Supporting Information

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1 Supporting Information Exploring the N-terminal region of C-X-C motif chemokine 12 (CXCL12): Identification of Plasma-Stable Cyclic Peptides As Novel, Potent C-X-C Chemokine Receptor Type 4 (CXCR4) Antagonists Salvatore Di Maro, 1 Anna Maria Trotta, 2 Diego Brancaccio, 3,4 Francesco Saverio Di Leva, 3 Valeria La Pietra, 3 Caterina Ieranò, 2 Maria Napolitano, 2 Luigi Portella, 2 Crescenzo D Alterio, 2 Rosa Siciliano 5 Deborah Sementa, 3 Stefano Tomassi, 3 Alfonso Carotenuto, 3 Ettore Novellino 3 Stefania Scala 2,* and Luciana Marinelli 3,* 1 DiSTABiF, Second University of Naples, Via Vivaldi 43, Caserta, Italy. 2 Genomica Funzionale, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione Giovanni Pascale", IRCCS-ITALY, Via M. Semmola 52, Naples, Italy. 3 Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II", via D. Montesano 49, Naples, Italy. 4 Laboratory of Food Chemistry, Dipartimento di Agraria (QuaSic.A.Tec.), Università Mediterranea di Reggio Calabria, Reggio Calabria, loc. Feo di Vito, Reggio Calabria, Italy 5 Istituto di Scienze dell Alimentazione CNR, Via Roma 64, Avellino, Italy. S1

2 CONTENTS Figure S1. Inhibition of CEM (a) and HT29 (b) human cancer cells migration experiments. Figure S2. Migration inhibition curve of 10 in CCRF-CEM cells. Figure S3. C carbons r.m.s.d. of residues at positions 3 to 7 of peptides 2 (red plot) and 10 (black plot) along the MD production run. Figure S4. (a) Binding mode of 2 at the CXCR4 receptor obtained through over 100 ns long MD simulations and (b) superposition between 2 and 10 at the CXCR4 binding site. Table S1. NMR resonance assignments of 2 in SDS- d25 solution at 298 K. Table S2. NMR resonance assignments of 10 in SDS- d25 solution at 298 K. Table S3. NOE derived upper limit constraints of 2. Table S4. NOE derived upper limit constraints of 10. S2

3 Figure S1. Migration experiments on CEM (a) and HT29 (b) human cancer cells: Migrated cells on the lower surface were fixed, stained with H&E and counted microscopically. The results are expressed as the average of the absolute number of cells seen in each of the ten high-power field (magnification 400X) in each experimental point. Data are presented as bar graph showing mean ± S.D. S3

4 Figure S2. Migration inhibition curve of 10 in CCRF-CEM cells. Figure S3. C carbons r.m.s.d. of residues at positions 3 to 7 of peptides 2 (red plot) and 10 (black plot) along the MD production run. Prior to the r.m.s.d calculations, trajectory frames were aligned on the C carbons of the CXCR4 transmembrane helices. S4

5 Figure S4. (a) Binding mode of 2 (green sticks) at the CXCR4 receptor (grey cartoons) obtained through over 100 ns long MD simulations. Receptor amino acids and waters important for peptide binding are shown as sticks. Hydrogen-bonds are displayed as dashed black lines. Nonpolar hydrogens are omitted for clarity reasons. (b) Superposition between 2 (green sticks) and 10 (orange sticks) at the CXCR4 (grey cartoons) binding site. S5

6 Table S1. NMR Resonance Assignments a of 2 in SDS- d25 Solution at 298 K. residue NH ( 3 J N, - / T) b C H C H Others Arg , , 1.70( ); 3.22(δ); 7.24(ε); Ala (6.1, 6.5) Cyss (7.3, 6.7) , 3.30 Arg (8.2, 5.3) , , 1.12( ); 2.90(δ); 6.99(ε) Phe (9.0, 2.6) (δ); 7.18(ε) Phe (8.2, 3.3) , (δ); 7.24(ε) Cyss (7.8, 5.0) , 3.31 a Obtained at ph = 5.5, with TSP ( 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 ppm. b 3 J N coupling constants in Hz. - / T = temperature coefficients (ppb/k) calculated in the range 298K 313 K. S6

7 Table S2. NMR Resonance Assignments a of 10 in SDS- d25 Solution at 298 K. residue NH ( 3 J N, - / T) b C H C H Others Arg (9.7, 5.3) , ( ); 3.25(δ); 7.14(ε); Ala (7.4, 6.0) DCyss (6.4, 2.9) , 3.26 Arg (7.5, 4.0) , ( ); 2.93(δ); 6.97(ε) Phe (9.4, 3.4) (δ); 7.16(ε); 7.11(ζ) Phe (8.2, 3.8) , (δ); 7.25(ε); Cyss (b.s, 3.4) , 3.33 a Obtained at ph = 5.5, with TSP ( 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 ppm. b 3 J N coupling constants in Hz. / T = temperature coefficients (ppb/k) calculated in the range 298K 313 K. Further signals: CH 3 CO, 2.12 ppm. S7

8 Table S3. NOE derived Upper Limit Constraints of 2. 1 ARG+ HA 1 ARG+ HB ARG+ HA 1 ARG+ HB ARG+ HA 2 ALA HN ARG+ HA 2 ALA QB ARG+ HA 3 CYSS HN ARG+ HB2 1 ARG+ HE ARG+ HB2 2 ALA HN ARG+ HB2 3 CYSS HN ARG+ HB3 1 ARG+ HE ARG+ HB3 2 ALA HN ARG+ HB3 3 CYSS HN ARG+ QB 2 ALA HN ARG+ QB 3 CYSS HN ARG+ HG2 2 ALA HN ARG+ HG2 3 CYSS HN ARG+ HG3 2 ALA HN ARG+ HG3 3 CYSS HN ARG+ QG 3 CYSS HA ARG+ QD 2 ALA HN ALA HN 3 CYSS HN ALA HA 3 CYSS HN ALA HA 3 CYSS HB ALA HA 3 CYSS HB ALA QB 3 CYSS HN CYSS HN 3 CYSS HA CYSS HN 3 CYSS HB CYSS HN 3 CYSS HB CYSS HA 3 CYSS HB CYSS HA 3 CYSS HB CYSS HA 4 ARG+ HN CYSS HB2 4 ARG+ HN CYSS HB2 5 PHE HN CYSS HB3 4 ARG+ HN CYSS HB3 5 PHE HN CYSS QB 4 ARG+ HN CYSS QB 5 PHE HN ARG+ HN 4 ARG+ HB ARG+ HN 4 ARG+ HB ARG+ HN 4 ARG+ QB ARG+ HN 4 ARG+ HG ARG+ HN 4 ARG+ HG ARG+ HN 4 ARG+ QD ARG+ HN 5 PHE HN ARG+ HN 5 PHE QB ARG+ HN 5 PHE QD ARG+ HN 6 PHE HN ARG+ HA 4 ARG+ HB ARG+ HA 4 ARG+ HB ARG+ HA 5 PHE HN ARG+ HA 5 PHE QD ARG+ HA 6 PHE HN ARG+ HB2 4 ARG+ HE ARG+ HB2 5 PHE HN ARG+ HB2 5 PHE QB ARG+ HB2 5 PHE QD 7.12 S8

9 4 ARG+ HB2 5 PHE QE ARG+ HB2 6 PHE HN ARG+ HB3 4 ARG+ HE ARG+ HB3 5 PHE HN ARG+ HB3 5 PHE QB ARG+ HB3 5 PHE QD ARG+ HB3 5 PHE QE ARG+ HB3 6 PHE HN ARG+ QB 6 PHE HN ARG+ HG2 5 PHE HN ARG+ HG2 5 PHE QD ARG+ HG2 5 PHE QE ARG+ HG3 5 PHE HN ARG+ HG3 5 PHE QD ARG+ HG3 5 PHE QE ARG+ QD 5 PHE HN ARG+ QD 5 PHE QE ARG+ HE 5 PHE HN PHE HN 5 PHE QB PHE HN 5 PHE QD PHE HN 6 PHE HN PHE HN 6 PHE QD PHE HA 5 PHE QD PHE HA 6 PHE HN PHE QB 6 PHE HN PHE QB 6 PHE QB PHE QB 6 PHE QD PHE QD 6 PHE HN PHE HN 6 PHE HA PHE HN 6 PHE HB PHE HN 6 PHE HB PHE HN 6 PHE QB PHE HN 6 PHE QD PHE HA 6 PHE QD PHE HA 7 CYSS HN CYSS HN 7 CYSS HA CYSS HN 7 CYSS HB CYSS HN 7 CYSS HB CYSS HN 7 CYSS QB 2.68 S9

10 Table S4. NOE derived Upper Limit Constraints of ACE QH 1 ARG+ HA ACE QH 1 ARG+ QD ACE QH 2 ALA HN ACE QH 3 DCSS HN ACE QH 5 PHE QB ACE QH 5 PHE QD ACE QH 6 PHE QB ACE QH 6 PHE QD ARG+ HN 1 ARG+ HB ARG+ HN 1 ARG+ HB ARG+ HN 1 ARG+ QB ARG+ HN 1 ARG+ QG ARG+ HN 2 ALA HN ARG+ HA 1 ARG+ HB ARG+ HA 1 ARG+ HB ARG+ HA 1 ARG+ QD ARG+ HA 1 ARG+ HE ARG+ HA 2 ALA HN ARG+ HB2 1 ARG+ HE ARG+ HB2 2 ALA HN ARG+ HB2 3 DCSS HN ARG+ HB2 5 PHE QB ARG+ HB2 5 PHE QD ARG+ HB3 1 ARG+ HE ARG+ HB3 2 ALA HN ARG+ HB3 3 DCSS HN ARG+ HB3 5 PHE QB ARG+ HB3 5 PHE QD ARG+ QB 1 ARG+ HE ARG+ QB 2 ALA HN ARG+ QB 3 DCSS HN ARG+ QG 2 ALA HN ARG+ QG 3 DCSS HN ARG+ QG 5 PHE QD ARG+ QD 2 ALA HN ALA HN 2 ALA HA ALA HN 2 ALA QB ALA HN 3 DCSS HN ALA HA 3 DCSS HN ALA QB 3 DCSS HN ALA QB 3 DCSS HA ALA QB 3 DCSS HB ALA QB 3 DCSS HB ALA QB 6 PHE QD ALA QB 6 PHE QE DCSS HN 3 DCSS HB DCSS HN 3 DCSS HB DCSS HN 4 ARG+ HN DCSS HA 3 DCSS HB DCSS HA 3 DCSS HB DCSS HA 4 ARG+ HN DCSS HA 7 CYSS HB DCSS HA 7 CYSS HB DCSS HA 7 CYSS QB DCSS HB2 4 ARG+ HN 3.48 S10

11 3 DCSS HB2 7 CYSS HA DCSS HB3 4 ARG+ HN DCSS HB3 7 CYSS HA DCSS QB 5 PHE QB DCSS QB 7 CYSS HA ARG+ HN 4 ARG+ HB ARG+ HN 4 ARG+ HB ARG+ HN 4 ARG+ QB ARG+ HN 4 ARG+ QG ARG+ HN 4 ARG+ QD ARG+ HN 4 ARG+ HE ARG+ HN 5 PHE HN ARG+ HN 5 PHE QD ARG+ HA 4 ARG+ QD ARG+ HA 4 ARG+ HE ARG+ HA 5 PHE HN ARG+ HA 5 PHE QB ARG+ HA 5 PHE QD ARG+ HA 5 PHE QE ARG+ HA 6 PHE HN ARG+ HB2 5 PHE HN ARG+ HB2 5 PHE QB ARG+ HB2 5 PHE QD ARG+ HB2 5 PHE QE ARG+ HB2 5 PHE HZ ARG+ HB3 5 PHE HN ARG+ HB3 5 PHE QB ARG+ HB3 5 PHE QD ARG+ HB3 5 PHE QE ARG+ HB3 5 PHE HZ ARG+ QB 4 ARG+ HE ARG+ QG 5 PHE HN ARG+ QG 5 PHE QB ARG+ QG 5 PHE QD ARG+ QG 5 PHE QE ARG+ QG 5 PHE HZ ARG+ QD 5 PHE HN ARG+ QD 5 PHE QB ARG+ QD 5 PHE QD ARG+ QD 5 PHE QE ARG+ QD 5 PHE HZ PHE HN 5 PHE QB PHE HN 5 PHE QD PHE HN 5 PHE QE PHE HN 6 PHE HN PHE HN 6 PHE QD PHE HN 6 PHE QE PHE HA 5 PHE QD PHE HA 6 PHE QE PHE QB 6 PHE HN PHE QB 6 PHE HB PHE QB 6 PHE HB PHE QB 6 PHE QD PHE QB 6 PHE QE PHE QD 6 PHE HN PHE QD 6 PHE QB PHE HN 6 PHE HB S11

12 6 PHE HN 6 PHE HB PHE HN 6 PHE QB PHE HN 6 PHE QD PHE HN 6 PHE QE PHE HN 7 CYSS HN PHE HA 6 PHE QD PHE HA 7 CYSS HN PHE QB 7 CYSS HN PHE QD 7 CYSS HN PHE QE 7 CYSS HN CYSS HN 7 CYSS HB CYSS HN 7 CYSS HB CYSS HN 7 CYSS QB 2.87 S12