Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies Luis Gustavo Deus Teixeira a , Luciana Malavolta b , Patrícia Alessandra Bersanetti c , Shirley Schreier d , Adriana K. Carmona a , Clovis R. Nakaie a,⇑ a Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP, Brazil b Department of Physiological Sciences, Santa Casa de Sao Paulo, School of Medical Sciences, 01221-020 Sao Paulo, SP, Brazil c Department of Health Informatics, Universidade Federal de Sao Paulo, 04023-062 Sao Paulo, SP, Brazil d Department of Biochemistry, Institute of Chemistry, Universidade de Sao Paulo, 05513-970 Sao Paulo, SP, Brazil article info Article history: Received 5 July 2016 Revised 10 October 2016 Accepted 26 October 2016 Available online 27 October 2016 Keywords: Bradykinin Angiotensin I-converting enzyme ACE Toac Spin label Biological activity abstract This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-a mino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac 0 -BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac 3 -BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9 lM 1 min 1 for BK and Toac 3 -BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE- hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction Many applications have been reported since the inception of the non-coded paramagnetic amino acid-type and fluorescent quenching probe Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-a mino-4-carboxylic acid) in the chemistry of peptides, firstly at the N-terminal end [1,2] and later at any internal position of the sequence [3]. These applications mainly involve electron paramag- netic resonance (EPR) spectroscopy. This cyclic spin label presents great sensibility to detect the motion and orientation of coupled macromolecules. This unique property is based on the fact that Toac has a constrained C a,a -tetrasubstituted cyclic structure in which the rotation about side chain bonds is hampered by the presence of nitroxide nitrogen and C a ,C b , and C c atoms in its heterocyclic moiety. Different types of Toac applications in biology and chemistry have been described [4,5]. This unnatural spin label amino acid has been used to investigate the structure of different peptide seg- ments [6–8], for their correlation with biological activities [9–14], or to examine G-protein coupled receptor fragments [15–18]. Toac has also been used to evaluate membrane-mimetic environments [19–21], measure intermolecular distances [22,23], and optimize peptide synthesis in a polymeric matrix [24–29]. In structure-function studies of Toac-attached peptides, we have mainly focused on investigating important vasoactive pep- tides [2,30–33], such as angiotensin II (AngII, DRVYIHPF) [34–36] and bradykinin (BK, RPPGFSPFR) [37,38]. More recently, studies testing for novel uses of Toac-peptide investigated the catalytic effect of the metallopeptidase angiotensin I-converting enzyme (EC 3.4.15.1 or ACE) upon angiotensin I (AngI, DRVYIHPFHL) bear- ing Toac group at several positions [39,40]. This enzyme releases the strong vasoconstrictor AngII from AngI and inactivates BK, hydrolyzing its structure sequentially at scissile bonds 7–8 and 5–6 [41,42]. http://dx.doi.org/10.1016/j.bioorg.2016.10.006 0045-2068/Ó 2016 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail address: cnakaie@unifesp.br (C.R. Nakaie). Bioorganic Chemistry 69 (2016) 159–166 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg