Supporting Information

Transcription

1 Supporting Information Discovery of Pentacyclic Triterpenes as Potential Entry Inhibitors of Influenza Viruses Maorong Yu*,,, Longlong Si,, Yufei Wang, Yiming Wu, Fei Yu,, Pingxuan Jiao, Yongying Shi, Han Wang, Sulong Xiao, Ge Fu, Ke Tian #, Yitao Wang ±, Zhihong Guo, Xinshan Ye, Lihe Zhang and Demin Zhou*,, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing , China # Stanley Manne Children's Research Institute, Northwestern University, Chicago, Illinois, USA ± State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao Department of Chemistry and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Medical Faculty of Kunming University of Science and Technology, Kunming , China Table of Content HPLC data for target compounds S2 S Figure 1. Discovery of triterpene-glycoconjugates as potent anti-influenza virus agents S10 S Figure 2. Identification of the action mode and mechanism of the lead compounds S11 S Figure 3. Test of the antiviral specificity of lead compounds against Ad5-eGFP, VSV, EMCV S12 S Figure 4. Evaluation of toxicity of lead compound Y3 towards Balb/C mice via intranasal administration S13 S Table 1. The K D value (µm) of the lead compound to different HA proteins S14 S Table 2. The SPR responses of HA protein to sialic acid receptor in the presence of lead compound S15 S Table 3. The kinetics parameters of HA protein to sialic acid receptor in the presence of lead compound S16 Copies of 1 H, 1 C NMR spectra and HRMS S17 S1

2 HPLC data for target compounds General procedure for purity determination by HPLC. An Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, USA) comprised a quaternary solvent delivery system, an on-line degasser, an auto-sampler, a column temperature controller and DAD detector coupled with an analytical workstation. The column configuration was an Agilent Zorbax SB C 18 reserved phase column (5µm, 250 mm 4.6 mm) coupled with an Agilent Zorbax SB C 18 guard column (5µm, 10 mm 4.6 mm). The compounds were dissolved in methanol and injection volume was10 µl. Detection wavelength was set at 215 nm, the flow rate was 1.0 ml min 1 and the column temperature was maintained at 30 ºC. The mobile phase was gradient elution which was mixed with solvent A (water) and B (acetonitrile). For triterpene-glycoconjugates with acetylation of the glycoside and aglycone moiety (EA, A, UA), the gradient program was as follows: 0 40 min 75% B. For triterpene-glycoconjugates without acetylation of the glycoside, the gradient program was as follows: 0 40 min 45% B. The above gradient program would result in the retention time of between 10 and 40 minute. Results indicated that all the compounds used in our anti-influenza virus test had a purity of more than 95%, even 98%. S2

3 Table. HPLC purity data for target compounds Compound RT (min) Purity (%) Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Y >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% Q >95% EA >95% A >95% UA >95% S3

4 HPLC Figure Y1 Y2 Y3 Y4 Y5 S4

5 Y6 Y7 Y8 Y9 Y10 Y11 S5

6 Y12 Y13 Y14 Y15 Y16 Q1 S6

7 Q2 Q3 Q4 Q5 Q6 Q7 S7

8 Q8 Q9 Q10 Q11 Q12 Q13 S8

9 EA A UA S9

10 S Figure 1. Discovery of triterpene-glycoconjugates as potent anti-influenza virus agents. (A) The mini-library of EA-glycoconjugates used in this study. (B) The cytotoxic effect-based screen of EA-glycoconjugate mini-library (50 µm) using CellTiter-Glo Assay. DMS acted as negative control. Error bars indicate standard deviations of triplicate experiments. (C) The cytopathic effect-based screen of EA-glycoconjugate mini-library. MDCK was utilized as the host cell to test A/WSN/33 virus infection; DMS acted as negative control. Error bars indicate standard deviations of triplicate experiments. S10

11 S Figure 2. Identification of the action mode and mechanism of the lead compounds. (A) Comparisons of the different antiviral mechanism of lead compound Y3 (attachment-targeting), ribavirin (replication-targeting) and Tamiflu (viral release-targeting). (B) Characterization of the affinity between lead compound Y4 and HA protein, which was immobilized on a CM5 sensor chip, based on the surface plasmon resonance assay. The negative compound ribavirin (RBV) was also tested. Their Kd values were in labeled the corresponding curves. S11

12 S Figure 3. Test of the antiviral specificity of lead compounds (50 µm) against adenovirus (Ad5-eGFP), vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). They did not exert protective effect from infection by these viruses, but the pseudotyped influenza A virus (IAVpp). S12

13 S Figure 4. Evaluation of toxicity of lead compound Y3 towards Balb/C mice via intranasal administration. No differences in the body weight between the treated and untreated mice with Y3. Five female Balb/C mice in each group were administrated intranasally with Y3 at dose 0, , 0.125, 0.25 and 0.5mg/20g/d twice daily for five consecutive days. The body weights and overall health statuses of mice were monitored for 9 days. S13

14 S Table 1. The K D value (µm) of the lead compound to different HA proteins Strains a Compounds H3-HA H5-HA H7-HA B-HA Y Y NT Q NT 7.6 a Virus strains used are indicated as follows: H3: A/Wyoming/03/2003 (H3N2); H5: A/chicken/India/NIV33487/2006(H5N1); H7: A/Shanghai/1/2013(H7N9); B: B/Florida/4/2006. NT: not tested. S14

15 S Table 2. The SPR responses of HA protein to sialic acid receptor in the presence of lead compound. Concentration (µg/ml) SPR response (R.U.) (-) (+)Y (+)Curcumin (+)Arbidol S15

16 S Table 3. The kinetics parameters of HA protein to sialic acid receptor in the presence of lead compound Kinetics parameter K D (nm) Ka (1/s) Kd (1/Ms) (-) (+)Y (+)Curcumin (+)Arbidol S16

17 H H Ac Ac Ac Ac Y1 H H Ac Ac Ac Ac Y1 S17

18 H H Ac Ac Ac Ac Y1 H H H H H H Y2 S18

19 H H H H H H Y2 H H H H H H Y2 S19

20 H Ac Ac Ac Ac Y3 H Ac Ac Ac Ac Y3 S20

21 H Ac Ac Ac Ac Y3 H H H H H Y4 S21

22 H H H H H Y4 H H H H H Y4 S22

23 H Ac Ac Ac Ac Y5 H Ac Ac Ac Y5 Ac S23

24 H Ac Ac Ac Ac Y5 H H H H H Y6 S24

25 H H H H Y6 H H H H H Y6 H S25

26 H Ac Ac Ac Ac Y7 H H H H H Y8 S26

27 H H H H H Y8 H H H H H Y8 S27

28 H Ac Ac Ac Ac Y9 H Ac Ac Ac Ac Y9 S28

29 H Ac Ac Ac Ac Y9 H H H H H Y10 S29

30 H H H H H Y10 H H H H H Y10 S30

31 Ac Ac Ac Ac Y11 Ac Ac Ac Ac Y11 S31

32 Ac Ac Ac Ac Y11 H H H H Y12 S32

33 H H H H Y12 H H H H Y12 S33

34 Ac Ac Ac Ac Y13 Ac Ac Ac Ac Y13 S34

35 Ac Ac Ac Ac Y13 H H H H Y14 S35

36 H H H H Y14 H H H H Y14 S36

37 H Ac H Ac Ac Ac Y15 H Ac H Ac Ac Ac Y15 S37

38 H Ac H Ac Ac Ac Y15 H H H H H H Y16 S38

39 H H H H H H Y16 H H H H H H Y16 S39