Báo cáo Y học: Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt ppt

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Báo cáo Y học: Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt ppt

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Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt Athanasios Kazantzis 1 , Michaela Waldner 1 , John W. Taylor 2 and Aphrodite Kapurniotu 1 1 Physiological-chemical Institute, Department of Physical Biochemistry, University of Tu ¨ bingen, Germany; 2 Rutgers University, Department of Chemistry and Chemical Biology, Piscataway, NJ, USA Calcitonin (Ct) is a 32-residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our p revious studies have led to poten t, side-chain lactam-bridged human Ct (hCt) analogues [Kapurniotu, A. Kayed, R. , Taylor, J.W. & Voelter W. (1999) Eur. J. Biochem. 265, 606–618; Kapurniotu, A. & Taylor, J .W. (1995) J. Med. Chem. 38, 836–847]. We have hypothesized that a possibly type I b turn/b sheet confor- mation in the region 1 7–21 may play an important role in hCt bioactivity. To investigate this hypothesis, analogues of the potent hCt agonist cyclo17,21-[Asp17,Lys21]hCt (1) bearing type I (and II¢)orIIb turn-promoting substituents at positions 18 and 19 were designed, synthesized and their solution conformations, human Ct receptor binding affinities and in vivo hypocalcemic potencies were assessed. The novel analogues include cyclo17,21-[Asp17, D -Phe19, Lys21]hCt (2), cyclo17,21-[Asp17,Aib18,Lys21]hCt (3), cyclo17,21-[Asp17, D -Lys18,Lys21]hCt (4), corresponding partial sequence peptides containing the lactam-bridged region 16–22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas a nalogues 2–4 had similar r eceptor affinities to hCt. In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1, whereas 2 completely lost the high potency of 1, s uggesting that type I ( and II¢) b turn-promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analog ues 1–4 were markedly bsheet-stabilized com- pared to h Ct and indicated t he presence of distinct b turn conformeric populations in each of the analogues. Unexpectedly, the D -amino acid- o r Aib-containing cyclic analogues 2–4 but not 1 or hCt self-associated i nto SDS denaturation-stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the r esidues in sequence 17–21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for t he self association s tate of hCt. These results may assist to delineate the structure-function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone-disorder-related diseases. Keywords: Human calcitonin; b turn/b sheet conformation; dimerization; receptor binding; hypocalcemic activity. Calcitonins (Ct) are peptide hormones of 32 amino-acid residues that have been mainly known for their hypocalce- mic effect and the inhibition of bone-resorption [1,2]. Calcitonins are u sed therapeutically for the treatment of osteoporosis and other with bone disorder-related diseases [1,2]. A marked species-specific differenc e in hypocalcemic potencies is observed for the Cts. C ts of ultimobranchial origin, i.e. salmon Ct ( sCt), are the most potent ones, whereas the human hormone (hCt) h as a strongly reduced potency [1,2]. Therefore, sCt is the main Ct to be applied therapeutically to date. However, there is only a 50% sequence homology between sCt and hCt, which is the cause for immunogenic reactions in humans w hen t reated with sCt [3]. Therefore, the development of hCt analogues bearing high bioactivity and a close structural similarity to the hCt sequence still remains an important task. The b iologically active conformation of the Cts yet remain to be identified. It has been long proposed that the propensity of the Cts to f orm an amphiphilic a helix in the region 8–22 might strongly correlate with their bioactivit ies [4–10]. H owever, while several reports have suggested t hat this might b e the case for sCt, no evidence h as been presented for a direct l ink between helicity and bioactivity for t he human sequence. In contrast, there is increasing evidence, suggesting that other factors, including a b turn/ b sheet conformation in the middle region of h Ct, overall conformational flexibility, tertiary structure i nteractions and interactions of specific residues may be related to b ioactivity [7,10–15]. NMR studies in nonhelix-inducing media suggested short a ntiparallel b sh eets and b turns to b e Correspondence to A. Kapurniotu, Physiological-chemical Institute, University of Tu ¨ bingen, Hoppe-Seyler-Str. 4, D-72076 Tu ¨ bingen, Germany, Fax: + 49 7071 2978781, Tel. + 49 7071 2978781, E-mail: afroditi.kapurniotu@uni-tuebingen.de Abbreviations: Aib, 2-aminoisobutyric acid; BOP, (benzotriazol- 1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; Ct, calcitonin; DCM, dichloromethane; DMF, dimethylformamide; DIEA, N,N¢-diisopropylethylamine; DMS, dimethylsulfide; EDT, ethanedithiol; hCt, human calcitonin; MBHA, 4-methyl benzhydryl- amine resin; Mtt, 4-methyltrityl; Pip, 2-phenylisopropyl; OFm, fluoren-9-yl methylester; sCt, salmon calcitonin, TBTU, O-benzotriazole-N,N,N¢,N¢-tetramethyluronium tetrafluoroborate; tBu, tert butyl; TIS, t riisopropylsilan; TFE, 2,2,2-t rifluoroethanol; TFMSA, trifluoromethanesulfonic acid; T rt, trityl. (Received 15 A ugust 2001, revised 14 November 2001, accepted 19 November 2001) Eur. J. Biochem. 269, 780–791 (2002) Ó FEBS 2002 present in the middle region of the Cts, while an ahelical conformation was f ound to be significantly populated only in alcohol-containing solvents [16–21]. As the Cts are short-sequence peptides of high c onfor- mational flexibility, their b ioactive conformation may b e completely different from t hat observed in the media used i n the N MR studies [22]. Introduction of conformational constraints has been often proven to be a necessary strategy towards ÔlockingÕ a p eptide into a bioactive conform ation [22,23]. The (i,i + 4) side chain-to-side ch ain c yclization approach has been successfully used for the stabilization o f bioactive a helical conformations of several medium-size peptide hormones [ 24,25]. We have previously applied this approach to constrain t he potentially bioactive, a helical conformation of hCt [14]. These studies unexpectedly led to the discovery of the potent but nonhelical hCt agonist, cyclo17,21-[Asp17,Lys21]hCt (1). Base d on our structure- activity results and previously published NMR data [19], we have suggested that a type I bturn/ b sheet conformation between residues 17 and 21, t hat might have been stabilize d by the introduced lactam bridge, may play an essential role in hCt bioactivity [14]. T his b turn could be centered at amino acids Lys18 and Phe19 a ccording t o the NMR data [14,19]. To investigate t he importance o f the conformational and topological features of the region between amino-acid residues 17 and 21 for hCt bioactivity and the b turn hypothesis, we have followed two strategies: in the first one, we prepared a s eries of ring-size analogues of 1 to study the effect of ring-size o n b turn/ b sheet stabilization a nd hCt bioactivity [ 15]. T hese studies led v ery r ecently to the discovery of the superpotent b turn/b sheet-stabilized, hCt- agonist cyclo17,21-[Asp17,Orn21]hCt [15]. In the second strategy, w hich we present in this r eport, we designed and synthesized a nalogues of 1 bearing t ype I- ( and II¢-) and type II-stabilizing amino acid substitutions for Lys18 and Phe19, corresponding partial sequence p eptides containing the lactam-bridged r egion 16–22, and also nonbridged control peptides (Scheme 1) and studied the e ffect o f these substi- tutions on conformation, self-assembly state, hCt receptor binding affinity, and in vivo hypocalcemic activity. MATERIALS AND METHODS Materials Protected amino acids, r esins for pep tide synthesis, BOP, and TBTU were purchased from Bachem, Novab iochem and Rapp P olymere. Solvents a nd miscellaneous chemicals for syntheses, HPLC purifications, SDS/PAGE, and CD studies were from Merck a nd Aldrich, and were of the highest purity grade available [15]. S ynthetic hCt and sCt for CD and bioactivity studies were from Novabiochem. The saline solution ( 0.9%, w/v) for the hypocalcemic assay w as from Delta P harma a nd BSA (99%) from Sigma. Medium and a ll reagents f or cell culture were from G ibco BRL. Insulin and h ydrocortisone for t he cell culture were from Sigma (tissue culture grade). Salmon Tyr22 125 I-labelled calcitonin ( 125 I-labelled sCt) w as from Amersham Pharma- cia Biotech. Peptide synthesis, purification, and characterization Solid phase peptide synthesis of 1–6 was performed as recently described on MBHA with N a -Boc-protected amino acids [14,15,25]. F ollowing deprotection and cleavage from the resin using HF and scavengers a ccording to our recently published procedure disulfide bridge formation was achieved by air oxidation of the crude peptides at 10 )4 M in 0.1 M NH 4 CO 3 [15] in the presence of 0 .5–1 M GdnHCl to improve solubilities and oxidation yields and its c ompletion was followed b y HPLC. Cru de, oxidized peptides were purified by reverse phase HPLC on a C 18 Nucleosil 250/8 column (Grom) with a length of 25 cm, an internal diameter of 8 mm and a 7-lm particle size. The flow rate was 2.0 mLÆmin )1 and e luting buffers were: A, 0 .058% (v/v) trifluoroacetic acid in water and B , 0.05% (v/v) trifluoro- acetic acid in 90% (v/v) C H 3 CN and w ater. T he elution program was: 7 min at 30% B, followed by a gradient from 30% to 60% B over 3 0 min. Peptides 2–6 were also synthesized by the Fmoc/tBu strategy on Rink–MBHA resin with N a -Fmoc-protected amino acids and standard protection of the side-chains [Asp(OtBu), Glu(OtBu), G ln(Trt), Cys( Trt), H is(Trt), Lys(Boc), Tyr(tBu) and Thr(tBu)], with the exception of residues Lys21 and Asp17 of the cyclic peptides 2– 4,for which the Mtt, respectively, the P ip groups were applied. For the side chain-to-side chain cyclization, th ese g roups were selectively cleaved following treatment o f the peptide resin with a mixture of 1% t rifluoroacetic acid and 5% triisopropylsilan (TIS; v/v) in dichloromethane ( DCM; 2 · 2min and 6· 10 min) [26]. Cyclizations were performed with fourfold excess (benzotriazol-1-yloxy)-tris- Scheme 1. Amin o-acid sequences of h Ct and analogues 1–6. Ami no- acid residues are presented with th e one letter code except for Asn17 and T hr21 and th e introduced substitut es. Numbers above the hCt sequence indicate positions of the sub stituted residues. Th e amino termini and the C -terminal amide groups o f hCt and the a nalogues are not shown. Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 781 (dimethylamino)phosphonium hexafluorophosphate (BOP) and N,N¢-diisopropylethylamine (DIEA) [ 15], and were usually performed twice (1 · 4hand1· overnight). Pep- tide resins were then acetylated. P rotected amino acids (fourfold exces s) were c oupled using O-benzotriazole- N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU; fourfold excess) and DIEA (sixfold excess). The final cleavage of the peptide and t he side chain p rotecting groups from the resin was performed with trifluoroacetic a cid/H 2 O/ thioanisol/EDT/phenol (10/0.5/0.5/0.25/0.5) (v/v w ith the exception o f p henol) [ 27]. F ormation of the d isulfide bridges of the crude peptides and HPLC purification were performed as described above. Identity of the HPLC purified synthetic peptides 1–6 was verified by matrix a ssisted laser desorption ionization mass spectrometry (MALDI-MS) with a Kratos Compact MALDI I (Shimadzu Europe, D uisburg, Germany) and a-cyano-4-hydroxycinnamic acid as matrix. Purity of the HPLC purified peptides were also confirmed by analytical HPLC analyses. The fo llowing results of MALDI-MS were obtained for the synthesized peptides by the Boc- and the Fmoc- protection strategy, respectively: Cyclo17,21-[Asp17,Lys21]hCt (1): MH+ of 3427.5 (calcu- lated 3428.9); cyclo17, 21-[Asp17, D -Phe19,Lys21]hCt (2): MH+ o f 3427.1 (3428.1, respectively) (calculated 3428.9); cyclo17, 21-[ Asp17,Aib18,Lys21]hCt (3): MH+ of 3387.3 (3386.5, respectively) (calculated 3386.9); cyclo17, 21-[Asp17, D -Lys18,Lys21]hCt (4): MH+ of 3427.0 (3449.3 (Na+ adduct), respectively) (calculated MH+ 3428.9 and calculated for M+ N a+ 3450.9); [ D -Phe19]hCt (5): MH+ of 3416 .8 (3418.9, respectively) (calculated 3418.9); [ D -Lys18]hCt (6): MH+ of 3 418.8 (3417.9, respectively) (calculated 3418.9). Solid phase peptide synthesis of the partial sequence peptides 1a, 1b , 2a,and3a, their cleavages from the resin, and HPLC purifications were performed as recently described on MBHA with N a -Boc-protected amino acids [15]. T he correct masses of HPLC purified peptides were assessed b y F AB-MS: cyclo17,21-[Asp17,Lys21]hCt (16–22)-NH 2 (1a): MH+ o f 949.4 (calculated ¼ 949.5); [Asp17,Lys21]hCt(16–22)-NH 2 (1b): MH+ of 967.5 (cal- culated 967.15); cyclo17,21-[Asp17, D -Phe19,Lys21]hCt(16– 22)-NH 2 (2a): MH+ of 9 49.4 (calculated 949.5); cyc lo17, 21-[Asp17,Aib19,Lys21]hCt(16–22)-NH 2 (3a): MH+ of 906.4 (calculated 906.5). Far-UV CD spectropolarimetry CD spectra were obtained with a J-720 spectropolarimeter (JASCO) at room temperature. Spectra were measured at 0.2 intervals (0.5 n m for the p artial sequence peptides), with a spectral ban d width of 1 nm, a scan speed of 20 nmÆmin )1 (50 nmÆmin )1 for the partial sequence peptides), a response time of 4 s (8 s for the partial peptides), and represent the average of three s cans in the range of 195–250 nm (185– 250 nm for the p artial sequence p eptides). Spectra were measured in 10 m M aqueous sodium phosphate buffer (pH 7.4) and in 10 m M sodium phosphate buffer (pH 7.4) diluted 1/1 (v/v) with TFE and peptides were diluted directly from their stock solutions into the buffer at the indicated concentrations. UV a bsorbance a t 274.5 nm was used to exactly determine the concentrations of the stock solutions of analogues 1–6 ( 500 l M )in1m M HCl, using e 274.5 ¼ 1440 M )1 Æcm )1 [15]. Stock solutions of the partial sequence analogues 1a, 1b, 2a,and3a (10 m M )were prepared in 10 m M HCl. The spectra are presented as plots of the m ean residue e llipticity ([h]) vs. the wavelength with the spectra of the buffer solution alone already subtracted. Analysis of secondary structure contents Secondary structure analyses o f the spectra were performed by multilinear r egression analysis using the program LINCOMB and t he r eference spectra of Brahms & Brahms [28] and Perczel et al. [29]. SDS/PAGE SDS/PAGE was performed with 18% homogeneous poly- acrylamide gels using the MINI-PROTEAN II electro- phoresis system (Bio-Rad) a s previously described [30]. To obtain comparable results to the CD concentration depen- dence studies, p eptide solutions were prepared by the same procedures as for C D, and assembly states were investigated at final peptide concentrations of 50 l M . For SDS/PAGE, peptide s tock solutions (500 l M in 1 m M HCl ( see under CD part) were diluted i nto 1 0 m M sodium phosphate buffer, pH 7.4, at a concentration of 100 l M ,aswasalso done for the CD experiments. The peptide solutions were then diluted with sample buffer [ 30] to a final concentration of 50 l M , boiled for 5 min, and electrophoresed. Cell culture T47D cells were obtained f rom the American Tissue Culture Collection and were cultured in RPMI 1640 containing 10% heat inactivated fetal bovine serum, 1% streptomycin/ penicillin, 0.1 l M insulin, and 0.1 l M hydrocortisone in 5% CO 2 and 37 °C. The latter hormones were omitted from the medium when subculturing cells that were to be used for the receptor binding assay 1–3 days later. Subculturing was performed with t rypsin/EDTA as described [31,32] and for the binding experiments cells were subcultured in 12-well dishes. Receptor b inding experiments w ere performed when cells reached 90% confluence (1–3 d ays after s ubculture). Receptor binding assay The assay was performed based on previously established protocols [14,31–33]. Briefly, cells in the 12-well dishes were washed with NaCl/P i (1 mL) at ambient temperature and then prewarmed (37 °C) assay buffer that consisted of RPMI 1640 and 0.1% (w/v) BSA w as added to t he cells (930 lL). 125 I-labelled s Ct (5 lCi, specific activity 2000 lCiÆmmol )1 ) in i ts lyo philized form was reconstituted in 100 m M HCl (200 lL), aliquoted at 4 °C in e ppendorf tubes (15 lL each), that w ere thereafter kept at )20 °C, and for e ach 12-well plate one tube was thawed a t room temperature, diluted with a ssay buffer ( 245 lL) and used immediately. Twenty microliters of the 125 I-labelled sCt solution(14.4pmol)werethenaddedtoeachwell,andthe wells were mixed b y gentle shaking. Thereafter, solutions (50 lL) of different concentrations of the peptides in assay buffer were added to t he cells, and following gentle mixing cells were incubated for 1 h at room temperature. Peptide solutions were freshly m ade p rior to each experiment by 782 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002 diluting peptide s tocks ( 500 l M in 1 m M HCl [14]) in assay buffer. Binding was terminated by aspiration of the medium and w ashing of the cells with NaCl/P i three times. Cells were then removed from the wells by s hort treatment (1 min) with 0.5 M NaOH (2 · 0.5 mL) and bound radio- activity was assessed by c-counting (counter efficiency  70%). Nonspecific binding was determined a s t he binding of 100 n M sCt. This was assessed from 13 independent experiments to b e 12.94% ( ± 3.59). Specific binding was the difference between total binding (tracer alone) and nonspecific binding. In vivo hypocalcemic assay The in vivo hypocalcemic assay in mice w as performed as described previously [14,15]. Hypocalcemic activities are plotted as percent reduction of [Ca +2 ] (mean ± SEM of 3– 10 mice) relative to control (3–8 mice). Basal [Ca +2 ] (mean ± SEM of 84 mice) was 1 0.11 ± 0 .06 mgÆdL )1 .In good agreement with our previous findings [14,15], maximum hypocalcemic e ffect w as caused by 2 lgofhCt and D[Ca 2+ ] ¼ )2.01 mgÆdL )1 ([Ca 2+ ] ¼ 8.10 ± 0.15 mgÆdL )1 ), which corresponded to a [Ca 2+ ] reduction of 19.90% ± 0 .81 (mean ± S EM of eight mice treated with peptide vs. 7 control mice). Statistical significance of the hypocalcemic effects o f the analogue s vs. the effects of the respective doses of hCt was assessed using ANOVA . S ignifi- cant statistical significance (P < 0.05) was found for the effects of 1, 3, and 4 at doses of 10–0.1 ng that corresponded to the linear parts of the curves as compared to t he respective hCt effects, whereas the effects o f 2, 5, and 6 were very similar t o h Ct. O f note, the low maximum hypocalcemic effects of 2 and 5 also differed significantly from the maximum effect of hCt (P < 0.05 and < 0.01, respectively). In addition, the effects of the 100 ng doses of 2 and 5 also differed significantly from the effect of hCt (P <0.05). Effective concentrations at 50% o f the maximal e ffect (EC 50 ) were estimated by nonlinear regression analyses of the data using the software PRISM (GraphPad Software, Inc.) RESULTS AND DISCUSSION Design of the analogues A b turn conformation strongly depends on the n ature a nd chirality of the amino-acid residues a t its corner positions and even small changes of these residues may dramatically affect the type a nd stability o f the turn [34,35]. To investigate the importance of t he type of the postulated turn for hCt b ioactivity, the i +1turn-residue L -Phe19 of 1 was r eplaced by D -Phe19 t o giv e cy clo17,21- [Asp17, D -Phe19,Lys21]hCt (2) ( Scheme 1). This substitu- tion was expected to stab ilize a type II btur n conformation [34,35] which, according to our hypothesis [14,15], should have a negative effect on bioactivity. Next, cyclo17,21- [Asp17,Aib18,Lys21]hCt (3) was designed (Scheme 1 ). The substitute Aib18 for Lys18 w as chosen. Due to its s trong conformational space restriction [34], t he Aib residue should favor the postulated type I b turn conformation centered at the Lys–Phe bond and was expected to result in a bioactive analogue [34,36–38]. Next, cyclo17,21-[Asp17, D -Lys 18,Lys21]hCt (4) was designed in which the chirality of the putative i + 1 turn-residue of 1, or Lys18, was inversed (Scheme 1). T his substitution was expected to stabilize a type II¢ b turn, which places the side chains of the corner residues at roughly th e same position a s a type I b turn [34,35,39]. Thus, this substitution was expe cted to maintain or increase the bioactivity of (1) [34]. Enhancement of potency through s tabilization o f a type II ¢ turn has been previously reported f or somatostatin [34,40]. Importan tly, studying t he structure–activity relationships of a type I I¢- stabilized analogue of 1 would offer direct information about the role of the topographical features of the side chains of the residues i n region 17–21 for receptor binding and in vivo bioactivity. Residues Lys18 and Phe19, that were elected to be substituted, had not previously been known to be important for hCt bioactivity or i ts overall co nformation. However, to be able to s eparately evaluate effects of the substitutes alone vs. the introduced confo rmational restrictions, the nonbrid- ged peptides [ D -Phe19]hCt (5)and[ D -Lys18]hCt (6)were also synthesized and s tudied (Scheme 1). CD spectroscopy describes the average conformation of polypeptides and the contribution o f a local confor- mational feature such as a f our-residue b turn to the CD spectrum of a polypeptide of 32 amino acids will usually remain unrecognised due to o ther secondary s tructure elements [41]. Therefore, t o be able to obtain more detailed information about a potential bturn stabilization we also synthesized a nd studied the conformation of cyclo17,21- [Asp17,Lys21]hCt(16–22)-NH 2 (1a), cyclo17,21-[Asp17, D -Phe19,Lys21]hCt(16–22)-NH 2 (2a), cyclo17,21-[Asp17, Aib19, Lys21]hCt(16–22)-NH 2 (3a), and [Asp17,Lys21] hCt(16–22)-NH 2 (1b) that comprise mainly the lactam bridge-containing region 16–2 2 of analogues 1, 2 and 3, respectively, and als o a linear control peptide for 1a , analogue 1b. Conformational analyses by CD: studies of hCt and the analogues in aqueous buffer, pH 7.4 CD spectra of hCt and analogues 1–6 (Fig . 1A) were measured at concentrations of 5 l M where all peptides were found in preliminary CD concentration-dependence studies to be in a monomeric state (data not shown) [14,15]. Visual inspection of the spectra indicated that all bicyclic analogues had s imilar o verall conformations. Secondary structure analyses of the spectra with the reference spectra of Brahms & Brahms [ 28] suggested b sheet contents of about 40% for 1–4, the rest being predominantly random coil. hCt contained 27% bs heet, the r est consisting mainly of random coil. These results indicated an about 50% increase of b sheet contents in 1–4 compared to hCt. Since the peptides were monomers, t his fi nding suggested they were b turn stabilized. O n t he other hand, the similarity of the spectra of 2–4 to the spectrum o f 1 suggested that the introduced substitutes d id not affect the o verall conformation of 1. Interestingly, also 5 and 6 contained 50% more b sheet t han hCt, which suggested that nature and c hirality of residues 18 and 19 are strongly associated with b sheet stabilization of hCt. The CD s pectra of 1a , 1b,and3a exhibited a strong negative b and between 185 and 190 nm that is characteristic for both turn-types (type I and II) [42]. T he spectrum of 2a did not exhibit such a minimum. Its shape indicated t hat the peptide was in a c onformeric e quilibrium state and that it Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 783 contained contributions of three previously repo rted bturn reference s pectra: one class C CD spectrum [42] (a negative band between 200 and 210 nm, a weak negative b and at about 220 nm, and a possitive band between 180 and 195 nm), one spectrum correponding to an op en or ÔZÕ conformation (one minimum at 195–200 nm) [43], and the third component could be the type I and II bturn spe ctrum according t o Brahms a nd Brahms [28] that exhibits a characteristic minimum at a bout 225 nm and a maximum at 210–220 nm. The spectra of 1a and 1b were very similar to each other. The spectra of both peptides showed positive bands at about 220 nm (Fig. 1B) th at most likely arise from coupling between the phenylalanyl (there are three P he residues in sequence 1 6–22) and the amide chromophores [44,45]. This suggestion w as further s upported by t he observation t hat the intensity of the 220 band, that most likely corresponds to the phenylalanyl La band [42,45], was significantly le ss in 1b and its maximum was blue shifted compared to 1a [44,45]. The similarity between the CD curves of 1a and 1b suggested that the lactam bridge of 1a did not significantly constrain t he aqueous conformation of 1b . However, the intense positive band of 1a at 220 nm suggest ed that cyclization may have resulted in topological changes of the side chain of one or more Phe r esidue(s). Interestingly, these positive bands were not present in 2a and 3a suggesting a significant effect of residues D -Phe19 and Aib18 on back- bone conformation and t opography of the Phe side chain(s) in sequence 16–22. The s pectrum of 3a had, except for t he minimum at 185–190 nm, a lso a strong maximum at about 198 nm and a marked negative band at a bout 212 nm. This shape is i ndicative of an equilibrium of the t wo forms o f type I b turn conformers that have been shown to be populated by cyclic bturn model peptides [43]. Together, the results of C D s pectroscopy under aqueous conditions, pH 7.4, suggested that all c yclic peptides had a stabilized b turn/b sheet structure as c ompared to hCt and that distinct bturn populations and their mixtures were present in each of them. This latter finding su ggested that the lactam bridge did not completely restrict the confor- mational flexibility of the analogues and was consistent with results o f s everal NMR and CD studies o n cyclic model peptides [42,43,46]. Conformational analyses by CD: studies of hCt and the analogues in 50% TFE in aqueous buffer, pH 7.4 Fifty percent aqueous TFE is a solvent system that is applied as a structure-inducing agent to short polypeptide sequences that usually exhibit strong conformational flex- ibility in pure aqueous buffers [23,47–49]. The conformeric states that are stabilized und er these conditions have been often related to bioactive, i.e. receptor-bound, con forma- tions [50]. In a ddition, TFE is a solvent that is able to stabilize the a helical conformation in a flexible polypeptide chain that has an a helical propensity [51]. Fig. 1. Far -UV CD spectroscopy of hCt and analogues 1–6 (A,C) and 1a, 1b, 2a, and 3a (B,D) in aqueous buffer (A,B) and in 50% aqueous TFE (C,D). The spectra were recorded in 10 m M aqueous sodium phosphate buffer, pH 7 .4 (A,B) and in 50% TFE in aqueous phosphate buffer, pH 7.4 (C,D) at a peptide concentration of 5 l M (for hCt and 1–6)and1m M (for 1a, 1b, 2a ,and3a) and at room temperature. 784 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002 As shown in F ig. 1C, TFE had a strong structuring and a helix inducing effect on hCt and the nonbridged peptides 5 and 6 that conta ined 40–50% a helical components accord- ing to s econdary structure a nalysis by t he reference s pectra of Brahms and Brahms [28]. This was consistent with the long described a helix-forming propensity of the middle region of the calcitonin sequence [4,16,52,53]. In contrast, nearly no a helical contents were found for all bridged analogues w hich contained i nstead 40–50% b sheet struc- ture, t he rest being mainly r andom coil. Thu s, it appears that the a helix-inducing e ffect of TFE on 1–4 was not as strong as on hCt, 5, and 6, most likely b ecause 1–4 were already significantly constrained by t he lactam bridges. Of note, the CD spectra of 1–4 were very similar to each other. Taken together, the results in 50% TFE were consistent to the ones under pure aqueous conditions and suggested that the introduction of the substituents at positions 18 and 19 of analogue 1 did not affect its o verall con formation. In addition, the studies in 50% TFE confirmed our earlier observations [14] that the 20- membered Asp17 t o Lys21 lactam bridge had an a helix-destabilizing a nd a bsheet- stabilizing effect on hCt. It has been reported that (i,i +4) Asp/Lys bridges may result in both stabilization [ 54] and destabilization [55] of a helices. Together with t hese reports, our results suggest that the effect of such bridges on a helix stabilization strongly depends on the particular peptide sequence the bridges a re being i ntroduced into [14]. CD spectra of 1a , 1b, 2a,and3a in 50% TFE were measured next (Fig. 1D). All three cyclic analogues, but not the line ar 1b, exhibited a marked positive p-p*bandat about 195 nm that has been observed in type I and type II¢ b turn models [42,43]. The CD spectra of these t hree peptides differed strongly from each other, however, in the region between 200 and 250 nm: the spectrum of 1a had a clear minimum at about 208 nm and its shape was reminiscent of a class C spectrum (see above), or a type I b turn, as has been found for cyclic model peptides [42,43]. The spectrum of 2a had a clear minimum at 225 nm. Together with the maximum a t  195 nm, this minimum suggested an conformeric equilibrium between a type I b turn [28] with another b turn population [ 35]. The spectrum of 3a had a pronounced minimum a t  215 nm and a shoulder at  225 nm that, together with the maximum a t 195 nm, indicated t he presence of the two type I b turn conformeric forms that have been described by Perczel et al. [43]. I mportantly, the 50% TFE solvent system allowed for a clear distinction between the conformation of 1a vs. 1b which showed only weak CD b ands. Such distinction was not possible under pure aqueous conditions (see above). Together with the results in aqueous buffer, the TFE-data suggested that the lactam b ridge stabilized a specific conformeric population in 1a, which, h owever, retained also a h igh degre e of flexibility. Oligomerization studies by CD and SDS/PAGE analysis For the ab ove d escribed CD studies , peptide concentrations as low a s 5 l M were applied which are clo se to physio- logically relevant concentrations. Confirming previous findings [14,15], no con centration dependence of t he CD spectra or aggregation was found between 5 and 100 l M for hCt and also for 1 in aq ueous buffer, pH 7.4. This suggested that the conformations observed by CD were adopted by monomeric peptides. However, there was a striking con- centration dependence of the CD spectra o f 2–4 between 5 and 100 l M (Fig. 2 A), that was indicative of peptide self association [5]. The mean residue ellipticities at 202 nm ([h] 202 ), that corresponded t o the minima of the C D spectra, Fig. 2. Studies on t he oligomerization propensity o f h Ct and 1–6. (A) CD concentration dependence studies: the concent ration depen- dence ( 5–100 l M ) of t he mean residue ellipticity at 202 nm ([ h] 202 ) for analogues 2, 3,and4 in aqueous buffer is shown. In the inset the line ar regression analysis of the data points of 2 th at were intro- duced to t he eq uation [([ h] 202(observed) ) [h] 202(mono) )/[analogue]] 1/2 ¼ [2/K d ([h] 202(dimer) ) [h] 202(mono) )] 1/2 ([h] 202(dimer) ) [h] 202(observed) )ispre- sented. CD measurements at various analogue concentrations ([ana- logue]) were perfo rmed in 10 m M sodium phosphate buffer, pH 7.4, and at room t emperature. (B) and (C) SDS/PAGE analysis and silver staining of hCt and an alogues 1–6. Molec ular mass markers (in kDa, lane 1). (B) Lane 2, hCt; lane 3, 6;lane4,2;lane5,3;lane6,4;lane7,1, and (C) lane 1, molecular mass markers; lane 2, hCt; lane 3, 5;lane4,2. 100 l M solutions of hCt and the analogu es in 10 m M phosphate buffer, pH 7.4 w ere diluted 1 : 1 with sample buffer containing 2 % SDS, boiled and electrophoresed as described under Materials and methods. Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 785 decreased with increasing concentrations, s uggesting that the peptides b ecame more ordered during s elf association [42]. Plateau values were reached at 50 l M (Fig. 2 A). The change of [h] 202 was b est fitted to an equation describing peptide cooperative dimerization [5]. Dimerization was also confirmed by SDS/PAGE (see below). [h] 202 for the monome rs ([h] 202(mono) )were obtained at 5 or 1 l M , where all analogues were essentially monome ric, and were )6059 (2), ) 6802 (3)and) 7680 deg.cm 2 /dmol (4). Plots of the observed [ h] 202(obs) vs. {([h] 202(obs) ) [h] 202(mono) )/[analogue]} 1/2 (i.e. Figure 2A, inset) gave dissociation constants o f 1 .44 · 10 )5 ,1.10 · 10 )5 and 2.47 · 10 )5 M for the dime rs of 2, 3, and 4, respectively. Human Ct belongs to the family of aggregating and amyloid-forming polypeptides [56,57]. Fibrillation of aque- ous hCt solutions strongly hampers its therapeutic use for the t reatment of bone-disorder-related diseases [ 58]. Sec- ondary structure analysis of spectra corresponding to monomeric and dimeric popu lations suggested that dimer- ization occured at the expense o f unordered structures a nd was accompanied b y a s ignificant antiparallel b sheet stabilization. The l atter one was most likely due to intermolecular, structure-stabilizing interactions [59,60]. According t o the reference spectra of Perczel et al.[29], dimerization of 2–4 was accompanied by increases in antiparallel b sheet contents of 18%, 14%, and 20%, respectively. These results are consistent with a recently proposed model of hCt aggregation at pH 7.4 into fibrils via formation and stacking of antiparallel b sheets [57]. As observ ed for hCt a nd 1, CD concentration depen- dence studies of 5 and 6 showed that these analogues also did not aggregate. This suggested that self-assembly of 2, 3, and 4 was related to both the confor mational r estriction, i.e. the b turn/b sheet stabilization that had been achieved by the lactam bridge, and the topological features of the side chains of residues 18 a nd 19. It has b een previously suggested that several hydrophobic residues, that may occupy the one face of the putative a helical region 8–21 of hCt, participate in the initial helix–helix association step [61]. This s tep i s then followed by f ormation of b sheet aggregates [61]. Thus, a reason for the increased b sheet formation and oligomerization propensity of 2–4 could be the changed topogr aphy of the side chains of r esidues 18 and 19 in 2–4. This may have led to formation of an hydrophobic face in the lac tam bridge-stabilized b sheet a n d an increase d dimerization and oligomerization propensity [55,62]. Association of b sheets i nto multimers and fibrils would be consistent with models of hCt fibrils [56,57]. To further study the self-assembly states of 2–4,50l M solutions of hCt and the analogues were next subjected to SDS/PAGE analysis (Fig. 2B,C). B ased on the results of the CD studies, 2, 3, and 4 were expected to predominantly consist o f (noncovalent) dimers. Stability of the dimers towards S DS treatment conditions (2% S DS, 100 m M 2-mercaptoethanol, 100 °C f or 5 min [30]) was not known. In fact, mixtures of peptide monomers and dimers at a ratio of about 40/60 were observed in 2, 3, and 4 (Fig. 2B), whereas hCt, 1, 5, and 6 mainly consisted of monomers (Fig. 2C). These results were in good agreement with the dimerization propensity of 2–4 as observed by CD. O f note, the dimeric forms o f these analogues were resistent to the denaturating SDS/PAGE conditions, indicating an unusu- ally strong self association potential. Such strong aggrega- tion potential has been described for other amyloid polypeptides, including hu man islet amyloid polypeptide (IAPP), which shares a receptor and hypocalce mic activity with hCt [30,63–66]. Receptor binding affinities hCt exerts its biological effe cts v ia binding to a receptor that belongs to the family of seven-tran smembrane G -protein coupled re ceptors [10]. C t r eceptors are localiz ed in bone and k indney and also in the central nervous system, i.e. the brain [10]. In addition, specific high affinity receptors for Ct have been found in several cancer cell lines including the human breast cancer cell line T47D [10,31]. We have used the T47D cell line t o assess human receptor binding affinities of the synthetic analogues as compared to sCt, which is the strongest known naturally occuring Ct ligand, and h Ct which is a weak ligand [33]. Binding affinities were assessed via the competitive i nhibition of the Fig. 3. Hu man Ct receptor binding of hCt, sCt and analogues 1–6 to T47D cells assessed via displacement o f bound 125 I-labelled sCt. Cells were prepared and incubated with 125 I-labelled sCt as described under Materials and meth- ods. Specific r adioligand binding is plotted vs. the concentration of c ompeting sCt, hCt, and 1–6 as indicated. Data for 1–6 represent the mean ± SD for three to five independen t experiments and data for h Ct and sCt are t he mean of 13 and 1 4, respectively, assays. 786 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002 specific binding of the r adioligand 125 I-labelled sCt that binds with high affinity and selectivity to the Ct receptors of this cell line [33]. As shown in Fig. 3, 1 sh owed increased binding affinity compared to hCt. Receptor binding affinity of 1 (IC 50 ¼ 2n M ) was threefold lower than the a ffinity of sCt (IC 50 ¼ 630 p M ), that was about 6 times more potent than hCt ( IC 50 ¼ 4n M ) [33]. The higher binding affinity of 1, compared to hCt, was consistent with both its high binding affinity to the rat brain Ct receptor and its increased in vivo hypocalcemic potency compared to hCt [14,15]. Analogues 2–4 had nearly i ndistinguishable binding isotherms to hCt (IC 50 ¼ 4n M ) suggesting that chirality of residues 1 8 and 19 plays a crucial role for hCt binding affinity to t he T47D receptors. This result was c onfirmed by the results of the binding studies o f 5 and 6; 5 showed a significantly reduced binding affinity (IC 50 ¼ 18 n M )as compared to hCt (IC 50 ¼ 4n M ), while 6 showed almost no binding. Together, the obtained receptor binding d ata showed that (a) the introduction of the Asp17,Lys21-lactam bridge in hCt resulted in a significant increase in human Ct receptor binding affinity (b) r esidues 18 and 19 of hCt and their chirality are strongly associated with receptor binding affinity (c) inversement of the chirality of residues 18 and 19 strongly reduces the binding affinity of hCt and (d) the Asp17,Lys21-lactam br idge leads to a partial inversement of the latter effect. In vivo hypocalcemic activities To directly assess the biological relevance of the introduced substitutions, we n ext studied in vivo hyp ocalcemic potencies in mice [1–3,14,15]. Analogues 3 and 4 exhib ited identical bioactivity to 1 [14], which was 5 times more potent than hCt (Fig. 4). However, 2 was co mpletely devoid of the increased bioactivity of 1.Analogue2 hadanEC 50 of 25 ng that corresponded to a hypocalcemic potency that was even lower than t he potency of hCt (EC 50 , 20 ng). Of note, 2 was unable to reach the maximum hypocalcemic effect o f hCt (20%) ( caused by 2 lg hCt), even when 10-fold higher doses were applied. The maximum effect of 2 was caused by the 20-lg dose a nd was at 16.1%. A nalogue 5 had t he same dose–response c urve as 2. This i ndicated that t he confor- mational restriction was not capable of r eversing the negative e ffect of the inversement of ch irality of Phe19 on in vivo bioactivity of 1. In contrast, 6 had the same potency and maximum effe ct as hCt which suggested that inverse- ment of chirality of L ys18 was w ell tolerated. Correlation of solution conformations with receptor binding affinities and in vivo bioactivities of hCt and analogues 1–6 The results of the CD studies and the studies on receptor binding affinities and hypocalcemic potencies in vivo of hCt, sCt, analogues 1–6 and/or 1a, 1b, 2a,and3a are summa- rized in Table 1. Our findings that 3 and 4, that contain type IandII¢ b-turn-promoting substitutes, had the same hypocalcemic potency as 1,whereas2, that contains the type II b-turn-promoting substitute, lost t he high potency of 1 supported the suggestion that a type I b turn/ b sheet i n the region 17–21 of hCt may play an important role in in vivo bioactivity [14,15]. Because 3 and 4 were also designed to contain the same side-chain topology in the turn-corner residues as 1, the above findings also indicated t hat a type I b turn side-chain topography in region 17–21, might be fully compatible with in vivo bioactivity. Our CD studies showed that the overall secondary structure of 2 was very s imilar to the ones of 1, 3,and4.In contrast, the CD studies of the partial sequence analogues showed that the Asp17,Lys21-lactam bridge and the substitutes r esulted i n a stabilization o f d istinct bturn conformeric populations in each one of the s hort a nalogues. In particular, t he studies in 50% TFE indicated that type I b turn conformers were the mostly populated b turn conformers in 1a and 3a, whereas confo rmeric e quilibria of several turn-types w ere p redominant under pure a queous conditions. Importantly, t he CD studies both under pure aqueous and in 50% TFE indicated that 2a populated distinct bturn conformeric states compared to 1a,and3a. Fig. 4. Hyp ocalcemic potencies of hCt and the analogues 1–6. Serum calcium levels were measured in gro ups of 3–10 mice pe r dose and 3–8 control mice 1 h a fter subcutane ous injection of the peptide s olution or vehicle alone. Hypocalce mic activities of ea ch dose are expressed as percent reduction of calcium (mean ± S EM) caused by the p eptide relative to control. Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 787 Furthermore, 2 was t he only analogue with reduced in vivo activity as compared to hCt, suggesting a crucial role of the topological features of the side chain of Phe19 i n confor- mation and in vivo bioactivity o f hCt. In the T47D receptor bindin g studies, only 1 showed a higher binding affinity than hCt, whereas 2–4 were equally potent t o hCt. Analogues 5 and 6 showed decreased binding affinities as compared to hCt. Thus, t hese studies demon- strated the cr ucial role of residues 18 a nd 19 and their chirality for human receptor binding. M oreover, these findings suggested that the side chains of residues 18 and 19 and/or of other residues i n r egion 1 7–21 may be d irectly involved in receptor binding. T hese results we re consistent with a recent model of ligand–Ct-receptor interaction and activation. According to this model, all three regions of the Ct sequence, including the N-terminal l oop 1–7, the potential ahelica l region 8 –22, and t he C-terminal region 22–32 interact with distinct domains of the Ct receptor [10,67]. Accordingly, even small or local changes in confor- mational and topographical features in Ct, may r esult i n dramatic changes in binding affinities an d efficacies [10]. Taken together, our CD and bioactivity studies suggested that both r eceptor binding affinity and in vivo bioactivity of hCt are associated with specific local conformational features of the backbone and with topological features of side chains of residues within the region 17–21. Previous studies have shown that replacement of Phe19 by Leu does not affect the in vivo hyp ocalcemic potency of hCt [1]. The aromatic rings of the three P he residues Phe16, Phe19, and Phe22 in hCt have been suggested to occupy the hydro- phobic side o f the potential amphiphilic a helical region of hCt [5,18]. Based on this model, inversement of chirality of Phe19 of hCt, as performed in 2, would disrupt the hydrophobic face of the putative b ioactive a helical confor- mation. This could be one plausible explanation for the observed strong decrease of in vivo bioactivity in 2 as compared to 1 [5,18]. It is noteworthy that the CD spectrum of 1a indicated interactions between phenylalanyl and amide chromophores, whereas no such interactions were observed in the spectrum of 2a. For analogues 1 and 2, we observed a clear correlation between hypocalcemic activity and receptor binding affinity. In contrast, n o such correlation was observed for 3 and 4. These latter a nalogues had lost the high receptor binding affinity of 1 and w ere similarity potent t o hCt, whereas they maintained the increased in vivo hypocalcemic potency of 1. Similarly, 5 and 6 had reduced binding affinity to the T47D receptor compared to hCt and the same hypocalcemic potency to hCt. It is believed that the hypocalcemic activity of the Cts is the result of a receptor-me diated inhibition of bone resorption via a direct effect of Ct on osteoclasts and of the c alciuretic effect of Ct on kidney [ 10]. Therefore, in vivo bioactivity of the Cts is d etermined by many different factors including receptor binding, signal transduction, receptor regulation, as also bioavailability and biodegrada- bility of the ligand [9,10,68]. The analogues 2–6 presented here differ from 1 and hCt only in the chirality o f residues 19 and/or 18 and/or the p resence of Aib instead of Lys18. Thus, these analogues are expected to have in vivo ahigher proteolytic stability t han 1 and hCt [69]. Therefore, the Table 1. S ummary of the results of the CD s tudies, the receptor binding affinities, and the hypocalcemic potencies in vivo of hCt, sCt, analogues 1–6 and/or the partial sequence analogues 1a, 1b, 2a, and 3a. The CD data of the partial sequence analogues 1a, 1b, 2a,and3a are presented, because there w ere no differences between the spectra of the respective complete s equence peptides. CD spectra were measured in 1 0 m M phosphate buffer, pH 7.4 and in 50% TFE in 10 m M phosphate buffer, pH 7.4, at room temperature. Peptide concentrations were 1 m M (for 1a, 1b, 2a,and3a)and 5 l M (for hCt, sCt, and 1–6). Exp. turn, expected stabilized turn based o n the analogue design strategy. Min., minimum of CD spectrum; max., maximumofCDspectrum. Analogue (exp. turn) Conformational analysis of partial sequence peptides by CD Receptor binding affinities (in vivo) Hypocalcemic potencies In aqueous solution In 50% aqueous TFE 1 Min., 190 nm; max., 220 nm: type I and II b turn conformers; Min., 208 nm; max., 195 nm: Threefold higher than hCt Fivefold more potent than hCt interactions of phenylalanyl with type I b turn amide chromophores 2 (type II) Max., 185 nm; min., 208 nm and 222–224 nm: type I and II b turn conformers Min., 225 nm; max., 195 nm: equilibrium: type I b-with another b turn conformer Same as hCt Less potent than hCt; has 80% of hCt maximum effect 3 (type I) Min., 185–190 and 212 nm; max., 198 nm: Min., 215 nm; max., 195 nm: equilibrium two type I conformers of two type I conformers Same as hCt Same as hCt 4 (type II¢) ND ND Same as hCt Same as hCt 5 ND (5 random coil as hCt) ND (5 a helix as hCt) Fivefold lower than hCt As 2 6 ND (6 random coil as hCt) ND (6 a helix as hCt) Nearly no binding As hCt hCt Spectrum of 1b: similar to 1a; less maximum at 220 than in 1a Spectrum of 1b: very weak bands Sixfold lower than sCt the strongest potency; sCt sCt: mainly random coil a sCt: a helix (more than hCt) a The strongest binding (IC 50 ¼ 630 p M ) 95-fold lower than sCt; 95-fold higher than hCt [15] a The sCt data were not shown in this work (see also references [4,12]). 788 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002 results of our studies support the notion that the in vivo hypocalcemic potency of the C ts is directly associated to a distinct bioactive con formeric population r ather than t o differences in proteolytic degradation rates [1,7,14,15,18, 67,70–72]. In conclusion, our structure activity studies supported the suggestion that a type I bturn/ b sheet conformation in the region 17–21 may play a n important role in hCt b ioactivity and showed that the conformation and the topological features of the side chains o f amino acid residues 18 a nd 19 are strongly associated with the self-assembly state, the human receptor binding affinity and the in vivo hypocalce- mic potency of hCt. ACKNOWLEDGEMENTS We are grateful to J. Bernhagen for his he lp with the receptor binding and the hypocalcemic assay. We thank H. R. Rackwitz and M. Schno ¨ lzer for help with the HF cleavage and D. Finkelmeir f or excellent technical assistance with the cell culture and the receptor binding assay. We thank N. Greenfield for the CD programs. We thank S. Stoeva and and her group for the the MALDI-MS and H. Bart holoma ¨ and R. Mu ¨ ller for FAB-MS. We thank K. T enidis, R. Kayed , and K. Sweimeh for their contributions to c ertain experimental parts of this work. We thank W. V oelter for supporting this work . This w ork was supported by the Deutsch e Forschungs- gemeinschaft (DFG) grant numbers Ka 979/2-1 an d -2. REFERENCES 1. Guttman n, S. (1981) Chemistry and structure-activity relationship of natural a nd synthetic calcitonins. In Calcitonin 1980 Chemistry Physiology Pharmacology and Clinical Aspe cts (Pecile, A., ed.), pp. 11–24. Excerpta Medica, Amsterdam. 2. Azria, M. (1989) Calcitonins in therapeutic use. In The Calcito- nins: Physiology and Pharmacology (Azria, M., ed.), pp. 133–143. Karger, Basel. 3. Azria, M. (1989) Introduction. 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Conformational analyses by CD: studies of hCt and the analogues in aqueous buffer, pH 7.4 CD spectra of hCt and analogues 1–6. the partial sequence analogues 1a, 1b, 2a, and 3a. The CD data of the partial sequence analogues 1a, 1b, 2a, and 3a are presented, because there w ere no differences between the spectra of the respective

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