Peptide Therapeutics DOI: 10.1002/ange.201200984 Orally Active Peptidic Bradykinin B 1 Receptor Antagonists Engineered from a Cyclotide Scaffold for Inflammatory Pain Treatment** Clarence T. T. Wong, Dewi K. Rowlands, Chi-Hang Wong, TheodoreW.C. Lo, Giang K. T. Nguyen, Hoi-Yeung Li, and James P. Tam* Chronic pain is a universal health issue associated with numerous medical conditions, for example after severe burns or following major surgery. [1] Compelling evidence suggests that bradykinin (BK) antagonists could be useful in treating chronic pain and inflammatory pain. [2] Bradykinin and its homolog kallidin (lysyl-BK or KD), which are collectively known as kinins, participate in many pathophysiological insults. They are short-lived peptide mediators and the most potent endogenous pain inducers. [3] Kinins are released during tissue injury or noxious stimulation and modulate pain through the activation of both the B 1 and the B 2 receptor, which are two G-protein-coupled receptors; the carboxypep- tidase metabolites of kinins, des-Arg 9 -BK and des-Arg 10 -KD, activate the B 1 receptor. [2b, 4] The B 1 receptor stimulates the chronic phase of the inflammatory pain response, while the B 2 receptor stimulates the acute phase owing to their differ- ences in ligand dissociation, receptor desensitization, down- regulation as well as internalization. [2c, 5] Emerging evidence also suggests that the B 1 receptor mediates various chronic pain responses through the activation of phospholipase C, thereby leading to the production of diacylglycerol and inositol triphosphate, which further activate protein kinase C and Ca 2+ mobilization. [3] Numerous BK-antagonist peptides have been discovered from natural sources and structure–activity studies. [6] Kine- statin isolated from frog skin and helokinestatin from lizard venom are examples of natural BK-antagonist peptides. [7] Structure–activity studies have shown that removing the C- terminal Arg residue and concurrently replacing the penulti- mate residue Phe to Leu of bradykinin to des-Arg 9 -[Leu 8 ]- bradykinin or kallidin to des-Arg 10 -[Leu 9 ]-kallidin (DALK) changes a bradykinin B 2 receptor agonist to a B 1 receptor antagonist (Figure 1). [8] To increase potency and in vivo stability, several laboratories also developed BK antagonists modified with unnatural amino acids. [6] An example is Icatibant (HOE140), an injectable B 2 receptor antagonist, which has recently gained approval by the European Com- mission for treating hereditary angioedema. [9] Thus far, no peptidic B 1 receptor antagonist has been approved for clinical use, and developing a BK-antagonist peptide as a long-lived and orally active therapeutic remains a major challenge. The peptide-grafting strategy by inserting a bioactive BK- antagonist peptide into a proteinaceous natural-product scaffold is an attractive approach to develop an orally active Figure 1. Scheme of engineered BK antagonists and their amino acid sequences. The peptidic B 1 receptor antagonists, DALK (kal) and DAK (kin), were used to replace the entire loop 6 of kalata B1 (ckb) in the design of two cyclic analogues ckb-kal and ckb-kin, respectively. A linear analogue of ckb-kin, kb-kin, was also synthesized as a compar- ison. [*] C.T. T. Wong, Dr. C. H. Wong, [+] G. K. T. Nguyen, Prof. H. Y. Li, Prof. J. P. Tam School of Biological Sciences, Nanyang Technological University 60 Nanyang Drive, Singapore 637551 (Singapore) E-mail: jptam@ntu.edu.sg Dr. D. K. Rowlands, Dr. T. W. C. Lo Laboratory Animal Service Centre, Faculty of Medicine The Chinese University of Hong Kong Prince of Wales Hospital, Hong Kong (China) [ + ] Current address: State Key Laboratory in Oncology in South China Sir YK Pao Centre for Cancer, Department of Clinical Oncology Hong Kong Cancer Institute, The Chinese University of Hong Kong (China) [**] This research was supported by Biomedical Research Council (BMRC 09/1/22/19/612) of A*STAR and Academic Research Fund (ARC21/08) of Ministry of Education in Singapore. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201200984. A ngewandte Chemi e 1 Angew. Chem. 2012, 124,1–6  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü