Peptide - peptoid hybrids based on (1-11)-parathyroid hormone analogs

Article abstract of DOI:10.1002/psc.1265

A series of peptide - peptoid hybrids, containing N-substituted glycines, were synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har- NH₂ (Har = Homoarginine) as the parent parathyroid hormone (1-11) analog. The compounds were pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor. Copyright

Full text of DOI:10.1002/psc.1265

Research Article
Received: 15 January 2010
Revised: 19 May 2010
Accepted: 20 May 2010
Published online in Wiley Interscience: 13 July 2010
(www.interscience.com) DOI 10.1002/psc.1265
Peptide–peptoid hybrids based on
(1–11)-parathyroid hormone analogs
A. Caporale,∗ E. Schievano and E. Peggion
A series of peptide–peptoid hybrids, containing N-substituted glycines, were synthesized based on the H-Aib-Val-Aib-Glu-Ile-
Gln-Leu-Nle-His-Gln-Har-NH2 (Har = Homoarginine) as the parent parathyroid hormone (1–11) analog. The compounds were
c
pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor. Copyright ꢀ 2010 European
Peptide Society and John Wiley & Sons, Ltd.
Keywords: N-substituted glycine; peptidomimetics; PTH analogs; conformation; bioactivities
Introduction
model. The N-terminal (1–14) segment interacts with the
7-transmembrane (7-TM) helical domain embedded in the
membrane and the C-terminal (19–34) segment with the
N-terminal extracellular domain of the receptor [24,25]. These
two domains, which are structured in α-helices, are separated by
two hinge-like motifs located around positions 12 and 19 [26,27].
Studiesonreduced-size(1–34)-PTHmolecules[28,29]haveshown
that analogs of the (1–11)-PTH fragment containing residues
capable of enhancing its helicity yielded potent agonists capable
ofreproducingthebiologicalresponsescharacteristicofthenative
intact PTH [23,30,31]. The lack of high-resolution structural data for
GPCRsledtothedesignofmimeticmoleculestogainmoredetailed
information on the receptor/ligand complex. In this work, we have
addressed the role and orientation of the Val2, Nle8 and Arg11 side
chains in the interaction of short PTH fragments with the PTH1R
receptor using three peptomers (Table 1), in which only one amino
acid residue at the time is replaced by the corresponding peptoid
residue. Their agonist activity is compared with the N-terminally
modified (1–11)-PTH, peptide 1 (Table 1) as reference 32,33.
Although bioactive peptides are known to play decisive roles
in many physiological processes via both inter- and intracellu-
lar communication and signal transduction mediated by various
classes of receptors, the development of peptides into drugs
for human medicine still represents a difficult task because of
their poor bioavailability and fast clearance rates. Nonetheless,
synthetic replicates of naturally occurring bioactive peptides and
related analogs are excellent tools to characterize biochemically
and biologically receptor systems for their further development
into diagnostic or therapeutic drugs by improving their phar-
macological properties often applying peptidomimetic strategies
[1–4].
Among peptide mimicries peptoids consisting of N-substituted
glycines (NSGs) represent an interesting class of synthetic
molecules suitable for drug discovery because of their potential
biological activities and high-proteolytic stability [5]. Indeed,
peptoids were found to act as efficient receptor ligands [6–9]
and to retain the biological activity of the parent natural peptides
despite the shift of the amino acid side chains to the nitrogen
atom [10–15].
The versatility and efficiency of the synthetic methods devel-
oped over the past years including the use of microwaves allow
for efficient preparation of peptoid oligomers containing the
most diversified side chains for proper mimicry of their peptide
counterparts [16–20]. A special class of these molecules is pep-
toid–peptide hybrids (peptomers) in which only one or several
amino acid residues are substituted by NSGs [21]. Such peptomers
allow to explore the peptide tolerance toward transformation
into peptoids in terms of potential bioactive structures and thus
receptor binding affinities [16].
Parathyroid hormone (PTH) is an 84-amino acid peptide
hormone secreted by the parathyroid glands. It acts primarily
on bones and kidneys to maintain extracellular calcium levels
within normal limits. The study of reduced-size PTH(1–34) agonist
and antagonist analogs has been the subject of extensive research
for the development of safer and non-parental bone anabolic
drugs [22–24].
Materials and Methods
Materials
All solvents were purchased from commercial sources and used
without purification. Reagent grade materials were obtained from
GL Biochem (Shangai, China) and Inalco-Novabiochem (Milano,
Italy). Molecular weights of final peptides were determined
by ESI-MS-TOF on a Perseptive Biosystems MARINER API-TOF
spectrometer (Foster City, CA, USA). RP purification was routinely
performed on a Shimadzu LC-8A equipped with a Shimadzu SPD-
6A UV detector on a Deltapak Waters C18-100 Å silica HPLC column
(19 mm × 30 cm, part no. 11799) at a flow rate of 17 ml/min by
elution with a linear gradient 20–45% (v/v) of solvent B in solvent
∗
Correspondence to: A. Caporale, Department of Chemical Sciences, Institute
of Biomolecular Chemistry, CNR, University of Padova, via Marzolo 1, 35131
Padova, Italy. E-mail: caporaleandrea74@gmail.com
The interaction of (1–34)-PTH with its parathyroid hormone 1
receptor (PTH1R), a receptor of the G-protein coupled receptor
(GPCR) family, has been postulated to follow a ‘two-domain’
Department of Chemical Sciences, Institute of Biomolecular Chemistry, CNR,
University of Padova, Padova, Italy
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Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

3 PTH(1–11) ANALOGUES WERE SYNTHESIZED AND TESTED AT PTH1R
Table 1. Activity of analogs versus parent peptide
Chemical
formula
[M+H]+
Rt
a
Entry
Peptide
MWcalc
found
(min)b
EC50 (nM)c
1
2
3
4
H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (parent peptide)
C59H103N19O15 1317.74
1318.73
1304.56
1304.46
1318.62
12.07
13.85
12.31
15.38
1.1 0.1
2130 160
Not active
Not active
H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-NArg-NH2 (NArg11-PTH(1–11)) C58H101N19O15 1303.74
H-Ala-NVal-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (NVal2-PTH(1–11))
C58H101N19O15 1303.74
C59H103N19O15 1317.74
H-Aib-Val-Aib-Glu-Ile-Gln-Leu-NNle-His-Gln-Har-NH2
a Monoisotopic (m.i.) molecular weight calculated for chemical formula.
b Rt was determined with a linear gradient of 20–45% (v/v) B over 20 min (A: water + 0.1% TFA; B: 90% acetonitrile + 0.1% TFA).
c EC50 (effective concentration 50) is the result of the average on at least three values. EC50 is defined as the half maximal effective concentration and
is referred to the concentration of peptide that induces a response halfway between the baseline and the maximum.
Figure 1. Scheme of strategic substitution of amino acids with NSG residues to generate peptomers.
A over 20 min. Homogeneity of the products was assessed by
analytical RP-HPLC using a Vydac C18 column (218TP510), UV
detection at 214 nm, flow rate 1 ml/min with a linear gradient
20–45% (v/v) of solvent B in solvent A over 20 min (solvent A:
water + 0.1% TFA; solvent B: 90% acetonitrile + 0.1% TFA in
water).
6 Transfection Reagent and CRE-luc plasmid, according to the
manufacturer’s procedure; 18 h after transfection, the cells were
incubated for 4 h at 37 ◦C yielding maximal response to luciferase.
Each peptide concentration was tested in triplicate. Luciferase
activity was measured on a Lumat LB 9507 luminometer (EG&G
Berthold, Bad Wildbad, Germany). The mean read-out from three
wells with identical peptide concentration was used to present
the data.
Peptide Synthesis
Peptomers were prepared by Fmoc solid-phase peptide synthesis
for both proteinogenic amino acid residues and NSG residues
on 0.1 mmol of Rink-amide-MBHA resin (0.73 mmol/g substitution
grade) from Inalco-Novabiochem. The NSG residues, NVal, NNle
and NArg, were synthesized according to the literature procedures
[8,34,35]. The coupling of the suitably protected amino acids was
performed with HOBt, HBTU and DIPEA, except for Aib, Val and
the amino acid residues N-terminal to NSG where HOAt, HATU
and collidine were used [36]. Fmoc cleavage was performed
with 20% piperidine (v/v) in DMF. The resin-bound peptides
were cleaved/deprotected with TFA/TIS/water (95 : 2.5 : 2.5 v/v/v)
at room temperature for 2 h. After filtration, the filtrate was
concentrated under nitrogen stream and precipitated with methyl
tert-butyl ether. All crude peptide–peptoid hybrid analogs were
purified and analytically characterized using RP C18 HPLC and
molecular weights were determined by ESI-MS. The average yield
of a complete synthesis after purification and lyophilization was
10%.
Circular Dichroism
CD measurements were carried out on a JASCO J-715 spectropo-
larimeter interfaced with a PC. The CD spectra were acquired and
processed using the J-700 program for Windows. All experiments
were carried out at room temperature using HELLMA quartz cells
with Suprasil windows and optical path lengths of 0.01 and 0.1 cm.
All spectra were recorded using a bandwidth of 2 nm and a time
constant of 8 s at a scan speed of 20 nm/min. The signal to noise
ratio was improved by accumulating eight scans. Measurements
were carried out in the 190–250 nm wavelength range and the
peptide concentration was in the range of 0.07–1.07 mM. The
peptides were analyzed in aqueous solutions containing 20% TFE
(v/v). The spectra are reported in terms of mean residue molar
ellipticity (deg · cm2 · d/mol). The helical content for each peptide
was estimated according to the literature [37].
NMR Measurements
Biological Tests
NMR spectra were recorded at 298 K on a BRUKER AVANCE DMX-
600spectrometer.TheexperimentswerecarriedoutinH2O/TFE-d3
(4 : 1, v/v). The sample concentration was approximately 1 mM
in 600 µl solution. The water signal was suppressed by pre-
saturation during the relaxation delay. The spin systems of all
Adenylyl cyclase assays for agonistic activities were performed
with C20 HEK293 cells stably expressing the PTH1R and seeded
at 106 cells/well in collagen-coated 24-well plates as previously
reported [23]. After 24 h, the cells were treated with FuGENE
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J. Pept. Sci. 2010; 16: 480–485 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

CAPORALE, SCHIEVANO AND PEGGION
Figure 2. Synthetic scheme for analogue 2. All analogues were synthesized in a similar way.
amino acid residues were identified using standard DQF (double-
quantum-filtered)-COSY [38] and clear TOCSY [39] spectra. In the
latter case, the spin-lock pulse sequence was 70 ms long. The
sequence-specific assignment was accomplished using the ROESY
experiment, with a mixing time of 150 ms. In all experiments, the
spectra were acquired by collecting 400–512 experiments, each
one consisting of 32–256 scans and 4K data points.
observed reduced potency supports the strategic role of arginine
or homoarginine, which replaces leucine of native PTH, in
anchoring (1–11)-PTH to the receptor [41] either by stabilizing
the bioactive conformation [28] or by specific interactions with
the receptor binding pocket [29]. Our previous work on molecular
modeling suggests that this role might involve the insertion of the
guanidinium side-chain group between the extracellular ends of
TM1 and TM7 [27].
Spectral processing was carried out using XWINNMR. Spectra
were calibrated against the TMS signal.
A CD study of the synthesized peptides was performed to
compare their conformation with the results of the biological
tests. The conformational properties of the new analogs were
investigated first in 20% TFE/water [23,32] and then in the
presence of increasing TFE concentrations [42]. The CD spectra
of the (1–11)-PTH analogs are reported in Figure 3. Peptomer
2 shows a typical α-helical CD spectrum, similar to that of the
parent peptide (Figure 3a), and the helix content increases at TFE
concentrations higher than 10% (Figure 4). This is consistent with
thepresenceofbiologicalactivityfor2(Table 1),albeitthreeorders
of magnitude lower than that of the parent peptide. By increasing
the TFE concentration, the CD spectra become compatible with
the presence of some 310-helix or β-turn, according to the
low-intensity ratio between the bands at 222 and 208 nm [43].
Anyway, peptides 2 and 3 have the identical helix-propensity,
as demonstrated in Figure 4, although the hybrid 3 (Figure 3b)
is not biologically active (Table 1). The introduction of NVal2 at
the N-terminal position might disrupt the nucleation of the helix,
or the side chain of NVal assumes an inadequate orientation for
interaction with the receptor.
The peptide–peptoid hybrid 4 exhibits a CD spectrum typical of
a flexible and extended structure even at high percentages of TFE
(not shown). The absence of any structural effect of TFE (Figure 4)
indicates that the introduction of an NSG in position 8 disrupts the
stability of the helix, which leads to the loss of bioactivity (Table 1).
To prove CD results, NMR analysis of secondary chemical shifts
of the Cα protons was performed on all analogs (Figure 5). The
secondary chemical shifts of peptide 4 are very close to zero
or positive, which confirms the absence of ordered structure as
Results and Discussion
The peptoid–peptide hybrids were designed as outlined in
Figure 1 and the resulting primary structures are reported in
Table 1. As reference, the mostactive (1–11)-PTH analog peptide 1
[22,31]wasusedinaccordancewithourpreviousstudies[32,33,40],
although for a very similar peptide no activity was observed [23].
The most plausible explanation for this surprising finding may be
the presence of the homoarginine residue in C-terminal position.
Indeed, very recently we could demonstrate that the distance of
the positive charge may play a decisive role in receptor binding
affinity[40].Moreover,Gardellaet al.[28,29]haverecentlyreported
how modifications of (1–11)-PTH are very often additive, such as
thereplacementofAsn10 byGln10 thatleadstoenhancedpotency.
The syntheses were performed by a suitable combination of
standard Fmoc solid-phase peptide synthesis for proteinogenic
amino acid residues [36] and of the submonomer approach for
NSG residues [8,16,34,35]. The synthesis of peptide 2 is reported
in Figure 2 as an example. For peptide 3, Aib in position 1 has
been replaced by Ala to avoid steric hindrance caused by NVal.
The analytical data are reported in Table 1.
Peptides 3 and 4, with NVal and NNle in positions 2 and 8,
respectively, showed no biological activity on the PTH receptor.
The substitution of Arg in position 11 with the corresponding
NSG maintained a low activity with an EC50 value of 2.1 × 10−6
M, according to a previous observation [40]. Specifically, the
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3 PTH(1–11) ANALOGUES WERE SYNTHESIZED AND TESTED AT PTH1R
(a)
(b)
Figure 4. % Helix versus % TFE calculated according to Greenfield and
Fasman [44].
are not very informative because random coil chemical shifts for
NSG are not available in the literature. Nevertheless, the effect in
the central part of the molecule is similar for these two analogs.
In these peptomers, the shift of the side chains from the
α-carbon to the amide nitrogen creates tertiary amides, thus
favoring cis–trans isomerism and preventing intramolecular
H-bonds, destabilizing the secondary structure [38] or placing
the side chain in an unfavorable topological position [45,46].
Conclusions
Figure 3. CD spectra of 2 (a) and 3 (b) at different concentrations of TFE
in water (from 0 to 50%). In both panels, the CD spectrum of the parent
peptide in 20% TFE is reported as a reference.
The hybrid compounds 2–4 demonstrate that replacing Val2, Nle8
and Har11 with peptoid residues leads to decreased potency at the
PTH1 receptor, although the magnitude of such decrease depends
on the site of substitution. Compound 2 (substitution of Har) was
the only hybrid that retained functional activity, although the
potency was reduced when compared with the reference peptide
1. The residual activity of NArg11 suggests that the positive charge
at the end of the peptide is important for binding, but its distance
from the peptide backbone is significantly affecting recognition
by the receptor [40]. The structural role of residues 2 and 8 was
determined by CD analysis. The crucial role of Nle in position 8
in stabilizing the α-helix from the N-terminal to the C-terminal
residue has just been confirmed in our preceding study [32]. Both
hybrid peptides 2 and 3 show partially ordered structures. In the
C-terminus, thehelicalstructureisbettermaintainedincompound
2 than in 3. The secondary chemical shifts in the N-terminal region
Figure 5. Secondary chemical shifts (ꢀδ = δmeasured − δrandom coil) of peptomers. Numbering is according to Table 1.
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J. Pept. Sci. 2010; 16: 480–485 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

CAPORALE, SCHIEVANO AND PEGGION
again confirmed [24]. Indeed, disruption of helicity is possibly the
main cause for the inactivity of the peptomer containing NNle8,
whereas the side-chain orientation of NVal2 is most probably
the main cause of the inactivity of the corresponding hybrid. A
significant reduction in the α-helical content was found in all the
analogs studied, showing that a stable helix is necessary, although
not sufficient, for PTH-like ligand interaction with the receptor.
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This study was supported by MIUR, Ministry of Education and
University of Italy, and CNR, National Council of Research of Italy.
The authors thank Dr Barbara Biondi for assistance in synthesis and
mass spectroscopy, Prof. Laura Biondi for stimulating suggestions,
and Dr Iwona Woznica for the biological tests carried out at the
Department of Physiology, Tufts University School of Medicine,
Boston University.
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