Supplementary Data Potent Complement C3a Receptor Agonists Derived from Oxazole Amino Acids: Structure–Activity Relationships. Ranee Singh†, Anthony N. Reed, Peifei Chu, Conor C. G. Scully, Mei-Kwan Yau, Jacky Y. Suen, Thomas Durek, Robert C. Reid*, David P. Fairlie* a Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia b† Present address: Dr. Ranee Singh, Department of Pharmacology & Toxicology, Faculty of Veterinary Medicine, Khon Kaen University, Thailand Table S1. Structures, C3aR binding and agonist activity of compounds 29-52 ................................................ S1 Figure S1. Normalised intracellular calcium release in HMDM cells for compounds 1-5, 7, 9 ........................ S2 Figure S2 Intracellular calcium release in HMDM cells for compounds 1-10, 13, 14, 16, 17, 19 .................... S2 Figure S3 Selectivity over C5aR by competitive binding with [125I]-C5a for all compounds at 20 µM ........... S3 Figure S4. Competitive binding with [125I]-C5a of compounds 2-4, 22, 29 ....................................................... S4 Figure S5. C3a receptor desensitisation calcium mobilization plots for compounds 1-5, 7, 9, 13, 22 ............... S4 Compound characterization NMR and HRMS data ................................................................................................ S5-17 Assay protocols ........................................................................................................................................................ S18-19 S1 Table S1. Acyl-leucine-5-methyl-oxazole-arginine compounds with C3aR-mediated agonist activity in HMDM. %[125I-C3a % %[125I]-C3a % Compound R Compound R binding a agonist b binding a agonist b 29 19 84 41 41 87 30 14 62 42 40 26 31 26 56 43 44 103 32 31 77 44 44 44 33 29 68 45 N/A 134 34 N/A 109 46 65 67 35 39 107 47 42 69 36 26 69 48 N/A 64 37 44 70 49 61 50 38 N/A 95 50 49 48 39 61 50 51 32 68 40 43 81 52 N/A 105 a % of [125I]-C3a remaining bound in the presence of compounds (20 µM) compared to the binding of [125I]-C3a with cells in the absence of compound 100%. Smaller number indicates stronger binding. b % Ca2+ response of compounds (tested at 10 µM in HMDM) relative to the activity of 100 nM C3a. Larger number indicates greater agonist activity. S2 Figure S1 Intracellular calcium release in HMDM cells induced by either hC3a (s, n=11), 1 (q, n=4), 2 (, n=4), 3 (p, n=3), 4 (®, n=3), 5 (l, n=7), 7 (n, n=7) or 9 (r, n=3). The results are expressed as a percentage of maximum change in fluorescence ± S.E.M. Figure S2. Intracellular Ca2+ mobilisation dose response induction by hC3a (l, n=11), SB290157 («, n=6) and variable concentrations of C3a non-peptide agonists. 1 (£, n=4), 2 (s, n=4), 3 (p, n=3), 4 (r, n=3), 5 (, n=7), 6 (, n=3), 7 (£, n=7), 9 (Î, n=3), 10 (, n=3), 13 (®, n=3), 14 (p, n=3), 15 (¯, n=2), 17 (q, n=3), 19 (r, n=2). The results are expressed as a percentage of maximum change in fluorescence induced by hC3a (100 nM) ± S.E.M. S3 Figure S3. Competitive binding between [125I]-C5a and C3a non-peptide ligands in human monocyte derived macrophages. Human monocyte derived macrophage cells (1.5x106 cells/mL) were incubated for 1 h at room temperature with constant concentration (20 pM) of [125I]-C5a and non-peptide ligands and known C5aR antagonist 3D53 at a concentration of 20 µM. The buffer represents the total binding of [125I]-C5a to HMDM (~ 4000 cpm). Data is expressed as mean [125I]-C5a binding ± S.E.M. with n=2 and shows that only 3D53 binds to C5aR. 6000 hC5a ) m 3D53 p 4000 c TR4401-25 ( a TR4167-72 5 C TR4401-16 - ] I 2000 PC95_1 5 2 1 [ BR64 0 -14 -12 -10 -8 -6 -4 -2 Log [ ligands] (M) Figure S4 Competitive [125I]-C5a binding of hC5a (l, n=3), 3D53 (n, n= 3) and C3aR-binding non-peptide ligands 2 (q), 3 (w), 4 (p), 22 (n), 29 (l), n=3 on human monocyte derived macrophage cells. Human monocyte derived macrophage cells (1.5x106 cells/mL) were incubated for 1 h at room temperature with 20 pM of [125I]-C5a and increasing concentrations of non-labelled C3a (concentration 1 pM to 0.3 µM) and non-peptidic ligands (concentration 0.1 nM to 100 µM). Each point represents the mean percentage specific binding ± S.E.M. Only C5a and 3D53 bind to C5aR on HMDM. S4 Figure S5 C3a receptor desensitisation calcium mobilization plots performed on human monocyte derived macrophages (HMDM) for selected non-peptidic agonists. HMDM were first treated with hC3a (3 µM at time = 10 s) which induced a calcium response that was allowed to dissipate over a further 240 s prior to addition of a second agonist. If these compounds induced calcium via another receptor we would expect to see a second spike after 250s for Ca2+ release, as Comment [II1]: Sure this wasn’t 300 seconds? for 9. Comment [RR2]: Jacky explained that C3a was added after 10 s (1st red arrow) then, agonist was added after 250 s (2nd red arrow). Therefore, the time for signal to dissipate would be 240 s, being the difference between the red arrows. Comment [II3]: o.k. but in case of 9 the spike is showing up at 300 secs. S5 Boc-leucine oxazole-arginine-OH (Compound 1) + + HRMS MH calculated for C20H34N6O6 455.2613 found 455.2752. 1 H NMR (400 MHz, DMSO-d6): d 8.58 (1H, s, Ox), 8.16 (1H, d, J = 7.8 Hz, Leu-NH), 7.56-7.54 (2H, m, Arg-NHe), 4.76-4.70 (1H, m, Leu-Ha), 4.43-4.37 (1H, m, Arg-Ha), 3.46 (2H, q, J = 7.2 Hz, Arg- Hd), 1.89-1.45 (7H, m, Leu-Hg, Leu-Hb2, Arg-Hb2, Arg-Hg2), 1.37 (9H, s, boc), 0.89 (6H, dd, J = 12, 6.4 Hz, Leu-Hd). 13 C NMR (100 MHz, DMSO-d6): d 173.5, 165.2, 160.4, 157.1, 155.7, 142.5, 135.8, 78.8, 51.6, 47.3, 41.6, 28.6, 28.3, 25.8, 24.6, 23.2, 21.9. (S)-2-(2-((1S,2S)-1-(tert-butoxycarbonylamino)-2-methylbutyl)oxazole-4-carboxylic acid Arginine OH (Compound 2) + HRMS calculated for C20H35N6O6 455.2613, found 455.2616. 1 H NMR (400 MHz, DMSO-d6): d 8.59 (1H, s, Ox), 8.17 (1H, d, J = 5.2 Hz, Arg-NH), 7.58 (1H, d, J = 5.6 Hz, Ile-NH), 7.46 (1H, t, J = 3.8, eNH), 4.52 (1H, t, J = 5.6 Hz, Ile-Ha), 4.41-4.38 (1H, m, Arg- Ha), 3.14-3.05 (2H, m, Arg-Hd), 1.91-1.84 (2H, m, Ile-Hb + Arg- Hb1), 1.80-1.38 (1H, m, Arg-Hb2), 1.52-1.46 (2H, m, Arg-Hg), 1.36 (9H, s, Boc), 1.25-1.16 (2H, m, Ile-Hg), 0.84 (3H, t, J = 4.8 Hz, Ile- 13 Hd), 0.72 (3H, d, J 4.8 Hz, Ile-Hg CH3). C NMR (100 MHz, DMSO-d6): d 173.0, 163.9, 159.9, 156.6, 153.3, 142.0, 135.2, 78.4, 53.3, 51.2, 37.1, 28.1, 25.3, 24.9, 15.3, 10.7. 3-Indole carboxylic acid-Leucine-Oxazole-Arginine-OH (Compound 3) + HRMS calculated for C24H32N7O5 498.2459, found 498.2461. 1 H NMR (400 MHz, DMSO-d6): d 9.96 (1H, d, J = 8.4 Hz, Arg- NH), 8.61 (1H, s, Ox), 8.19 (1H, d, J = 7.8 Hz, Leu-NH), 7.87- 7.85 (1H, m, Indole -NH), 7.54-7.46 (4H, m, Ar), 5.10-5.06 (1H, t, J = 4.6 Hz, Leu-Ha), 4.42-4.36 (1H, m, Arg-Ha), 3.11- 3.06 (2H, m, Arg-Hd), 2.21-2.15 (1H, m, Leu-Hg), 1.87-1.71 (2H, m, Arg- Hb), 1.62-1.45 (3H, m, Leu-Hb1, Arg-Hg2), 1.32-1.21 (1H, m, Leu-Hb, Leu-Hb), 0.87 (3H, d, J = 6.6 Hz, Leu-Hd), 0.80 (3H, d, J = 6.6 Hz, Leu-Hd). 13C NMR (100 MHz, DMSO-d6): d 173.0, 166.5, 163.6, 159.9, 156.6, 142.1, 135.3, 133.7, 131.5, 128.3, 127.6, 52.1, 52.0, 51.2, 36.5, 27.9, 25.3, 25.0, 15.6, 10.5. 5-bromonicotinic acid-leucine-oxazole-arginine-OH (Compound 4) 79 + HRMS calculated for C21H29 BrN7O5 538.1408, found 538.1405. 1 H NMR (400 MHz, DMSO-d6): d 9.29 (1H, d, J = 8 Hz, Arg- NH), 8.98 (1H, d, J = 1.6 Hz, Ar), 8.87 (1H, d, J = 2 Hz, Ar), 8.64 (1H, s, Ox), 8.48 (1H, t, J = 2 Hz, Ar), 8.22 (1H, d, J = 8 Hz, Ile- NH), 7.53 (1H, t, J = 5.6 Hz, Arg-Nhe), 5.06 (1H, t, J = 8.6 Hz, S6 Ile-Ha), 4..42-4.36 (1H, m, Arg-Ha), 3.09 (2H, q, J = 6.4 Hz, Arg-Hd), 2.19-2.12 (1H, m, Ile-Hb), 1.92-1.72 (2H, m, Arg-Hb2), 1.62-1.46 (3H, m, Ile-Hg1, Arg-Hg2), 1.31-1.23 (1H, m, Ile-Hg1), 0.88 13 (3H, t, J = 4.8 Hz, Ile-Hd), 0.82 (2H, d, J = 4.8 Hz, Ile-Hg, Isopropyl CH3). C NMR (100 MHz, DMSO-d6): d 173.0, 163.7, 163.0, 159.9, 156.6, 152.8, 147.4, 147.3, 142.3, 137.6, 135.3, 130.7, 120.0, 52.2, 52.1, 51.3, 36.7, 27.9, 25.4, 25.0, 15.5, 10.6.