol) LXRβ/4 (kal/mol) ΔEvdw a (2.29) (2.55) ΔEele b (4.50) (3.33) ΔEepb c (3.04) (2.48) ΔGgas d (4.09) (3.18) ΔGsolv e (3.04) (2.48) ΔGbinding f (2.94) (3.15) a Nonbonded van der Waals. b Nonbonded electrostatics. c Polar component to solvation. d Total gas phase energy. e Sum of nonpolar and polar contributions to solvation. f Final estimated binding free energy calculated from the terms above. Standard deviation values are shown in parentheses. 50