Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity. 1. Introduction Cysteine-rich peptides such as conotoxins and insulin-like peptides are an increasingly important class of biomolecules. They usually possess intricately folded, sometimes knotted, structures and some have been developed as treatments for a variety of conditions, such as pain [1, 2], cancer [3], diabetes mellitus [4], and heart failure [5, 6]. As such, much work is being undertaken to optimise their pharmacological properties so that new lead compounds are developed for preclinical evaluation. Disulfide bonds play a critical role in maintaining the peptide conformation and biological activity of these molecules. However, they are susceptible to reduction in vivo, as part of the normal degradative process which, in turn, can disrupt the three-dimensional structure and lead to loss of activity. In order to stabilise peptide structures, numerous disulfide bond mimics have been developed. Guo et al. substituted a diselenide for a disulfide bond in a sunflower trypsin inhibitor which retained high potency [7]. Armishaw et al. also applied this further to an α-conotoxin, which maintained full biological activity and had enhanced stability under biologically reducing conditions [8]. This same model peptide was also
References
[1]
N. Satkunanathan, B. Livett, K. Gayler, D. Sandall, J. Down, and Z. Khalil, “Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones,” Brain Research, vol. 1059, no. 2, pp. 149–158, 2005.
[2]
G. P. Miljanich, “Ziconotide: neuronal calcium channel blocker for treating severe chronic pain,” Current Medicinal Chemistry, vol. 11, no. 23, pp. 3029–3040, 2004.
[3]
M. T. Ma, J. A. Karas, J. M. White, D. Scanlon, and P. S. Donnelly, “A new bifunctional chelator for copper radiopharmaceuticals: a cage amine ligand with a carboxylate functional group for conjugation to peptides,” Chemical Communications, no. 22, pp. 3237–3239, 2009.
[4]
F. G. Banting, C. H. Best, J. B. Collip, W. R. Campbell, and A. A. Fletcher, “Pancreatic extracts in the treatment of diabetes mellitus,” Canadian Medical Association Journal, vol. 12, no. 3, pp. 141–146, 1922.
[5]
J. R. Teerlink, M. Metra, G. M. Felker et al., “Relaxin for the treatment of patients with acute heart failure (Pre-RELAX-AHF): a multicentre, randomised, placebo-controlled, parallel-group, dose-finding phase IIb study,” The Lancet, vol. 373, no. 9673, pp. 1429–1439, 2009.
[6]
J. R. Teerlink, G. Cotter, B. A. Davison et al., “Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial,” The Lancet, vol. 381, no. 9860, pp. 29–39, 2013.
[7]
X. Guo, J. Shi, Z. Tang, D. Cui, and Y. Zhang, “Synthesis and biological activity of seleno sunflower trypsin inhibitor analog,” Chemical Biology and Drug Design, vol. 68, no. 6, pp. 341–344, 2006.
[8]
C. J. Armishaw, N. L. Daly, S. T. Nevin, D. J. Adams, D. J. Craik, and P. F. Alewood, “α-selenoconotoxins, a new class of potent α7 neuronal nicotinic receptor antagonists,” The Journal of Biological Chemistry, vol. 281, no. 20, pp. 14136–14143, 2006.
[9]
Z. Dekan, I. Vetter, N. L. Daly, D. J. Craik, R. J. Lewis, and P. F. Alewood, “α-conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure,” Journal of the American Chemical Society, vol. 133, no. 40, pp. 15866–15869, 2011.
[10]
K. Holland-Nell and M. Meldal, “Maintaining biological activity by using triazoles as disufide bond mimetics,” Angewandte Chemie—International Edition, vol. 50, no. 22, pp. 5204–5206, 2011.
[11]
M. Empting, O. Avrutina, R. Meusinger et al., “‘Triazole bridge’: disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles,” Angewandte Chemie—International Edition, vol. 50, no. 22, pp. 5207–5211, 2011.
[12]
J. Elaridi, J. Patel, W. R. Jackson, and A. J. Robinson, “Controlled synthesis of (S,S)-2,7-diaminosuberic acid: a method for regioselective construction of dicarba analogues of multicystine-containing peptides,” Journal of Organic Chemistry, vol. 71, no. 20, pp. 7538–7545, 2006.
[13]
A. S. Cuthbertson, M. Husbyn, M. Engebretsen et al., “Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide,” Journal of Medicinal Chemistry, vol. 40, no. 18, pp. 2876–2882, 1997.
[14]
C. A. MacRaild, J. Illesinghe, B. J. van Lierop et al., “Structure and activity of (2,8)-dicarba-(3,12)-cystino α-ImI, an a-conotoxin containing a nonreducible cystine analogue,” Journal of Medicinal Chemistry, vol. 52, no. 3, pp. 755–762, 2009.
[15]
J. DiMaio, J. Jaramillo, D. Wernic, L. Grenier, E. Welchner, and J. Adams, “Synthesis and biological activity of atrial natriuretic factor analogues: effect of modifications to the disulfide bridge,” Journal of Medicinal Chemistry, vol. 33, no. 2, pp. 661–667, 1990.
[16]
M. A. Hossain, K. J. Rosengren, S. Zhang et al., “Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity,” Organic and Biomolecular Chemistry, vol. 7, no. 8, pp. 1547–1553, 2009.
[17]
S. Zhang, R. A. Hughes, R. A. D. Bathgate et al., “Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2,” Peptides, vol. 31, no. 9, pp. 1730–1736, 2010.
[18]
S. C. Koerber, J. Gulyas, S. L. Lahrichi et al., “Constrained corticotropin-releasing factor (CRF) agonists and antagonists with i-(i+3) Glu-Xaa-DXbb-Lys bridges,” Journal of Medicinal Chemistry, vol. 41, no. 25, pp. 5002–5011, 1998.
[19]
M. D. Lanigan, M. W. Pennington, Y. Lefievre, H. Rauer, and R. S. Norton, “Designed peptide analogues of the potassium channel blocker ShK toxin,” Biochemistry, vol. 40, no. 51, pp. 15528–15537, 2001.
[20]
M. Dong, J. A. Te, X. Xu et al., “Lactam constraints provide insights into the receptor-bound conformation of secretin and stabilize a receptor antagonist,” Biochemistry, vol. 50, no. 38, pp. 8181–8192, 2011.
[21]
P. Grieco, P. M. Gitu, and V. J. Hruby, “Preparation of “side-chain-to-side-chain” cyclic peptides by Allyl and Alloc strategy: potential for library synthesis,” Journal of Peptide Research, vol. 57, no. 3, pp. 250–256, 2001.
[22]
D. Limal, J. P. Briand, P. Dalbon, and M. Jolivet, “Solid-phase synthesis and on-resin cyclization of a disulfide bond peptide and lactam analogues corresponding to the major antigenic site of HIV gp41 protein,” Journal of Peptide Research, vol. 52, no. 2, pp. 121–129, 1998.
[23]
B. Hargittai, N. A. Solé, D. R. Groebe, S. N. Abramson, and G. Barany, “Chemical syntheses and biological activities of lactam analogues of α-conotoxin SI,” Journal of Medicinal Chemistry, vol. 43, no. 25, pp. 4787–4792, 2000.
[24]
C. Thurieau, P. Janiak, S. Krantic et al., “A new somatostatin analog with optimized ring size inhibits neointima formation induced by balloon injury in rats without altering growth hormone release,” European Journal of Medicinal Chemistry, vol. 30, no. 2, pp. 115–122, 1995.
[25]
P. Grieco, A. Carotenuto, R. Patacchini, C. A. Maggi, E. Novellino, and P. Rovero, “Design, synthesis, conformational analysis, and biological studies of urotensin-II lactam analogues,” Bioorganic and Medicinal Chemistry, vol. 10, no. 12, pp. 3731–3739, 2002.
[26]
M. A. Fázio, V. X. Oliveira Jr., P. Bulet, M. T. M. Miranda, S. Daffre, and A. Miranda, “Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability,” Biopolymers, vol. 84, no. 2, pp. 205–218, 2006.
[27]
M. J. Spinella, A. B. Malik, J. Everitt, and T. T. Andersen, “Design and synthesis of a specific endothelin 1 antagonist: effects on pulmonary vasoconstriction,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 16, pp. 7443–7446, 1991.
[28]
W. M. Abraham, A. Ahmed, A. Cortes, M. J. Spinella, A. B. Malik, and T. T. Andersen, “A specific endothelin-1 antagonist blocks inhaled endothelin-1-induced bronchoconstriction in sheep,” Journal of Applied Physiology, vol. 74, no. 5, pp. 2537–2542, 1993.
[29]
E. E. Büllesbach and C. Schwabe, “Replacement of disulfides by amide bonds in the relaxin-like factor (RLF/INSL3) reveals a role for the A11-B10 link in transmembrane signaling,” Biochemistry, vol. 51, no. 20, pp. 4198–4205, 2012.
[30]
S. Zimmermann, G. Steding, J. M. A. Emmen et al., “Targeted disruption of the Insl3 gene causes bilateral cryptorchidism,” Molecular Endocrinology, vol. 13, no. 5, pp. 681–691, 1999.
[31]
F. Shabanpoor, F. Separovic, and J. D. Wade, “The human insulin superfamily of polypeptide hormones,” Vitamins and Hormones, vol. 80, pp. 1–31, 2009.
[32]
R. A. Bathgate, S. Zhang, R. A. Hughes, K. J. Rosengren, and J. D. Wade, “The structural determinants of insulin-like peptide 3 activity,” Frontiers in Endocrinology, vol. 3, article 11, 2012.
[33]
D. J. Scott, T. N. Wilkinson, S. Zhang et al., “Defining the LGR8 residues involved in binding insulin-like peptide 3,” Molecular Endocrinology, vol. 21, no. 7, pp. 1699–1712, 2007.
[34]
M. A. Hossain, K. J. Rosengren, L. M. Haugaard-J？nsson et al., “The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors,” The Journal of Biological Chemistry, vol. 283, no. 25, pp. 17287–17297, 2008.
[35]
M. P. Del Borgo, R. A. Hughes, R. A. D. Bathgate, F. Lin, K. Kawamura, and J. D. Wade, “Analogs of insulin-like peptide 3 (INSL3) B-chain are LGR8 antagonists in vitro and in vivo,” The Journal of Biological Chemistry, vol. 281, no. 19, pp. 13068–13074, 2006.
[36]
F. Shabanpoor, R. A. Hughes, R. A. D. Bathgate et al., “Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions,” Bioconjugate Chemistry, vol. 19, no. 7, pp. 1456–1463, 2008.
[37]
S. Zhang, F. Lin, M. A. Hossain, F. Shabanpoor, G. W. Tregear, and J. D. Wade, “Simultaneous post-cysteine(S-Acm) group removal quenching of iodine and isolation of peptide by one step ether precipitation,” International Journal of Peptide Research and Therapeutics, vol. 14, no. 4, pp. 301–305, 2008.
[38]
L. V. Najbar, D. J. Craik, J. D. Wade, D. Salvatore, and M. J. McLeish, “Conformational analysis of LYS(11–36), a peptide derived from the β-sheet region of T4 lysozyme, in TFE and SDS,” Biochemistry, vol. 36, no. 38, pp. 11525–11533, 1997.
[39]
K. J. Rosengren, F. Lin, R. A. D. Bathgate et al., “Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3,” The Journal of Biological Chemistry, vol. 281, no. 9, pp. 5845–5851, 2006.
[40]
A. K. Tickler, A. B. Clippingdale, and J. D. Wade, “Amyloid-β as a “difficult sequence” in solid phase peptide synthesis,” Protein and Peptide Letters, vol. 11, no. 4, pp. 377–384, 2004.
