Peptide synthesis - MATERIALS AND METHODS

II. MATERIALS AND METHODS

2. Peptide synthesis

The truncated rPTH(1-12) analogues were synthesized by modifying their 7^th, 10^th, and 11^th residues serially. The substitution of these residues is known to be more tolerated than the substitution of the first 6 residues of the N-terminus, as described by Shimizu et al²¹. In addition, rPTH(1-11) analogues were also synthesized and made substitutions at the 1^st, 7^th or 8^th residues (Table 1). All synthesized peptides had an amide group at the C-terminus. The peptides used in this study were synthesized at the Korea Basic Science Institute (Seoul, Korea) using the solid phase approach and purified by HPLC. Peptide sequences were assembled with a Milligen 9050 (Fmoc Chemistry). MS was used to confirm the molecular masses of the synthesized peptides. For deprotection, a reagent mixture (88% trifluoroacetic acid, 5% phenol, 2% triisopropylsilane, 5% H2O; 2h) was used. The raw peptides formed were purified by HPLC (Delta PAK 15 C18 300 3.9×150mm column, with a 240 nm detector).

3. Subcloning of human PTH receptor cDNAs into mammalian expression vector

Human PTH1R cDNA, kindly provided by the Mogam Biotech. Institute in korea, was cloned into the expression vector pcDNA3 neo. By using a PCR cloning technique, they were able to obtain a 1.8 kb hPTH1R cDNA, which included the total open reading frame of hPTH1R, from the human kidney library.

4. Preparation of mammalian cell lines stably expressing the human PTH1 receptor (hPTH1R)

For receptor expression, LLC-PK1 porcine kidney cells were grown in Medium-199 with 3% fetal bovine serum (FBS) in 95% O2/5% CO2 and stably transfected with hPTH1R-pcDNA3 neo using the calcium phosphate method.

Briefly, 5∼7×10⁵ cells (approximately 80% confluent cultures) were plated per 10cm culture dish the day before transfection. Cells were fed with fresh complete culture medium containing 20mM Hepes and incubated in 95%

O2/5% CO2. After 3×4h, the medium was discarded and 5 ml of calcium phosphate-DNA precipitate containing, 25 μg DNA, 124 mM CaCl2, 140mM NaCl, 25 mM Hepes and 1.41 mM Na2HPO4 (pH 7.12) was added. The cells were then incubated for 4h in 95% O2/5% CO2, washed with NaCl/Pi (137mM NaCl, 2.68 mM KCL, 4.3 mM N2HPO4, 1.47 mM KH2PO4, pH 7.12), shocked with glycerol buffer (15% glycerol, 140mM NaCl, 25mM Hepes and 1.41mM Na2HPO4, pH7.12), rewashed with NaCl/Pi, and incubated for an additional

36-48h in complete Medium-199. Cells were then cultured in complete Medium-199 containing 0.5 mg/ml G418 (Geneticin, Life Technologies) until G418-resistant colonies appeared, and these were then picked out and subcultured for at least 10∼14 days. Human PTH1R-expressing cells were identified by screening more than 30 colonies and confirmed using the hPTH(1-34) induced cAMP accumulation assay, as described below. The highest reactivity LLC-PK1 porcine kidney cell, stably transfected with human PTH1R cDNA, was selected for further study. The number of hPTH1 receptors was determined to be 800,000 receptors/cell, by competitive radioligand binding assay using ¹²⁵I-[Nle^8,18Tyr³⁴]bPTH(1-34) and Scatchard plotting, as previously described²⁹.

5. cAMP generating assay

LLC-PK1 cells expressing the receptors were grown to confluence in 48-well plates. Cell culture medium was changed to complete Medium-199 containing 3% FBS 3∼4hrs before the cells were treated with peptides. For the assays, the medium was first removed and the cells were washed with 0.25 ml of cAMP-generating medium containing 3% FBS, 2 mM 3-isobutyl-1-methyl-xanthine, 0.1% BSA, 20mM Hepes, and 0.002% ascorbic acid in complete Medium-199. cAMP-generating media (0.15 ml) containing various concentrations of peptides were added and the cells were incubated for 30mins at 37℃. The media were then completely discarded, and the cells frozen at -70℃ for 30min and thawed at room temperature for 15∼20mins;

this freeze-thawing process was repeated twice. The cells were then

detached from the plates with 0.5 ml of 50mM HCl solution per well, transferred to a 1.5ml Eppendorf tube, and centrifuged at 1900×g for 10min.

The supernatants were then diluted 50-fold with radio-immunoassay (RIA) buffer and the cAMP concentration was measured using a cAMP ¹²⁵I RIA Kit (bti, USA), according to the manufacturer's instructions. The mean values of the data were fitted to a sigmoid curve with a variable slope factor using the nonlinear squares regression technique in the GRAPHPAD PRISM. EC50 (nM) values are represented by means SE. All of the cAMP assays were performed in triplicate wells and repeated twice. (p<0.05 was considered significant).

6. Circular Dichroism Spectroscopy

Circular Dichroism spectra of the peptide samples in H2O with various concentrations of trifluoroethanol (TFE) were recorded at room temperature using a Jasco J-715 spectropolarimeter. The concentrations of the peptides used ranged from 79M to 81M in 50mM phosphate buffer, at pH 7.0, and the CD spectra were recorded from 190 to 250nm at a scanning rate of 50nmmin^-1 with a time constant of 0.5s. CD data were obtained from an average of 7 scans with a resolution of 0.2nm and bandwidth of 2.0nm. A standard noise reduction was applied to the final spectrum. Ellipticity is reported as the mean residue molar ellipticity, [θ]r.

7. NMR Spectroscopy and modeling calculations

NMR spectroscopy was carried out in both 70% H2O(D2O)/30% 2,2,2- trifluoro-(d3)-ethanol (TFE) and 90% H2O/10% D2O at pH 7.0. The peptide concentrations were:-2.2 mM for [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1-12)NH2, and 3.2 mM for [Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2 and [Ala^3,10Phe⁷Arg¹¹] rPTH(1-11)NH2, in 50mM of sodium phosphate buffer. NMR spectra were recorded at 298K on a Bruker DRX-500 spectrometer equipped with a triple resonance probe, and triple axis gradient coils. The spectra were also recorded over the temperature range 10-25℃, to calculate the temperature coefficients. Pulsed-field gradient (PFG) techniques were used in all H2O experiments to suppress solvent signals. Two-dimensional total correlation spectroscopy (TOCSY)³⁰, with an MLEV-17 mixing pulse of 69.7ms, and two-dimensional nuclear Overhauser effect spectroscopy (NOESY)³¹, with mixing times of 100∼600 ms, was also performed. Two-dimensional double-quantum-filtered (DQF) COSY³² spectra were collected in H2O to obtain the vicinal coupling constants. A series of one-dimensional NMR measurements were obtained to identify the slowly exchanging amide hydrogen resonances in freshly prepared D2O solutions after the H2O sample had been lyophilized. All NMR experiments were performed in the phase-sensitive mode using the time proportional phase incrementation (TPPI) method³³ with 2048 data points in the t2, and 256 points in the t1 domains. NMR data were processed using XWIN-NMR (Bruker Instruments) software, and the processed data further analyzed using Sparky 3.60 software, which was developed at UCSF on a Silicon Graphics Indigo² workstation. Starting structures were generated using distance geometry (DG) by employing a refinement protocol using distance restraints assigned as strong, medium, or

weak, on the basis of cross-peak volumes in the NOESY spectra. All distances had a lower limit of 1.8, with upper limits of 2.7, 3.3, or 5.0 for strong, medium or weak intensities, respectively. The dihedral angle constraints were also deduced from the³ JHNα coupling constants from the 2D DQF-COSY spectra in H2O. Structure calculations were performed using hybrid distance geometry and the dynamically simulated-annealing protocol^34-37 using the X-PLOR 3.81 (Biosym/Molecular Simulations, Inc.) program on a Silicon Graphics Indigo² workstation.

III. RESULTS

1. Synthesis of short-length amino terminal PTH analogues

2. Preparation of LLC-PK1 cell lines stably expressing the hPTH1R

With stably transfected by the calcium phosphate method, the G418-resist

Figure 1. Selection of the LLC-PK1 cell lines stably over- expressing hPTH1R by measuring the level of PTH(1-34)-stimulated cAMP accumulation.

0 50 100 150 200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Clone number

cAMP level (folds)

-ant cell lines were selected by of PTH(1-34)-induced cyclic AMP accumulation. As shown in Figure 1, compared to the vehicle-treated group, the 4^th cell colony in hPTH1R exhibited the most significant increasement of intracellular levels.

All peptides have an amide group on the C-terminus. The main substituted

3. Effects of the synthesized peptides on cAMP accumulation in LLC- PK1 cells stably expressing hPTH1R

LLC-PK1 cells stably transfected with human PTH1R cDNA were stimulated with the synthesized PTH analogues substituting at 7^th, 8^th, 10^th, and 11^th positions. Parental LLC-PK1 cells, which do not express hPTH1 receptors, were unresponsive to hPTH(1-34) or any of the PTH analogues used in this study. However human PTH1R-expressing cells were responsive to hPTH (1-34) (EC50=50.9±30.13pM) and the synthesized PTH analogues, and they showed a basal activity that was similar to that of untransfected LLC-PK1 cells, without hPTH(1-34) treatment. A typical dose-dependent induction of cAMP generating activity was observed upon increasing the concentrations of the PTH analogues, and the EC50 values calculated from the dose-response curves proved reproducible over several experiments; results are summarized in Table 2.

4. Effect of substituting the 10^th and 11^th residues

Most of the [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 analogues, in which Leu⁷ was substituted by Phe⁷, showed lower cAMP formation, except [Ala^3,10,12 Leu⁷Lys¹¹]rPTH(1-12)NH2, [Ala^3,12Leu⁷Asp¹⁰Lys¹¹]rPTH(1-12)NH2, and [Ala^3,12 Leu⁷Gln¹⁰Lys¹¹]rPTH(1-12)NH2 (Table 2). All of the oligopeptides in which Arg¹¹ of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1-12)NH2 were substituted by Met¹¹ or Lys¹¹ displayed lower cAMP accumulation. In particular, the substitution of Arg¹¹ by Ile¹¹ led to remarkable decrease in cAMP formation, even at high oligopeptide concentrations (10^-4M). The order of the potency of the

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 analogues substituted at the 11^th residue was Arg¹¹≈ Met¹¹> Lys¹¹> Ile¹¹ (Figure 2A), and that of the [Ala^3,10,12Phe⁷Arg¹¹] rPTH(1-12)NH2 analogues was Arg¹¹≈ Lys¹¹> Met¹¹> Ile¹¹ (Figure 2B).

All peptides have an amide group at the C-terminus. The main substituted amino-acid residues are shown in bold italics. The amino acid sequence of rPTH(1-12) is AVSEIQLMHNLG. EC50 values were obtained by computer analysis of the respective dose-response curves on hPTH1R stably transfected LLC-PK1 cell lines.

"-", the mature cAMP generation failed to reach the maximum levels achieved by other ligands. Data are represented as means±SE of three independent experiments.

Table 2. EC50 values (means ± SEM) of the rPTH(1-12) and rPTH(1-11) analogues used in this study

Peptide EC50(nM) Peptide EC50(nM)

Leu7 oligopeptides Phe7 oligopeptides

rPTH(1-12) analogues

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH₂ 1404 ± 82.278 [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH₂ 7845 ± 507.64 [Ala^3,10,12Leu⁷Lys¹¹]rPTH(1-12)NH₂ 22330 ± 493.55 [Ala^3,10,12Phe⁷Lys¹¹]rPTH(1-12)NH₂ 14560 ± 892.79 [Ala^3,10,12Leu⁷Ile¹¹]rPTH(1-12)NH₂ 171600 ± 5683.34 [Ala^3,10,12Phe⁷Ile¹¹]rPTH(1-12)NH₂ 546300 ± 38888.9 [Ala^3,10,12Leu⁷Met¹¹]rPTH(1-12)NH2 3358 ± 146.263 [Ala^3,10,12Phe⁷Met¹¹]rPTH(1-12)NH2 108200 ± 5016.56 [Ala^3,12Leu⁷Asp¹⁰Arg¹¹]rPTH(1-12)NH2 1969 ± 122.455 [Ala^3,12Phe⁷Asp¹⁰Arg¹¹]rPTH(1-12)NH2 6159 ± 483.69 [Ala^3,12Leu⁷Asp¹⁰Lys¹¹]rPTH(1-12)NH2 28890 ± 514.39 [Ala^3,12Phe⁷Asp¹⁰Lys¹¹]rPTH(1-12)NH2 13240 ± 884.63 [Ala^3,12Leu⁷Asp¹⁰Ile¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Phe⁷Asp¹⁰Ile¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Leu⁷Asp¹⁰Met¹¹]rPTH(1-12)NH₂ 3759 ± 559.62 [Ala^3,12Phe⁷Asp¹⁰Met¹¹]rPTH(1-12)NH₂ 48850 ± 7626.96 [Ala^3,12Leu⁷Gln¹⁰Arg¹¹]rPTH(1-12)NH₂ 2301 ± 240.562 ^[Ala^3,12^Phe⁷Gln¹⁰Arg¹¹]rPTH(1-12)NH₂ 4257 ± 445.11 [Ala^3,12Leu⁷Gln¹⁰Lys¹¹]rPTH(1-12)NH2 27531 ± 5033.62 [Ala^3,12Phe⁷Gln¹⁰Lys¹¹]rPTH(1-12)NH2 9463 ± 946.52 [Ala^3,12Leu⁷Gln¹⁰Ile¹¹]rPTH(1-12)NH2 _ [Ala^3,12Phe⁷Gln¹⁰Ile¹¹]rPTH(1-12)NH2 _

[Ala^3,12Leu⁷Gln¹⁰Met¹¹]rPTH(1-12)NH₂ 3163 ± 698.62 [Ala^3,12Phe⁷Gln¹⁰Met¹¹]rPTH(1-12)NH₂ 19490 ± 3207.54 [Ala^3,12Leu⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH₂ 10890 ± 2339.33 [Ala^3,12Phe⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH₂ 22030 ± 4037.15 [Ala^3,12Leu⁷Glu¹⁰Lys¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Phe⁷Glu¹⁰Lys¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Leu⁷Glu¹⁰Ile¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Phe⁷Glu¹⁰Ile¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Leu⁷Glu¹⁰Met¹¹]rPTH(1-12)NH₂ ^_ [Ala^3,12Phe⁷Glu¹⁰Met¹¹]rPTH(1-12)NH₂ ^_ rPTH(1-11) analogues

[Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2 1529 ± 81.695 [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 2132 ± 126.24 [Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH₂ ^_ [Ser¹Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH₂ 4230 ± 136.26 [Ala^3,10dPhe⁷Arg¹¹]rPTH(1-11)NH₂ ^_ [Ala³Phe⁷Asp¹⁰Arg¹¹]rPTH(1-11)NH₂ ^_ [Ala^3,10Val⁷Arg¹¹]rPTH(1-11)NH₂ ^_ [Ala³Phe⁷Gln¹⁰Arg¹¹]rPTH(1-11)NH₂ ^_ [Ala^3,10Ile⁷Arg¹¹]rPTH(1-11)NH2 _ [Ala^3,10Phe⁷Leu⁸Arg¹¹]rPTH(1-11)NH2 _

[Ala^3,10Phe⁷Nle⁸Arg¹¹]rPTH(1-11)NH2 3256± 752.62

-9 -8 -7 -6 -5 -4

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10,12Leu⁷Lys¹¹]rPTH(1-12)NH₂ [Ala^3,10,12Leu⁷Met¹¹]rPTH(1-12)NH2

[Ala^3,10,12Leu⁷Ile¹¹]rPTH(1-12)NH2

peptide(log[M])

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10,12Phe⁷Lys¹¹]rPTH(1-12)NH2

[Ala^3,10,12Phe⁷Met¹¹]rPTH(1-12)NH2

[Ala^3,10,12Phe⁷Ile¹¹]rPTH(1-12)NH2

peptide(log[M])

-9 -8 -7 -6 -5 -4

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Leu⁷Asp¹⁰Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Leu⁷Gln¹⁰Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Leu⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH2

peptide(log[M])

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Phe⁷Asp¹⁰Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Phe⁷Gln¹⁰Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Phe⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH2

peptide(log[M]) cAMP-generating activity was reduced so remarkably, I determined their solution structures by CD and NMR spectroscopy later.

-9 -8 -7 -6 -5 -4

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 [Ala^3,10,12Leu⁷Ile¹¹]rPTH(1-12)NH2 [Ala^3,12Leu⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH2 [Ala^3,12Leu⁷Glu¹⁰Ile¹¹]rPTH(1-12)NH2

pe ptide (log[M ])

[Ala^3,10,12Phe⁷Arg¹¹]rP TH(1-12)NH2

[Ala^3,10,12Phe⁷Ile¹¹]rPTH(1-12)NH2 [Ala^3,12Phe⁷Glu¹⁰Arg¹¹]rPTH(1-12)NH2

[Ala^3,12Phe⁷Glu¹⁰Ile¹¹]rPTH(1-12)NH2

pe ptide (log[M ])

rPTH(1-12)NH2 into either Leu⁷ or Phe⁷ analogues. The induction of cAMP formation by [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 was found to be comparable to that of [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2, though the substitution of Leu⁷ by Phe in most of the PTH(1-12) analogues reduced cAMP generating activity.

Surprisingly, when Ser was introduced as the first residue of [Ser¹Ala^3,10Leu⁷ Arg¹¹]rPTH(1-11)NH2, cAMP generating activity was almost undetectable, but the substitution of the Ala1 of [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 by Ser¹

[Ala^3,10dPhe⁷Arg¹¹]rPTH(1-11)NH2 [Ala^3,10Val⁷Arg¹¹]rPTH(1-11)NH2 [Ala^3,10Ile⁷Arg¹¹]rPTH(1-11)NH2 [Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2 [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2

Peptide (log[M])

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2

Peptide (log[M])

8. Circular dichroism

In order to determine why the biological activity of the rPTH(1-11) and rPTH(1-12) analogues differ according to the presence of Leu or Phe at the 7^th residue, I determined the solution structures of [Ala^3,10,12Leu⁷Arg¹¹]rPTH (1-12)NH2, [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2, [Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2

and [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 by circular dichroism. CD spectra were acquired for the rPTH analogues under different conditions. (CD) spectra in aqueous solutions were measured at a peptide concentration of 20uM under benign conditions, i.e., in 50mM sodium phosphate, pH 7.0, 25℃. Figure 7 shows that the peptides possess a degree of helical structure in physiological

-9 -8 -7 -6 -5 -4

0 50 100 150 200 250 300

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2

[Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH₂ [Ser¹Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2

Pe ptide (log[M])

cAMP Levels (Fold of Basal)

Figure 6. Comparison of cAMP stimulating activity induced by [Ala^3,10,12 (Leu⁷/Phe⁷)Arg¹¹]rPTH(1-12)NH2, [Ala^3,10(Leu⁷/Phe⁷)Arg¹¹]rPTH(1-11)NH2, and [Ser¹Ala^3,10(Leu⁷/Phe⁷)Arg¹¹]rPTH(1-11)NH2 in LLC-PK1 cells stably transfected with hPTH1R. Each experiment was performed in duplicate and repeated three times. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in Materials and Methods. The amino acid sequence of native rPTH(1-12) is AVSEIQLMHNLG.

environments, based on the ellipticity at 222nm, and the characteristic α -helical transition, which was observed in the presence of TFE (Figure 7): i.e., a typical double minimum (n-π* transition) at 208 and 222nm, and a maximum (π-π* transition) at 192nm. The exact mechanism by which TFE stabilizes secondary structure is unknown, but more recent studies suggest that TFE interacts preferentially with the helical conformation of a peptide to

190 200 210 220 230 240 250

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 H2O (pH 7.0)

190 200 210 220 230 240 250

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 H2O (pH 7.0)

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

Wavelength (nm)

H₂O (pH 7.0) { TFE30%

z TFE50%

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH₂

-25

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

Wavelength (nm)

H₂O (pH 7.0) { TFE30%

z TFE50%

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH₂

-25

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

190 200 210 220 230 240 250

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 H2O (pH 7.0)

190 200 210 220 230 240 250

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 H2O (pH 7.0)

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

Wavelength (nm)

H₂O (pH 7.0) { TFE30%

z TFE50%

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH₂

-25

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

Wavelength (nm)

H₂O (pH 7.0) { TFE30%

z TFE50%

[Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH₂

-25

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

190 200 210 220 230 240 250

[θ]x10-3degree cm2/decimole

significant α-helix population.

190 200 210 220 230 240 250

Leu7Ala10Arg11

190 200 210 220 230 240 250

Phe7Ala10Arg11

190 200 210 220 230 240 250

Phe7Ala10Arg11

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ]x10-3degree cm2/decimole

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ]x10-3degree cm2/decimole

190 200 210 220 230 240 250

Leu7Ala10Arg11

190 200 210 220 230 240 250

Phe7Ala10Arg11

190 200 210 220 230 240 250

Phe7Ala10Arg11

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ]x10-3degree cm2/decimole

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ]x10-3degree cm2/decimole

Phe7

A B

C D

9. Resonance assignment and solution structure

Complete proton resonance assignments were possible using the standard sequential resonance assignment procedure³⁸. Having classified the individual spin systems, the backbone sequential resonances were assigned using dN(i,i+1) NOE connectivities in the 2D-nuclear Overhauser effect spectroscopy (NOESY) spectra. A number of well-resolved, intense dNN cross-peaks were thicknesses. The dashed line represents NOEs unresolved by resonance overlapping. Vicinal coupling constants (●; ³JHN <6 Hz) are indicated and

the sequential and short-range NOE connectivities observed for the rPTH analogues in 30% TFE. The observation of continuous dNN(i,i+1) contacts together with the characteristic dN(i,i+3) and the d(i,i+3) NOEs supports the existence of α-helices containing the Ala³-His⁹ residues in all of the rPTH analogues.

The NMR structures were calculated using the experimental restraints derived from 2D-NOESY and double-quantum-filtered correlated spectroscopy (DQF-COSY) spectra. A total of 50 distance geometry (DG) structures served as starting structures for the dynamic simulated-annealing (SA) calculations of the peptides in TFE solutions. The 20 lowest energy structures (<SA>k) from the 50 simulated-annealing structures were selected for detailed structural analysis (Table 3). The average structure (<SA>k) was calculated from the geometrical average of the 20 <SA>k structural coordinates, and restraint energy minimization (REM) was used to correct covalent bonds and angular distortions. In Figure 10, the final 20 structures are displayed as averaged structures (<SA>kr) of [Ala^3,12Leu⁷Glu¹⁰Arg¹¹] rPTH(1-12)NH2. In Figure 11(A/B), the final 20 structures are displayed as averaged structures (<SA>kr) of rPTH(1-12). A Ramachandran plot (data

Figure 10. NMR structure of the [Ala^3,12Leu⁷Glu¹⁰Arg¹¹]rPTH(1-12) NH2 analogue. Superposition of the final 20 <SA>k

structures upon the energy-minimized average structure (<SA>kr) of Ala³-His⁹, and ribbon drawings of the REM structures of [Ala^3,12Leu⁷Glu¹⁰Arg¹¹]rPTH (1-12)NH2.

not shown) for all 20 <SA>k structures showed that the, and angles of the finally simulated-annealing structures are distributed adequately in energetically acceptable regions. A best-fit superposition of the 20 <SA>k

structures along with the energy-minimized average structure (<SA>kr) of rPTH(1-11) are also shown in Figure 11(C/D). The main secondary structural feature of the rPTH analogues is the α-helix spanning residues of Ala³-His⁹ in 30% TFE/H2O solution.

Figure 11. NMR structures of the rPTH(1-12)NH2 and rPTH(1-11)NH2 analogues.

Superposition of the final 20 <SA>k structures upon the energy- minimized average structure (<SA>kr) of Ala³-His⁹, and ribbon drawings of the REM structures of [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 (A), [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 (B), [Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11) NH2 (C), and [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 (D) in an aqueous 30% TFE solution at pH 7.0 and 298K.

B A

C D

Table 3. Structural statistics for the 20 final simulated-annealing structures

<SA>k <SA>kr <SA>k <SA>kr <SA>k <SA>kr <SA>k <SA>kr

(A) RMSD from experimental distance restraints (A) all

dihedral angle restraints(deg) 0.4709 0.4689 0.4342 0.4259 0.4566 0.4457 0.4346 0.436 (C) Energies(kcal/mol)

IV. DISCUSSION

In this study, short oligopeptides, based on the first 11 or 12 amino acids of PTH, tend to form an α-helical structure, and that the destabilization of this α-helical structure remarkably reduces PTH/PTHrP receptor activation.

Furthermore, the activity differences associated with these short amino terminal analogues may be due to residue specificity.

In a previous study, the only residue tolerated in place of Leu⁷ was Phe⁷

21. Therefore, the synthesized oligopeptides were classified, based on rPTH(1-11) NH2 or on rPTH(1-12)NH2 into either Leu⁷ or Phe⁷ analogues. The induction of cAMP formation by [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 was found to be comparable to that of [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2, though the substitution of Leu⁷ by Phe in most of the PTH(1-12) analogues reduced cAMP generating activity. [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2, [Ala^3,10,12Phe⁷Arg¹¹] rPTH(1-12)NH2, [Ser¹Ala^3,10Leu⁷Arg¹¹]rPTH(1-11)NH2 and [Ala^3,10Phe⁷Arg¹¹] rPTH(1-11)NH2 peptides were found to have essentially helical conformations; however, their helix populations were very low in aqueous solution, as shown in Figure 7. In SDS solution, the CD spectra indicated the presence of typical helix conformations (data not shown), and in high concentrations of TFE (50%) solution the helical structures of [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 and [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

stabilized, as demonstrated by their negative ellipticities at 222nm. Moreover, their solution structures showed that these species differ in terms of their side chain orientations, and that it is attributed to the steric effects of the Leu⁷ and His⁹ residues (Figures 11 and 12). However, the reasons why such changes cause differences in biological activity are unknown. Based on the

solution structures of [Ser¹Ala^3,10Leu⁷ Arg¹¹]rPTH(1-11)NH2 and [Ala^3,10Phe⁷ Arg¹¹]rPTH(1-11)NH2, It is suggested that their functional differences could be associated with the hydropho- bicities of their side-chains as affected by the 7^th position, and the influence that these have on optimum binding in the receptor pocket (Figures, 11C and 11D).

When Ser was introduced as the first residue of [Ser¹Ala^3,10Leu⁷Arg¹¹] rPTH(1-11)NH2, cAMP generating activity was almost undetectable, but the substitution of the Ala¹ of [Ala^3,10Phe⁷Arg¹¹]rPTH(1-11)NH2 by Ser¹ restored cAMP activity (figure 6). In addition, both analogues revealed typical helix conformations. This result re-confirms that the 1^st∼3^rd or 5^th residues are important for receptor activation by the N-terminal of short length PTH, and also demonstrate the criticality of Ala¹ ³⁹. Furthermore, it also suggested that their functional differences could originate from the residue specificity of the first one rather than being due to the possession of an alpha helix backbone.

In terms of the [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1-12)NH2 derivatives based on substituting the 10^th residue with Ala, Asp, Gln, or Glu, their order of potencies in terms of cAMP accumulation was Ala ≈ Asp ≈ Gln > Glu (Figure 3). The lower Figure 12. Superimposed REM structure

of the rPTH(1-12)NH2 analog ues.

C-terminus

N-terminus

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2

Leu⁷/Phe⁷

C-terminus

N-terminus

[Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2

[Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2

Leu⁷/Phe⁷

potencies of the [Ala^3,12(Leu⁷/Phe⁷)Glu¹⁰Arg¹¹]rPTH(1-12)NH2 analogues versus the [Ala^3,12(Leu⁷/Phe⁷)Asp¹⁰Arg¹¹]rPTH(1-12)NH2 analogues is attributed to the fact that the Glu residue is larger than the Asp residue, though the two residues carry the same charge. The arginine residue was found to be the most suitable 11^th residue in the [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹] rPTH(1-12)NH2 derivatives, and the order of potency with respect to cAMP formation was Arg ≈ Lys > Met > Ile (Figure 2). It is also substituted to the 8^th Met residue with Leu or Nle, because the Met residue is vulnerable to oxidation. Substituting Met⁸ with Nle had little effect upon cAMP activity;

however, substituting it with Leu reduced cAMP activity 10 fold (Figure 5B), which may be due to the smaller size of the Leu residue.

In the present study, PTH analogues, which had the 10^th Ala residue in [Ala^3,10,12Leu⁷Arg¹¹]rPTH(1-12)NH2 or in [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 substituted by Glu, or the 11^th Arg residue by Ile, showed remarkably reduced cAMP generating activity. Furthermore, when both residues were substituted with Glu¹⁰ or Ile¹¹, the resulting peptides were almost inactive. Both [Ala^3,10,12 Leu⁷Arg¹¹]rPTH(1-12)NH2 and [Ala^3,10,12Phe⁷Arg¹¹]rPTH(1-12)NH2 have potentially helical conformations, as shown in Figure 11, even though their helix populations may be very low in aqueous solution. The exact mechanism by which TFE stabilizes secondary structure is unknown, but more recent studies suggest that TFE interacts preferentially with the helical

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