Supporting Information

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1 Supporting Information Discovery of Hydrocarbon-Stapled Short α-helical Peptides as Promising Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Fusion Inhibitors Chao Wang,, Shuai Xia,, Peiyu Zhang, #, Tianhong Zhang, Weicong Wang, ǁ Yangli Tian, Guangpeng Meng, # Shibo Jiang *,, and Keliang Liu *,,# State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, 27 Tai- Ping Road, Beijing, , China; Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences & Shanghai Public Health Clinical Center, Fudan University, 130 Dong-An Road, Shanghai , China; # Key Laboratory of Structure-based Drug Design & Discovery of the Ministry of Education, Shenyang Pharmaceutical University, Shenyang , China; Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA; ǁ Pharmaceutical Preparation Section, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , China; These authors contributed equally to this work. Corresponding Authors: K.L.: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing , China; Tel.: ; Fax: , keliangliu55@126.com. S.J.: Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences & Shanghai Public Health Clinical Center, Fudan University, 130 Dong-An Road, Shanghai , China; Tel.: ; Fax: ; shibojiang@fudan.edu.cn. Table of Contents Supplemental Tables and Figures Table S1. Inhibitory activity of peptides on MERS-CoV infection in Calu-3 cells S2 Table S2. Solubility of P21S8, P21S10 and P21S8Z......S2 Table S3. The biophysical properties and biological activity of uncyclized peptides S2 Table S4. Biophysical properties of the stapled peptides S3 Table S5. Summary of the SVA results of P21S8/HR1P and P21S10/HR1P complexes... S3 Table S6. HPLC method used for the purification of peptide compounds. S3 Table S7. HPLC method used for the analysis of peptide compounds.. S4 Figure S1. Stapled peptides as inhibitors of MERS-CoV infection S5 Figure S2. Cytotoxicity of the P21S8, P21S10, and P21S8Z peptides on Huh-7 cells S5 Figure S3. Cytotoxicity of the P21S8, P21S10, P21S8Z, and HR2P-M2 on Calu-3 cells S6 Figure S4. N-PAGE analysis of the stapled peptides and their complexes with HR1P S6 Pharmacokinetic assessments.... S7 Q-FT-ICR-MS and Analytical HPLC of designed peptides. S8 S1

2 Table S1. Inhibitory activity of peptides on MERS-CoV infection in Calu-3 cells a Compound EC 50 (µm) CC 50 (µm) P21S ± 0.62 >100 P21S ± 0.16 >100 P21S8Z 2.57 ± 0.24 >100 HR2P-M ± 0.05 >100 a MERS-CoV (EMC/2012) pseudovirus was used in this assay. Data were derived from the results of three independent experiments and are presented as the mean ± standard deviation. Table S2. Solubility of P21S8, P21S10 and P21S8Z Compd Solubility (mg/ml) PBS (ph 7.4) H 2 O P21S ± ± 0.08 P21S ± ± 0.1 P21S8Z 0.47 ± ± 0.06 Table S3. The biophysical properties of uncyclized peptides Compd Sequence a Helicity (%) P21L1 XDLTXEM LSLQQVV KALNESY not determined b P21L2 LXLTYXM LSLQQVV KALNESY not determined P21L3 LDLXYEM XSLQQVV KALNESY 5.3 P21L4 LDLTXEM LXLQQVV KALNESY 7.9 P21L5 LDLTYEM XSLQXVV KALNESY 14.4 P21L6 LDLTYEM LXLQQXV KALNESY 45.8 P21L7 LDLTYEM LSLXQVV XALNESY 18.1 P21L8 LDLTYEM LSLQXVV KXLNESY 37.4 P21L9 LDLTYEM LSLQQVV XALNXSY 26.8 P21L10 LDLTYEM LSLQQVV KXLNEXY 35.6 a These peptides have an acetyl group at the N-terminus and carboxyamide at the C-terminus. X indicates the position of S5 amino acids which left uncyclized. b Peptide too insoluble for accurate measurement. S2

3 Table S4. Biophysical properties of the stapled peptides a Compd Helicity (%) Complex with HR1P Helicity (%) Tm ( C) c P21S undetectable P21S2 not determined b not determined not determined P21S undetectable P21S ± 0.6 P21S ± 0.3 P21S undetectable P21S undetectable P21S ± 0.1 P21S ± 0.4 P21S ± 0.2 a CD spectra of each designed peptide were monitored in 50 mm PBS, ph 7.2. The final concentration of the peptide was 50 µm. b Peptide too insoluble for accurate measurement. c Numbers after ± are standard deviations from three measurements. Table S5. Summary of the SVA results of P21S8/HR1P and P21S10/HR1P complexes a Complex Sedimentation coefficient (s) Observed molecular mass (kda) Calculated molecular mass (kda) P21S8/HR1P 6-HB P21S10/HR1P 6-HB a SVA studies were performed at a concentration of 250 µm in PBS (ph 7.2) and a rotor speed of 60,000 rpm at 20 ºC. Table S6. HPLC method used for the purification of peptide compounds a Time (min) Solvent A (%) Solvent B (%) a The crude peptide products were purified by preparative reverse phase HPLC with a Waters preparative HPLC system (PrepLC 4000) on a Waters X-bridge C8 column (19.5mm 250mm, 10µm) at constant flow rate of 16 ml/min. Solvent A: 0.1% trifluoroacetic acid in H 2 O; Solvent B: 0.1% trifluoroacetic acid in 70%CH 3 CN/H 2 O. S3

4 Table S7. HPLC method used for the analysis of peptide compounds a Methods Time (min) Solvent A (%) Solvent B (%) Method A Method B a The peptide compounds were analyzed by analytical RP-HPLC was performed on a RP-C8 column (Zorbax Eclipse XDB-C8, mm, 5 µm) using two different solvent systems (Methods A and B), and a flow rate of 1 ml/min with detection at 210 nm. Solvent A: 0.1% trifluoroacetic acid in H 2 O; Solvent B: 0.1% trifluoroacetic acid in 70% CH 3 CN/H 2 O. S4

5 Figure S1. Stapled peptides as inhibitors of MERS-CoV infection. Inhibition of infection by pseudoviruses expressing (A) S protein of MERS-CoV Erasmus Medical Center (EMC)/2012 strain, or S protein with (B) Q1020H or (C) Q1020R in Huh-7 cells. (D) Inhibitory activity of peptides on pseudotyped MERS-CoV (EMC/2012) infection in Calu-3 cells. Figure S2. Cytotoxicity of the P21S8, P21S10, and P21S8Z peptides on Huh-7 cells. S5

6 Figure S3. Cytotoxicity of the P21S8, P21S10, P21S8Z, and HR2P-M2 on Calu-3 cells. Figure S4. N-PAGE analysis of the stapled peptides and their complexes with HR1P. Lane 1: P21S1 + HR1P, lane 2: P21S1, lane 3: P21S7 + HR1P, lane 4: P21S7, lane 5: P21S4 + HR1P, lane 6: P21S4, lane 7: P21S5 + HR1P, lane 8: P21S5, lane 9: P21S6 + HR1P, lane 10: P21S6. S6

7 Pharmacokinetic assessments Qualification assay. A Waters Acquity UPLC system was used for chromatographic analysis. The chromatography was performed using an Agilent SB-Aq RRHD column (1.8 µm, 100 mm 2.1mm) kept at ambient temperature. The mobile phase was composed of solvent M (water containing 1% formic acid) and solvent N (methanol containing 1% formic acid). The HPLC separation was performed by a gradient method of solvent B from 5% to 30% over 1 min, from 30% to 70% over 0.5 min, from 70% to 95% over 1.5 min, and holding the column at 95% B for 1 min. The sample injection volume was 10 µl. Chemical reagents and solvents were HPLC grade. AB Sciex QTRAP 5500 mass spectrometer equipped with an electrospray ion (ESI) source working in positive ion mode were used for mass spectrometric measurements. The curtain, nebulizer, and turbo-gas (all nitrogen) pressures were set at 20 psi, 60 psi, and 60 psi, respectively. The source temperature was 550 C and the ion spray needle voltage was 5500 V. The collision activated dissociation gas level was set at 9. Multiple reaction monitoring (MRM) were used to monitor the ion transitions at m/z for P21S8, m/z for P21S10, and m/z for HR2P-M2. Preparation of samples. By use of a simple protein precipitation method, a test compound was extracted from rat plasma. Plasma samples for standard curves were prepared by spiking 50 µl of rat plasma with 10 µl of various concentrations of each test compound ranging from 1 to 50 µg/ ml in methanol/water (50/50, v/v) and a constant volume (10 µl) of the internal standard (IS) (4 µg/ ml in methanol/water (50/50, v/v)). Calibration curves for testing compounds in plasma were linear in the concentration range of µg/ml, with correlation coefficients of > for all curves. To each tested plasma sample (50 µl), 10 µl methanol/water (50/50, v/v) and 10 µl of the same concentration of IS was added. After the mixture was vortexed and centrifuged at 13,000g for 10 min, the supernatant was transferred to autosampler vials, and 10 µl of the supernatant was injected into the UPLC-MS/MS system for analysis. In vivo animal test. Three male Sprague-Dawley rats (200 ± 10 g) were used in each study. Each of these rats was dosed with a testing compound at 4 mg/kg for i.v. administration. Blood samples were collected at 0, 5, 15, 30, 60, 120, and 240 min and were immediately centrifuged to separate the plasma fractions. The plasma samples obtained were stored at -20 C until analysis. Concentration-versus-time profiles were obtained for each analyte to calculate parameters. Data for each pharmacokinetic parameter were averaged and reported as mean ± standard deviation. S7

8 Q-FT-ICR-MS and analytical HPLC of designed peptides S8

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20 Analytical HPLC of P21S1 Analytical HPLC of P21S1 [97% purity, t R (Method A)= 13.3min] [98% purity, t R (Method B)=10.2 min] Analytical HPLC of P21S2 Analytical HPLC of P21S2 [98% purity, t R (Method A)= 20.9min] [99% purity, t R (Method B)= 17.1min] Analytical HPLC of P21S3 Analytical HPLC of P21S3 [98% purity, t R (Method A)= 13.2min] [97% purity, t R (Method B)=10.8 min] S20

21 Analytical HPLC of P21S4 Analytical HPLC of P21S4 [95% purity, t R (Method A)=12.9 min] [97% purity, t R (Method B)=9.6 min] Analytical HPLC of P21S5 [98%purity, t R (Method A)= 17.7min] Analytical HPLC of P21S5 [97% purity, t R (Method B)=13.6 min] Analytical HPLC of P21S6 Analytical HPLC of P21S6 [95% purity, t R (Method A)= 12.1min] [96% purity, t R (Method B)=10.5 min] Analytical HPLC of P21S7 Analytical HPLC of P21S7 [98% purity, t R (Method A)= 13.9min] [98% purity, t R (Method B)=10.8 min] Analytical HPLC of P21S8 Analytical HPLC of P21S8 [99% purity, t R (Method A)=13.2 min] [99% purity, t R (Method B)= 10.5min] S21

22 Analytical HPLC of P21S9 Analytical HPLC of P21S9 [97% purity, t R (Method A)= 12.6min] [97% purity, t R (Method B)= 9.6min] Analytical HPLC of P21S10 Analytical HPLC of P21S10 [97% purity, t R (Method A)=12.9 min] [99% purity, t R (Method B)=9.7 min] Analytical HPLC of P21R8 Analytical HPLC of P21R8 [99% purity, t R (Method A)=12.9 min] [99% purity, t R (Method B)= 9.1min] Analytical HPLC of P21S8Z Analytical HPLC of P21S8Z [98% purity, t R (Method A)=12.6 min] [99% purity, t R (Method B)= 9.7min] Analytical HPLC of P21S8F Analytical HPLC of P21S8F [96% purity, t R (Method A)= 14.8min] [97% purity, t R (Method B)=10.8 min] S22

23 Analytical HPLC of P21SZF Analytical HPLC of P21SZF [97% purity, t R (Method A)=15.5 min] [98% purity, t R (Method B)= 12.6min] Analytical HPLC of P21L1 Analytical HPLC of P21L1 [99% purity, t R (Method A)= 12.8min] [99% purity, t R (Method B)= 10.6min] Analytical HPLC of P21L2 Analytical HPLC of P21L2 [98% purity, t R (Method A)= 20.2min] [98% purity, t R (Method B)=16.4 min] Analytical HPLC of P21L3 Analytical HPLC of P21L3 [98% purity, t R (Method A)= 16.0min] [97% purity, t R (Method B)=12.9 min] Analytical HPLC of P21L4 Analytical HPLC of P21L4 [98% purity, t R (Method A)=13.5 min] [99% purity, t R (Method B)=11.3 min] S23

24 Analytical HPLC of P21L5 Analytical HPLC of P21L5 [98% purity, t R (Method A)= 12.0min] [99% purity, t R (Method B)= 8.5min] Analytical HPLC of P21L6 Analytical HPLC of P21L6 [99% purity, t R (Method A)=12.7 min] [98% purity, t R (Method B)=11.2 min] Analytical HPLC of P21L7 Analytical HPLC of P21L7 [96% purity, t R (Method A)= 17.9min] [98% purity, t R (Method B)=15.2 min] Analytical HPLC of P21L8 Analytical HPLC of P21L8 [99% purity, t R (Method A)= 12.8min] [99% purity, t R (Method B)= 9.5min] Analytical HPLC of P21L9 Analytical HPLC of P21L9 [98% purity, t R (Method B)=12.9 min] [96% purity, t R (Method A)= 12.6min] S24

25 Analytical HPLC of P21L10 Analytical HPLC of P21L10 [99% purity, t R (Method A)=13.2 min] [99% purity, t R (Method B)=11.5 min] Analytical HPLC of P21 Analytical HPLC of P21 [99% purity, t R (Method A)=13.5 min] [99% purity, t R (Method B)= 11.2min] S25