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

To investigate this hypothesis, analogues of the potent hCt agonist cyclo17,21-[Asp17,Lys21]hCt 1 bearing type I and II¢ or II b turn-promoting substituents at positions 18 and 19 were d

Conformationally constrained human calcitonin (hCt) analogues

reveal a critical role of sequence 17–21 for the oligomerization

state and bioactivity of hCt

Athanasios Kazantzis1, Michaela Waldner1, John W Taylor2and Aphrodite Kapurniotu1

1

Physiological-chemical Institute, Department of Physical Biochemistry, University of Tu¨bingen, Germany;2Rutgers 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 previous studies have led to potent,

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 17–21 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¢) or II b 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 analogues 2–4 had similar receptor 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, suggesting that type I (and II¢) b turn-promoting substituents are fully compatible with in vivo bioactivity CD spectroscopy showed that analogues 1–4 were markedly b sheet-stabilized com-pared to hCt and indicated the presence of distinct b turn conformeric populations in each of the analogues Unexpectedly, theD-amino acid- or Aib-containing cyclic analogues 2–4 but not 1 or hCt self-associated into SDS denaturation-stable dimers Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17–21 and in particular of residues

18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state 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 used therapeutically for the treatment of

osteoporosis and other with bone disorder-related diseases

[1,2] A marked species-specific difference in hypocalcemic

potencies is observed for the Cts Cts of ultimobranchial origin, i.e salmon Ct (sCt), are the most potent ones, whereas the human hormone (hCt) has 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 when treated 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 biologically active conformation of the Cts yet remain to be identified It has been long proposed that the propensity of the Cts to form an amphiphilic a helix in the region 8–22 might strongly correlate with their bioactivities [4–10] However, while several reports have suggested that this might be the case for sCt, no evidence has been presented for a direct link between helicity and bioactivity for the human sequence In contrast, there is increasing evidence, suggesting that other factors, including a b turn/

b sheet conformation in the middle region of hCt, overall conformational flexibility, tertiary structure interactions and interactions of specific residues may be related to bioactivity [7,10–15] NMR studies in nonhelix-inducing media suggested short antiparallel b sheets and b turns to be

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, triisopropylsilan; TFE, 2,2,2-trifluoroethanol;

TFMSA, trifluoromethanesulfonic acid; Trt, trityl.

(Received 15 August 2001, revised 14 November 2001, accepted 19

November 2001)

present in the middle region of the Cts, while an a helical

conformation was found to be significantly populated only

in alcohol-containing solvents [16–21]

As the Cts are short-sequence peptides of high

confor-mational flexibility, their bioactive conformation may be

completely different from that observed in the media used in

the NMR studies [22] Introduction of conformational

constraints has been often proven to be a necessary strategy

towards ÔlockingÕ a peptide into a bioactive conformation

[22,23] The (i,i + 4) side chain-to-side chain cyclization

approach has been successfully used for the stabilization of

bioactive a helical conformations of several medium-size

peptide hormones [24,25] We have previously applied this

approach to constrain the 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) Based on our

structure-activity results and previously published NMR data [19], we

have suggested that a type I b turn/b sheet conformation

between residues 17 and 21, that might have been stabilized

by the introduced lactam bridge, may play an essential role

in hCt bioactivity [14] This b turn could be centered at

amino acids Lys18 and Phe19 according to the NMR data

[14,19]

To investigate the importance of 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 series of ring-size analogues of 1 to study the

effect of ring-size on b turn/b sheet stabilization and hCt

bioactivity [15] These studies led very recently to the discovery of the superpotent b turn/b sheet-stabilized, hCt-agonist cyclo17,21-[Asp17,Orn21]hCt [15] In the second strategy, which we present in this report, we designed and synthesized analogues of 1 bearing type I- (and II¢-) and type II-stabilizing amino acid substitutions for Lys18 and Phe19, corresponding partial sequence peptides containing the lactam-bridged region 16–22, and also nonbridged control peptides (Scheme 1) and studied the effect of these substi-tutions on conformation, self-assembly state, hCt receptor binding affinity, and in vivo hypocalcemic activity

M A T E R I A L S A N D M E T H O D S

Materials Protected amino acids, resins for peptide synthesis, BOP, and TBTU were purchased from Bachem, Novabiochem and Rapp Polymere Solvents and miscellaneous chemicals for syntheses, HPLC purifications, SDS/PAGE, and CD studies were from Merck and Aldrich, and were of the highest purity grade available [15] Synthetic hCt and sCt for

CD and bioactivity studies were from Novabiochem The saline solution (0.9%, w/v) for the hypocalcemic assay was from Delta Pharma and BSA (99%) from Sigma Medium and all reagents for cell culture were from Gibco BRL Insulin and hydrocortisone for the cell culture were from Sigma (tissue culture grade) Salmon Tyr22 125I-labelled calcitonin (125I-labelled sCt) was 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 Na-Boc-protected amino acids [14,15,25] Following deprotection and cleavage from the resin using HF and scavengers according to our recently published procedure disulfide bridge formation was achieved by air oxidation of the crude peptides at 10)4M

in 0.1MNH4CO3[15] in the presence of 0.5–1MGdnHCl to improve solubilities and oxidation yields and its completion was followed by HPLC Crude, oxidized peptides were purified by reverse phase HPLC on a C18Nucleosil 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 eluting buffers were: A, 0.058% (v/v) trifluoroacetic acid in water and B, 0.05% (v/v) trifluoro-acetic acid in 90% (v/v) CH3CN and water The elution program was: 7 min at 30% B, followed by a gradient from 30% to 60% B over 30 min

Peptides 2–6 were also synthesized by the Fmoc/tBu strategy on Rink–MBHA resin with Na-Fmoc-protected amino acids and standard protection of the side-chains [Asp(OtBu), Glu(OtBu), Gln(Trt), Cys(Trt), His(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 Pip groups were applied For the side chain-to-side chain cyclization, these groups were selectively cleaved following treatment of the peptide resin with a mixture of 1% trifluoroacetic acid and 5% triisopropylsilan (TIS; v/v) in dichloromethane (DCM;

2· 2 min and 6· 10 min) [26] Cyclizations were performed with fourfold excess

(benzotriazol-1-yloxy)-tris-Scheme 1 acid sequences of hCt and analogues 1–6

Amino-acid residues are presented with the one letter code except for Asn17

and Thr21 and the introduced substitutes Numbers above the hCt

sequence indicate positions of the substituted residues The amino

termini and the C-terminal amide groups of hCt and the analogues are

not shown.

(dimethylamino)phosphonium hexafluorophosphate (BOP)

and N,N¢-diisopropylethylamine (DIEA) [15], and were

usually performed twice (1· 4 h and 1 · overnight)

Pep-tide resins were then acetylated Protected amino acids

(fourfold excess) were coupled using

O-benzotriazole-N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU;

fourfold excess) and DIEA (sixfold excess) The final

cleavage of the peptide and the side chain protecting groups

from the resin was performed with trifluoroacetic acid/H2O/

thioanisol/EDT/phenol (10/0.5/0.5/0.25/0.5) (v/v with the

exception of phenol) [27] Formation of the disulfide 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 assisted laser desorption ionization mass

spectrometry (MALDI-MS) with a Kratos Compact

MALDI I (Shimadzu Europe, Duisburg, Germany) and

a-cyano-4-hydroxycinnamic acid as matrix Purity of the

HPLC purified peptides were also confirmed by analytical

HPLC analyses The following 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+ of 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+ Na+ 3450.9); [D-Phe19]hCt (5): MH+

of 3416.8 (3418.9, respectively) (calculated 3418.9);

[D-Lys18]hCt (6): MH+ of 3418.8 (3417.9, respectively)

(calculated 3418.9)

Solid phase peptide synthesis of the partial sequence

peptides 1a, 1b, 2a, and 3a, their cleavages from the resin,

and HPLC purifications were performed as recently

described on MBHA with Na-Boc-protected amino acids

[15] The correct masses of HPLC purified peptides

were assessed by FAB-MS: cyclo17,21-[Asp17,Lys21]hCt

(16–22)-NH2 (1a): MH+ of 949.4 (calculated ¼ 949.5);

[Asp17,Lys21]hCt(16–22)-NH2 (1b): MH+ of 967.5

(cal-culated 967.15); cyclo17,21-[Asp17,D-Phe19,Lys21]hCt(16–

22)-NH2(2a): MH+ of 949.4 (calculated 949.5); cyclo17,

21-[Asp17,Aib19,Lys21]hCt(16–22)-NH2 (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 nm for the partial sequence peptides), with

a spectral band width of 1 nm, a scan speed of 20 nmÆmin)1

(50 nmÆmin)1for the partial sequence peptides), a response

time of 4 s (8 s for the partial peptides), and represent the

average of three scans in the range of 195–250 nm (185–

250 nm for the partial sequence peptides) Spectra were

measured in 10 mM aqueous sodium phosphate buffer

(pH 7.4) and in 10 mMsodium 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 absorbance at 274.5 nm was used to

exactly determine the concentrations of the stock solutions

of analogues 1–6 ( 500 l ) in 1 m HCl, using

e274.5 ¼ 1440M )1Æcm)1[15] Stock solutions of the partial sequence analogues 1a, 1b, 2a, and 3a (10 mM) were prepared in 10 mMHCl The spectra are presented as plots

of the mean residue ellipticity ([h]) vs the wavelength with the spectra of the buffer solution alone already subtracted Analysis of secondary structure contents

Secondary structure analyses of the spectra were performed

by multilinear regression analysis using the program LINCOMBand the reference 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) as previously described [30] To obtain comparable results to the CD concentration depen-dence studies, peptide solutions were prepared by the same procedures as for CD, and assembly states were investigated

at final peptide concentrations of 50 lM For SDS/PAGE, peptide stock solutions (500 lMin 1 mM HCl (see under

CD part) were diluted into 10 mM sodium phosphate buffer, pH 7.4, at a concentration of 100 lM, as was also done for the CD experiments The peptide solutions were then diluted with sample buffer [30] to a final concentration

of 50 lM, boiled for 5 min, and electrophoresed

Cell culture T47D cells were obtained from the American Tissue Culture Collection and were cultured in RPMI 1640 containing 10% heat inactivated fetal bovine serum, 1% streptomycin/ penicillin, 0.1 lMinsulin, and 0.1 lMhydrocortisone in 5%

CO2and 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 trypsin/EDTA as described [31,32] and for the binding experiments cells were subcultured in 12-well dishes Receptor binding experiments were performed when cells reached 90% confluence (1–3 days after subculture) 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/Pi(1 mL) at ambient temperature and then prewarmed (37°C) assay buffer that consisted of RPMI 1640 and 0.1% (w/v) BSA was added to the cells (930 lL) 125I-labelled sCt (5 lCi, specific activity

2000 lCiÆmmol)1) in its lyophilized form was reconstituted

in 100 mM HCl (200 lL), aliquoted at 4°C in eppendorf tubes (15 lL each), that were thereafter kept at)20 °C, and for each 12-well plate one tube was thawed at room temperature, diluted with assay buffer (245 lL) and used immediately Twenty microliters of the 125I-labelled sCt solution (14.4 pmol) were then added to each well, and the wells were mixed by gentle shaking Thereafter, solutions (50 lL) of different concentrations of the peptides in assay buffer were added to the cells, and following gentle mixing cells were incubated for 1 h at room temperature Peptide solutions were freshly made prior to each experiment by

diluting peptide stocks ( 500 lM in 1 mM HCl [14]) in

assay buffer Binding was terminated by aspiration of the

medium and washing of the cells with NaCl/Pithree times

Cells were then removed from the wells by short treatment

(1 min) with 0.5MNaOH (2· 0.5 mL) and bound

radio-activity was assessed by c-counting (counter efficiency

 70%) Nonspecific binding was determined as the binding

of 100 nM sCt This was assessed from 13 independent

experiments to be 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 was 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 10.11 ± 0.06 mgÆdL)1 In

good agreement with our previous findings [14,15],

maximum hypocalcemic effect was caused by 2 lg of hCt

and D[Ca2+] ¼ )2.01 mgÆdL)1 ([Ca2+] ¼ 8.10 ±

0.15 mgÆdL)1), which corresponded to a [Ca2+] reduction

of 19.90% ± 0.81 (mean ± SEM of eight mice treated

with peptide vs 7 control mice) Statistical significance of the

hypocalcemic effects of the analogues vs the effects of the

respective doses of hCt was assessed usingANOVA

Signifi-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 the respective

hCt effects, whereas the effects of 2, 5, and 6 were very

similar to hCt Of 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% of the maximal effect

(EC50) were estimated by nonlinear regression analyses of

the data using the softwarePRISM(GraphPad Software, Inc.)

R E S U L T S A N D D I S C U S S I O N

Design of the analogues

A b turn conformation strongly depends on the nature and

chirality of the amino-acid residues at its corner positions

and even small changes of these residues may dramatically

affect the type and stability of the turn [34,35]

To investigate the importance of the type of the

postulated turn for hCt bioactivity, the i + 1 turn-residue

L-Phe19 of 1 was replaced byD-Phe19 to give

cyclo17,21-[Asp17,D-Phe19,Lys21]hCt (2) (Scheme 1) This

substitu-tion was expected to stabilize a type II b turn conformasubstitu-tion

[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 was chosen Due to its strong

conformational space restriction [34], the 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) This substitution was expected to stabilize a type II¢ b turn, which places the side chains of the corner residues at roughly the same position as a type I

b turn [34,35,39] Thus, this substitution was expected to maintain or increase the bioactivity of (1) [34] Enhancement

of potency through stabilization of a type II¢ turn has been previously reported for somatostatin [34,40] Importantly, studying the structure–activity relationships of a type II¢-stabilized analogue of 1 would offer direct information about the role of the topographical features of the side chains of the residues in 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 its overall conformation However, to

be able to separately evaluate effects of the substitutes alone

vs the introduced conformational restrictions, the nonbrid-ged peptides [D-Phe19]hCt (5) and [D-Lys18]hCt (6) were also synthesized and studied (Scheme 1)

CD spectroscopy describes the average conformation

of polypeptides and the contribution of a local confor-mational feature such as a four-residue b turn to the CD spectrum of a polypeptide of 32 amino acids will usually remain unrecognised due to other secondary structure elements [41] Therefore, to be able to obtain more detailed information about a potential b turn stabilization we also synthesized and studied the conformation of cyclo17,21-[Asp17,Lys21]hCt(16–22)-NH2 (1a), cyclo17,21-[Asp17,

D-Phe19,Lys21]hCt(16–22)-NH2 (2a), cyclo17,21-[Asp17, Aib19, Lys21]hCt(16–22)-NH2 (3a), and [Asp17,Lys21] hCt(16–22)-NH2 (1b) that comprise mainly the lactam bridge-containing region 16–22 of analogues 1, 2 and 3, respectively, and also 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 lMwhere 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 similar overall 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% b sheet, the rest 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, this finding suggested they were b turn stabilized On the other hand, the similarity of the spectra of 2–4 to the spectrum of 1 suggested that the introduced substitutes did not affect the overall conformation of 1 Interestingly, also 5 and 6 contained 50% more b sheet than hCt, which suggested that nature and chirality of residues 18 and 19 are strongly associated with b sheet stabilization

of hCt

The CD spectra of 1a, 1b, and 3a exhibited a strong negative band between 185 and 190 nm that is characteristic for both turn-types (type I and II) [42] The spectrum of 2a did not exhibit such a minimum Its shape indicated that the peptide was in a conformeric equilibrium state and that it

contained contributions of three previously reported b turn

reference spectra: one class C CD spectrum [42] (a negative

band between 200 and 210 nm, a weak negative band at

about 220 nm, and a possitive band between 180 and

195 nm), one spectrum correponding to an open or ÔZÕ

conformation (one minimum at 195–200 nm) [43], and the

third component could be the type I and II b turn spectrum

according to Brahms and Brahms [28] that exhibits a

characteristic minimum at about 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) that most likely arise from coupling

between the phenylalanyl (there are three Phe residues in

sequence 16–22) and the amide chromophores [44,45] This

suggestion was further supported by the observation that

the intensity of the 220 band, that most likely corresponds to

the phenylalanyl La band [42,45], was significantly less 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 the aqueous conformation of 1b However, the

intense positive band of 1a at 220 nm suggested that

cyclization may have resulted in topological changes of the

side chain of one or more Phe residue(s) Interestingly, these

positive bands were not present in 2a and 3a suggesting a

significant effect of residuesD-Phe19 and Aib18 on

back-bone conformation and topography of the Phe side chain(s)

in sequence 16–22 The spectrum of 3a had, except for the minimum at 185–190 nm, also a strong maximum at about

198 nm and a marked negative band at about 212 nm This shape is indicative of an equilibrium of the two forms of type I b turn conformers that have been shown to be populated by cyclic b turn model peptides [43]

Together, the results of CD spectroscopy under aqueous conditions, pH 7.4, suggested that all cyclic peptides had a stabilized b turn/b sheet structure as compared to hCt and that distinct b turn populations and their mixtures were present in each of them This latter finding suggested that the lactam bridge did not completely restrict the confor-mational flexibility of the analogues and was consistent with results of several NMR and CD studies on 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 under these conditions have been often related to bioactive, i.e receptor-bound, conforma-tions [50] In addition, 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) and 1 m M (for 1a, 1b, 2a, and 3a) and at room temperature.

As shown in Fig 1C, TFE had a strong structuring and

a helix inducing effect on hCt and the nonbridged peptides 5

and 6 that contained 40–50% a helical components

accord-ing to secondary structure analysis by the reference spectra

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 which contained instead 40–50% b sheet

struc-ture, the rest being mainly random coil Thus, it appears

that the a helix-inducing effect of TFE on 1–4 was not as

strong as on hCt, 5, and 6, most likely because 1–4 were

already significantly constrained by the 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 overall conformation In

addition, the studies in 50% TFE confirmed our earlier

observations [14] that the 20-membered Asp17 to Lys21

lactam bridge had an a helix-destabilizing and a b

sheet-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 these reports,

our results suggest that the effect of such bridges on a helix

stabilization strongly depends on the particular peptide

sequence the bridges are being introduced into [14]

CD spectra of 1a, 1b, 2a, and 3a in 50% TFE were

measured next (Fig 1D) All three cyclic analogues, but not

the linear 1b, exhibited a marked positive p-p* band at

about 195 nm that has been observed in type I and type II¢

b turn models [42,43] The CD spectra of these three

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 at 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 at 215 nm

and a shoulder at  225 nm that, together with the

maximum at 195 nm, indicated the presence of the two

type I b turn conformeric forms that have been described by

Perczel et al [43] Importantly, the 50% TFE solvent system

allowed for a clear distinction between the conformation of

1a vs 1b which showed only weak CD bands 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 bridge stabilized a

specific conformeric population in 1a, which, however,

retained also a high degree of flexibility

Oligomerization studies by CD and SDS/PAGE analysis

For the above described CD studies, peptide concentrations

as low as 5 lM were applied which are close to

physio-logically relevant concentrations Confirming previous

findings [14,15], no concentration dependence of the CD

spectra or aggregation was found between 5 and 100 lMfor

hCt and also for 1 in aqueous 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 of 2–4 between

5 and 100 lM(Fig 2A), that was indicative of peptide self association [5] The mean residue ellipticities at 202 nm ([h]202), that corresponded to the minima of the CD spectra,

Fig 2 Studies on the oligomerization propensity of hCt and 1–6 (A) CD concentration dependence studies: the concentration depen-dence (5–100 l M ) of the mean residue ellipticity at 202 nm ([h] 202 ) for analogues 2, 3, and 4 in aqueous buffer is shown In the inset the linear regression analysis of the data points of 2 that were intro-duced to the equation [([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) ) is pre-sented CD measurements at various analogue concentrations ([ana-logue]) were performed in 10 m M sodium phosphate buffer, pH 7.4, and at room temperature (B) and (C) SDS/PAGE analysis and silver staining of hCt and analogues 1–6 Molecular mass markers (in kDa, lane 1) (B) Lane 2, hCt; lane 3, 6; lane 4, 2; lane 5, 3; lane 6, 4; lane 7, 1, and (C) lane 1, molecular mass markers; lane 2, hCt; lane 3, 5; lane 4, 2.

100 l M solutions of hCt and the analogues in 10 m M phosphate buffer, pH 7.4 were diluted 1 : 1 with sample buffer containing 2% SDS, boiled and electrophoresed as described under Materials and methods.

decreased with increasing concentrations, suggesting that

the peptides became more ordered during self association

[42] Plateau values were reached at 50 lM (Fig 2A) The

change of [h]202 was best fitted to an equation describing

peptide cooperative dimerization [5]

Dimerization was also confirmed by SDS/PAGE

(see below) [h]202 for the monomers ([h]202(mono)) were

obtained at 5 or 1 lM, where all analogues were essentially

monomeric, and were )6059 (2), ) 6802 (3) and ) 7680

deg.cm2/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 of 1.44· 10)5, 1.10· 10)5

and 2.47· 10)5Mfor the dimers 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 treatment of bone-disorder-related diseases [58]

Sec-ondary structure analysis of spectra corresponding to

monomeric and dimeric populations suggested that

dimer-ization occured at the expense of unordered structures and

was accompanied by a significant antiparallel b sheet

stabilization The latter one was most likely due to

intermolecular, structure-stabilizing interactions [59,60]

According to 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 observed for hCt and 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 conformational restriction, 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 and 19 It has been 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 step is then followed by formation of b sheet

aggregates [61] Thus, a reason for the increased b sheet

formation and oligomerization propensity of 2–4 could be the changed topography of the side chains of residues 18 and 19 in 2–4 This may have led to formation of an hydrophobic face in the lactam bridge-stabilized b sheet and

an increased dimerization and oligomerization propensity [55,62] Association of b sheets into multimers and fibrils would be consistent with models of hCt fibrils [56,57]

To further study the self-assembly states of 2–4, 50 lM solutions of hCt and the analogues were next subjected to SDS/PAGE analysis (Fig 2B,C) Based on the results of the

CD studies, 2, 3, and 4 were expected to predominantly consist of (noncovalent) dimers Stability of the dimers towards SDS treatment conditions (2% SDS, 100 mM 2-mercaptoethanol, 100°C for 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 Of note, the dimeric forms of 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 human islet amyloid polypeptide (IAPP), which shares a receptor and hypocalcemic activity with hCt [30,63–66]

Receptor binding affinities hCt exerts its biological effects via binding to a receptor that belongs to the family of seven-transmembrane G-protein coupled receptors [10] Ct receptors are localized in bone and kindney 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 to assess human receptor binding affinities of the synthetic analogues as compared to sCt, which is the strongest known naturally occuring Ct ligand, and hCt which is a weak ligand [33] Binding affinities were assessed via the competitive inhibition of the

Fig 3 Human Ct receptor binding of hCt, sCt and analogues 1–6 to T47D cells assessed via displacement of bound 125 I-labelled sCt Cells were prepared and incubated with125I-labelled sCt as described under Materials and meth-ods Specific radioligand binding is plotted vs the concentration of competing sCt, hCt, and 1–6 as indicated Data for 1–6 represent the mean ± SD for three to five independent experiments and data for hCt and sCt are the mean of 13 and 14, respectively, assays.

specific binding of the radioligand 125I-labelled sCt that

binds with high affinity and selectivity to the Ct receptors of

this cell line [33] As shown in Fig 3, 1 showed increased

binding affinity compared to hCt Receptor binding affinity

of 1 (IC50 ¼ 2 nM) was threefold lower than the affinity of

sCt (IC50 ¼ 630 pM), that was about 6 times more potent

than hCt (IC50 ¼ 4 nM) [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 vivohypocalcemic potency compared to hCt [14,15]

Analogues 2–4 had nearly indistinguishable binding

isotherms to hCt (IC50 ¼ 4 nM) suggesting that chirality

of residues 18 and 19 plays a crucial role for hCt binding

affinity to the T47D receptors This result was confirmed by

the results of the binding studies of 5 and 6; 5 showed a

significantly reduced binding affinity (IC50 ¼ 18 nM) as

compared to hCt (IC50 ¼ 4 nM), while 6 showed almost

no binding

Together, the obtained receptor binding data 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) residues 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 bridge leads to a partial inversement of

the latter effect

In vivo hypocalcemic activities

To directly assess the biological relevance of the introduced

substitutions, we next studied in vivo hypocalcemic potencies

in mice [1–3,14,15] Analogues 3 and 4 exhibited identical

bioactivity to 1 [14], which was 5 times more potent than

hCt (Fig 4) However, 2 was completely devoid of the

increased bioactivity of 1 Analogue 2 had an EC50of 25 ng

that corresponded to a hypocalcemic potency that was even

lower than the potency of hCt (EC50, 20 ng) Of note, 2 was

unable to reach the maximum hypocalcemic effect of 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 and was at 16.1% Analogue 5 had the same dose–response curve as 2 This indicated that the confor-mational restriction was not capable of reversing the negative effect of the inversement of chirality of Phe19 on

in vivobioactivity of 1 In contrast, 6 had the same potency and maximum effect as hCt which suggested that inverse-ment of chirality of Lys18 was well tolerated

Correlation of solution conformations with receptor binding affinities andin 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, and 3a are summa-rized in Table 1 Our findings that 3 and 4, that contain type

I and II¢ b-turn-promoting substitutes, had the same hypocalcemic potency as 1, whereas 2, that contains the type II b-turn-promoting substitute, lost the high potency of

1 supported the suggestion that a type I b turn/b sheet in 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 that 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 similar to the ones of 1, 3, and 4 In contrast, the CD studies of the partial sequence analogues showed that the Asp17,Lys21-lactam bridge and the substitutes resulted in a stabilization of distinct b turn conformeric populations in each one of the short analogues

In particular, the studies in 50% TFE indicated that type I

b turn conformers were the mostly populated b turn conformers in 1a and 3a, whereas conformeric equilibria

of several turn-types were predominant under pure aqueous conditions Importantly, the CD studies both under pure aqueous and in 50% TFE indicated that 2a populated distinct b turn conformeric states compared to 1a, and 3a

Fig 4 Hypocalcemic potencies of hCt and the

analogues 1–6 Serum calcium levels were

measured in groups of 3–10 mice per dose and

3–8 control mice 1 h after subcutaneous

injection of the peptide solution or vehicle

alone Hypocalcemic activities of each dose

are expressed as percent reduction of calcium

(mean ± SEM) caused by the peptide relative

to control.

Furthermore, 2 was the 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 in

confor-mation and in vivo bioactivity of hCt

In the T47D receptor binding studies, only 1 showed a

higher binding affinity than hCt, whereas 2–4 were equally

potent to hCt Analogues 5 and 6 showed decreased binding

affinities as compared to hCt Thus, these studies

demon-strated the crucial role of residues 18 and 19 and their

chirality for human receptor binding Moreover, these

findings suggested that the side chains of residues 18 and 19

and/or of other residues in region 17–21 may be directly

involved in receptor binding These results were 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 loop 1–7, the

potential a helical region 8–22, and the 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 result in

dramatic changes in binding affinities and efficacies [10]

Taken together, our CD and bioactivity studies suggested

that both receptor 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 hypocalcemic potency of hCt [1] The

aromatic rings of the three Phe residues Phe16, Phe19, and

Phe22 in hCt have been suggested to occupy the

hydro-phobic side of 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 bioactive 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, no such correlation was observed for 3 and 4 These latter analogues had lost the high receptor binding affinity of 1 and were similarity potent to 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-mediated inhibition of bone resorption via a direct effect of Ct on osteoclasts and of the calciuretic effect of Ct on kidney [10] Therefore, in vivo bioactivity of the Cts is determined 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 of residues 19 and/or 18 and/or the presence of Aib instead of Lys18 Thus, these analogues are expected to have in vivo a higher proteolytic stability than 1 and hCt [69] Therefore, the

Table 1 Summary of the results of the CD studies, 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, and 3a are presented, because there were no differences between the spectra of the respective complete sequence peptides CD spectra were measured in 10 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, and 3a) and

5 l M (for hCt, sCt, and 1–6) Exp turn, expected stabilized turn based on the analogue design strategy Min., minimum of CD spectrum; max., maximum of CD spectrum.

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]).

results of our studies support the notion that the in vivo

hypocalcemic potency of the Cts is directly associated to a

distinct bioactive conformeric population rather than to

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 b turn/b sheet conformation in the

region 17–21 may play an important role in hCt bioactivity

and showed that the conformation and the topological

features of the side chains of amino acid residues 18 and 19

are strongly associated with the self-assembly state, the

human receptor binding affinity and the in vivo

hypocalce-mic potency of hCt

A C K N O W L E D G E M E N T S

We are grateful to J Bernhagen for his help 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 for

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 Bartholoma¨ and R Mu¨ller for FAB-MS We thank K Tenidis,

R Kayed, and K Sweimeh for their contributions to certain

experimental parts of this work We thank W Voelter for supporting

this work This work was supported by the Deutsche

Forschungs-gemeinschaft (DFG) grant numbers Ka 979/2-1 and -2.

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