Supporting Information. Minimum active structure of insulin-like. peptide 5 (INSL5)

Transcription

1 Supporting Information Minimum active structure of insulin-like peptide 5 (INSL5) Alessia Belgi 1,2, Ross A.D. Bathgate *1,2,3, Martina Kocan *4, Nitin Patil 1,5, Suode Zhang 1, Geoffrey W. Tregear 1,2, John D. Wade #1,3,5 and Mohammed Akhter Hossain #1,3,5 1 Florey Institute of Neuroscience and Mental Health, 2 Department of Biochemistry and Molecular Biology, 3 Florey Department of Neuroscience and Mental Health, 5 School of Chemistry, The University of Melbourne, Victoria 3010, Australia, and 4 Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria, 3052, Australia *# Equal contribution Address correspondence to: Dr Mohammed Akhter Hossain, Tel: akhter.hossain@unimelb.edu.au Prof John D Wade, Tel: john.wade@florey.edu.au S1

2 Table of Contents 1 Protective groups used for Cys Biological assessment of analogues Analytical RP-HPLC and MALDI-TOF-MS of all compounds Table S1: Summary of peptide characterization by HPLC and MALDI TOF MS Peptide characterization by amino acid analysis Peptide Yield S2

3 1 Protective groups used for Cys The Cysteine residues in the A-chain analogues 3 and 4 (Table 1) were protected using orthogonal protecting groups to enable the sequential formation of the disulfide bonds to obtain the correct connectivity. The protective groups used are represented in Figure S1 below. Figure S1 Small protective groups used for the thiol group of cysteine: (a) tbu, (b) Acm, (c) acetamide. Cysteine is represented at the bottom (d). 2 Biological assessment of analogues 1-4 Competition binding assays were performed for analogues 1 and 2. The peptides showed no affinity for RXFP4 receptor in a concentration range from 0.01 nm to 1 µm as shown in Figure S2A and Table 1. We wanted to get the full activity profiles of the single chain analogues. Thus we performed activation assays even though their inability S3

4 to bind to RXFP4 receptor was a good indication of their inability to also activate it. Each B-chain analogue (1-2) was then tested for its ability to activate RXFP4 1. Analogue 1 was able to very weakly inhibit forskolin-stimulated camp at very high concentration (10 µm) though this effect was statistically not significant compared with native minsl5. However, analogue 2 was unable to show any activation even up to a concentration of 10 µm (Figure S2B, Table 1). The single A-chain analogues 3 and 4 were subjected to competition binding assay on CHO-K1/RXFP4 cells where none of them showed any affinity for the receptor in a concentration range from 0.01 nm to 1 µm (Figure S3A, Table 1). In the same concentration range and on the same cell-line, they showed no ability to inhibit forskolin-stimulated camp production (Figure S3B, Table 1). S4

5 Figure S2 In vitro RXFP4 receptor bioassays: (A) competition binding curve of analogues 1 and 2 and minsl5 acid competing with 5 nm of Eu-(A) minsl5; (B) camp inhibition assay of analogue 1 and 2 and minsl5 acid. S5

6 Figure S3 In vitro RXFP4 receptor bioassays of analogues 3 and 4: (A) competition binding curve of analogue 3, analogue 4 and minsl5 competing with 5 nm of Eu-(A) minsl5; (B) camp inhibition assay of analogue 3, analogue 4 and minsl5 acid. The data are the result of n= 3 independent experiments and are expressed as mean ± SEM. S6

7 3 Analytical RP-HPLC and MALDI-TOF-MS of all compounds Analytical RP-HPLC analysis of the purified compounds was performed with Waters RP-HPLC systems using Empower hardware for data collection, monitoring and analysis. The RP-HPLC profiles were acquired using a Vydac C18 analytical column (4.6 x 250 mm, pore size 300 Å, particle size 5 µm), at a constant flow rate of 1.5 ml/min, in a gradient mode with buffer A: 0.1% aq. TFA and buffer B: 0.1% TFA in acetonitrile, CH 3 CN, monitoring at a wavelength of 214 nm which is characteristic for the amide bond. MALDI-TOF-MS spectra were carried out on a Bruker Ultrafex II instrument (Bruker Daltonics, Germany) using sinapinic acid as matrix. Analogue 1 RP-HPLC carried out as described above using the elution gradient: buffer B 25-55% over 30 minutes, t20.36 min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S4 RP-HPLC profile of analogue 1 (left) and MALDI-TOF-MS (right). S7

8 Analogue 2 RP-HPLC carried out as described above using the elution gradient: buffer B 20-50% over 30 minutes, t18.26min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S5 RP-HPLC profile of analogue 2 (left) and MALDI-TOF-MS (right). Analogue 3 RP-HPLC carried out as described above using the elution gradient: buffer B 14-45% over 30 minutes, t21.12min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S6 RP-HPLC profile of analogue 3 (left) and MALDI-TOF-MS (right). S8

9 Analogue 4 RP-HPLC carried out as described above using the elution gradient: buffer B 14-45% over 30 minutes, t19.28min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S7 RP-HPLC profile of analogue 4 (left) and MALDI-TOF-MS (right). Analogue 5 RP-HPLC carried out as described above using the elution gradient: buffer B 20-50% over 30 minutes, t18.05min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S8 RP-HPLC profile of analogue 5 (left) and MALDI-TOF-MS (right). S9

10 Analogue 6 RP-HPLC carried out as described above using the elution gradient: buffer B 20-50% over 30 minutes, t18.26min. MALDI-TOF-MS [M+H]+ calculated , observed Figure S9 RP-HPLC profile of analogue 6 (left) and MALDI-TOF-MS (right). Analogue 7 RP-HPLC carried out as described above using the elution gradient: buffer B % over 30 minutes, t20.22 min. For this analogue a Vydac C4 analytical column (4.6 x 250 mm, pore size 300 Å, particle size 5 µm) was used. MALDI-TOF-MS [M+H]+ calculated , observed Figure S10 RP-HPLC profile of analogue 7 (left) and MALDI-TOF-MS (right). S10

11 4. Table S1: Summary of peptide characterization by MALDI-TOF MS and HPLC 1 Observed molecular weights were determined by MALDI-TOF MS of the purified peptides 2 Molecular weights were calculated from monoisotopic masses 3 HPLC analysis was performed as specified in section 3 of the Supporting Information 5. Peptide characterization by amino acid analysis The peptide content and the amino acid ratio for each compound was determined using vapor-phase acid hydrolysis in 6 M HCl containing 2% phenol at 110 C for 24 hours. Analogue 1 Peptide content 41.84%. Amino acid analysis: Arg ratio expected 3, ratio found 3.05; Ser ratio expected 3, ratio found 2.78; Gly ratio expected 1, ratio found 1.15; Asp/Asn ratio expected 1, ratio found 1.06; Glu/Gln ratio expected 1, ratio found 1.00; Thr ratio expected 2, ratio found 1.94; Ala ratio expected 3, ratio found S11

12 3.20; Lys ratio expected 1, Not Determined; Tyr ratio expected 2, ratio found 2.10; Val ratio expected 3, ratio found 2.85; Ile ratio expected 2, ratio found 1.92; Leu ratio expected 2, ratio found 2.13; Trp ratio expected 1, Not Determined. Analogue 2 Peptide content 74.76%. Amino acid analysis: Arg ratio expected 3, ratio found 3.21; Ser ratio expected 1, ratio found 0.90; Gly ratio expected 1, ratio found 1.06; Asp/Asn ratio expected 1, ratio found 0.90; Glu/Gln ratio expected 1, ratio found 0.93; Thr ratio expected 2, ratio found 1.92; Ala ratio expected 2, ratio found 1.97; Lys ratio expected 1, Not Determined; Tyr ratio expected 2, ratio found 2.07; Val ratio expected 3, ratio found 2.84; Ile ratio expected 2, ratio found 1.86; Leu ratio expected 2, ratio found Analogue 3 Peptide content 36.70%. Amino acid analysis: Arg ratio expected 2, ratio found 2.06; Ser ratio expected 2, ratio found 1.81; Gly ratio expected 1, ratio found 1.04; Asp/Asn ratio expected 1, ratio found 1.03; Glu/Gln ratio expected 3, ratio found 3.11; Thr ratio expected 1, ratio found 0.99; Ala ratio expected 1, ratio found 0.95; Cys ratio expected 4, Not Determined; Lys ratio expected 1, Not Determined; Met ratio expected 1, ratio found 0.99; Leu ratio expected 4, ratio found Analogue 4 Peptide content 64.55%. Amino acid analysis: Arg ratio expected 2, ratio found 2.16; Ser ratio expected 2, ratio found 1.88; Gly ratio expected 1, ratio found 1.06; Asp/Asn ratio expected 1, ratio found 0.93; Glu/Gln ratio expected 3, ratio found 2.97; Thr ratio expected 1, ratio found 0.96; Ala ratio expected 1, ratio found 0.97; Cys ratio expected 4, Not Determined; Lys ratio expected 1, Not Determined; Met ratio expected 1, ratio found 1.02; Leu ratio expected 4, ratio found S12

13 Analogue 5 Peptide content 90.60%. Amino acid analysis: Arg ratio expected 5, ratio found 5.68; Ser ratio expected 5, ratio found 4.99; Gly ratio expected 2, ratio found 2.32; Asp/Asn ratio expected 2, ratio found 1.37; Glu/Gln ratio expected 4, ratio found 2.96; Thr ratio expected 3, ratio found 2.70; Ala ratio expected 4, ratio found 3.28; Cys ratio expected 4, Not Determined; Lys ratio expected 2, Not Determined; Tyr ratio expected 2, ratio found 2.33; Met ratio expected 1, ratio found 1.07; Val ratio expected 3, ratio found 2.70; Ile ratio expected 2, ratio found 1.72; Leu ratio expected 6, ratio found 6.08; Trp ratio expected 1, Not Determined. Analogue 6 Peptide content 92.22%. Amino acid analysis: Arg ratio expected 4, ratio found 5.49; Ser ratio expected 5, ratio found 5.85; Gly ratio expected 2, ratio found 2.56; Asp/Asn ratio expected 1, ratio found 0.73; Glu/Gln ratio expected 3, ratio found 2.33; Thr ratio expected 3, ratio found 3.08; Ala ratio expected 2, ratio found 1.79; Cys ratio expected 4, Not Determined; Lys ratio expected 2, Not Determined; Tyr ratio expected 2, ratio found 3.11; Met ratio expected 1, ratio found 1.25; Val ratio expected 3, ratio found 3.07; Ile ratio expected 2, ratio found 1.97; Leu ratio expected 4, ratio found 4.46; Trp ratio expected 1, Not Determined. Analogue 7 Peptide content 32.5% Amino acid analysis: Not Determined. S13

14 6. Peptide Yield Native INSL5 peptides: The A and B-chains of native human and mouse INSL5 (acid) peptides were separately synthesized using standard SPPS protocols. The A-chains were conjugated to their respective native B-chain following our protocol 2 to give native hinsl5 (0.2 mg) and minsl5 (0.14 mg). The yield was 0.81% for hinsl5 and 2.17% for minsl5 as calculated from the purified B-chains as starting material. It is important to note that the yield of hinsl5 amide was found to be much higher (about 10%) 2b compared with native hinsl5 (acid). The single chain analogues: The single chain analogues 1-4 were synthesized using standard Fmoc SPPP methodology. The yield (1: 1.2 mg; 2: 2.62 mg; 3: 0.4 mg; 4: 0.27 mg) calculated from the crude material was in the range of 15-20%. Two chain minimized analogues 5, 6 and 7: The A and B-chains were separately synthesized using standard SPPS protocols. The A- chains were conjugated to the native minsl5 B-chain following a recently developed protocol 3 to give analogues 5 (0.32 mg), 6 (0.4 mg), and 7 (0.3 mg). The yield was 5-7% calculated from the purified B-chains as starting material. REFERENCES 1. Haugaard-Kedström, L. M.; Shabanpoor, F.; Hossain, M. A.; Clark, R. J.; Ryan, P. J.; Craik, D. J.; Gundlach, A. L.; Wade, J. D.; Bathgate, R. A. D.; Rosengren, K. J., Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3. J. Am. Chem. Soc. 2011, 133 (13), S14

15 2. (a) Belgi, A.; Hossain, M. A.; Shabanpoor, F.; Chan, L.; Zhang, S.; Bathgate, R. A. D.; Tregear, G. W.; Wade, J. D., Structure and function relationship of murine insulin-like peptide 5 (INSL5): Free C-terminus is essential for RXFP4 receptor binding and activation. Biochemistry 2011, 50 (39), ; (b) Hossain, M. A.; Bathgate, R. A. D.; Kong, C. K.; Shabanpoor, F.; Zhang, S.; Haugaard-Jönsson, L. M.; Rosengren, K. J.; Tregear, G. W.; Wade, J. D., Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5). ChemBioChem 2008, 9 (11), Shabanpoor, F.; Hossain, M. A.; Ryan, P. J.; Belgi, A.; Layfield, S.; Kocan, M.; Zhang, S.; Samuel, C. S.; Gundlach, A. L.; Bathgate, R. A. D., Minimization of relaxin- 3 leading to high affinity analogues with increased selectivity for relaxin-family peptide 3 receptor (RXFP3) over RXFP1. J. Med. Chem. 2012, 55 (4), S15