Discovery of a low affinity thyrotropin-releasing hormone (TRH)-like peptide that exhibits potent inhibition of scopolamine-induced memory impairment in mice

Article abstract of DOI:10.1039/c5ra06935a

TRH-like peptides were synthesized in which the critical N-terminus residue l-pGlu was replaced with various heteroaromatic rings, and the central residue histidine with 1-alkyl-l-histidines. All synthesized TRH-like peptides were evaluated in vitro as agonists in HEK mTRH-R¹ and HEK mTRH-R² cell lines, an expressing receptor binding assay (IC₅₀), and cell signaling assay (EC₅₀). The analeptic potential of the synthesized peptides was evaluated in vivo by using the antagonism of a pentobarbital-induced sleeping time. The peptides 6a, 6c and 6e were found to activate TRH-R² with potencies (EC₅₀) of 0.002 μM, 0.28 μM and 0.049 μM, respectively. In contrast, for signaling activation of TRH-R¹, the same peptides required higher concentration of 0.414 μM, 50 μM and 19.1 μM, respectively in the FLIPR assay. The results showed that these peptides were 207, 178 and 389-fold selective towards TRH-R² receptor subtype. In the antagonism of a pentobarbital-induced sleeping time assay, peptide 6c showed a 58.5% reduction in sleeping time. The peptide 6c exhibited high stability in rat blood plasma, a superior effect on the scopolamine-induced cognition impairment mice model, safe effects on the cardiovascular system, and general behavior using a functional observation battery (FOB).

Full text of DOI:10.1039/c5ra06935a

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DOI: 10.1039/C5RA06935A.
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Page 1 of 14
RSC Advances
View Article Online
DOI: 10.1039/C5RA06935A
Journal Name
RSC Publishing
Discovery of a low affinity thyrotropinꢀreleasing
hormone (TRH)ꢀlike peptide that exhibits potent
Cite this: DOI: 10.1039/x0xx00000x
inhibition
of
scopolamineꢀinduced
memory
impairment in mice
Received 00th January 2012,
Accepted 00th January 2012
Chhuttan L. Meena,a Shubdha Ingole,b Satyendra Rajpoot,b Avinash Thakur,b Prajwal P.
Nandeker,c Abhay T. Sangamwar,c Shyam S. Sharma,b and Rahul Jaina*
DOI: 10.1039/x0xx00000x
www.rsc.org/
TRHꢀlike peptides were synthesized in which critical
with various heteroaromatic rings, and the central residue histidine with 1ꢀalkylꢀ
N
ꢀterminus residue
L
ꢀpGlu was replaced
ꢀhistidines.
L
All synthesized TRHꢀlike peptides were evaluated in vitro as agonists at HEK mTRHꢀR1 and
HEK mTRHꢀR2 cell lines, expressing receptor binding assay (IC50), and cell signaling assay
(EC50). The analeptic potential of the synthesized peptides was evaluated in vivo by using the
antagonism of a pentobarbitalꢀinduced sleeping time. The peptides 6a
activate TRHꢀR2 with potencies (EC50) of 0.002 ꢁM, 0.28 ꢁM and 0.049 ꢁM
,
6c and 6e were found to
respectively In
,
.
contrast, for signaling activation of TRHꢀR1, the same peptides required higher concentration
of 0.414 ꢁM, 50 ꢁM and 19.1 ꢁM, respectively in the FLIPR assay. The results showed that
these peptides were 207, 178 and 389ꢀfold selective towards TRHꢀR2 receptor subtype. In the
antagonism of a pentobarbitalꢀinduced sleeping time assay, peptide 6c showed 58.5% reduction
in sleeping time. The peptide 6c exhibited high stability in rat blood plasma, superior effect on
the scopolamineꢀinduced cognition impairment mice model, safe effects on the cardiovascular
system, and general behavior using functional observation battery (FOB).
Introduction
functions. No function has as yet been shown to be mediated by
TRHꢀR2; however, as it is highly expressed in several brain
regions, and it is expected to mediate neurotransmitter effects.9ꢀ
Thyrotropinꢀreleasing hormone (TRH) is the first hypothalamic
peptide identified by Guillemin et al.1 and Schally et al.2 TRH is
a key factor responsible for proper brainꢀbody coordination,
synthesized mainly in the hypothalamus and acts as a
neurohormone, neurotransmitter and a neuromodulator. As a
neurohormone, TRH stimulates the release of thyrotropin
(thyroidꢀstimulating hormone, TSH) and prolactin from the
anterior pituitary gland. As
neuromodulator in the central nervous system (CNS), it
modulates various systems and exerts variety of
extrahypothalamic effects.3 TRH executes its activity in rodents
through two subtypes of Gꢀprotein coupled (7ꢀtransmembraneꢀ
spanning) receptors, TRH receptor type 1 and 2 (TRHꢀR1 and
TRHꢀR2). These receptors activate the same signaling
pathways, mediated primarily by coupling to Gq/11 proteins
with the subsequent activation of phosphoinositide specific
phosholipase C.4ꢀ5 Although TRHꢀR1 and TRHꢀR2 show
identical binding affinities and indistinguishable potencies for
TRH and some TRHꢀderived peptides, they exhibit different
basal signaling activities and different rates of internalization.6ꢀ7
It is to be noted that in humans only a single type of TRH
receptor is reported that is more similar to TRHꢀR1 than TRHꢀ
R2.8 The two TRH receptors show clear differences in their
anatomical distribution suggesting distinct biological roles.
TRHꢀR1 has been shown to mediate endocrine and CNS
10
The administration of TRH causes a number of CNS effects
including arousal, antidepressant activity, anxiolytic effects,
increase in locomotor activity, antagonism of pentobarbital
(PB)ꢀinduced sedation, thermoregulation, and cardiovascular
and gastrointestinal autonomic functions.11 The CNS effects
have been described in mice in which TRHꢀR1 or TRHꢀR2
receptors were "knockedꢀout". TRHꢀR1 knockout mice were
found to exhibit increased depressionꢀlike and increased
anxietyꢀlike behaviors. Whereas female, but not male, TRHꢀR2
knockout mice exhibited moderately increased depressionꢀlike
and reduced anxietyꢀlike phenotypes.12ꢀ13 Also, TRHꢀR1 but
not TRHꢀR2 knockout mice were found to be mildly
hypothyroid. Several previous studies described TRHꢀlike
peptide that exhibited neuroprotective activity in various animal
models. Most of the reported analogues have low binding
affinity for the TRH receptors and retain TSH secretory
activity. They are also limited by their poor selectivity for the
two types of TRH receptors, poor stability, poor permeability
and access to the CNS, and lesser efficacy with some unwanted
side effects. In recent studies, the susceptibility of TRH to
degradation by TRHꢀDE is recognized to be a major factor
undermining the investigation of the neurobiological functions
and its therapeutic use, and a large number of TRH analogues
and their various CNS applications are reported.14ꢀ15 Recently
a
neurotransmitter and
a
This journal is © The Royal Society of Chemistry 2015
J. Name., 2013, 00, 1-3 | 1

RSC Advances
Page 2 of 14
ARTICLE
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C5RA06935A
pGluꢀAsnꢀProꢀ
D
ꢀTyrꢀ
D
ꢀTrpNH2, was effective in vivo in producing
Kelly et al. reported a set of compounds that display ability to
both inhibit TRHꢀDE and bind preferentially to central TRH
receptors. This dual pharmacological activity within one
molecular entity was found through selective manipulation of
peptide stereochemistry.16ꢀ17 Earlier synthesized TRHꢀlike
peptides have been modified at all of the three amino acids of
TRH, i.e. pGlu, His and Pro.18 In this report, we synthesized
TRHꢀlike peptide that involve simultaneous replacement of the
and potentiating central actions of TRH without evoking release of
TSH. Specifically, this peptide displayed high stability and
combined potent inhibition of TRHꢀDE (Ki = 151 nM) with high
affinity binding to central TRH receptors (Ki = 6.8 nM). Moreover,
intraperitoneal injection of this peptide mimicked and augmented the
effects of TRH on behavioral activity in rat. Kelly et al. later
reported that pGluꢀAsnꢀProꢀDꢀTyrꢀD
ꢀTrpꢀNH2 binds to [3H][3ꢀMeꢀ
His2]TRHꢀlabeled sites in rat hippocampus and cortex but not
pituitary or heterologous cells expressing TRHꢀR1 or TRHꢀR2.24
Kelly et al. recently reported that the same peptide bound selectively
with high affinity to native TRH receptors in human hippocampal
tissues. They noted that these receptors are pharmacologically
distinct from TRH receptors in human pituitary, thus providing the
possibility of a new TRH receptor subtype.25 It is possible that the
inconsistent effects of administered TRH and its analogues in
humans may have been caused by their rapid degradation of and
poor bloodꢀbrain barrier penetration.26
critical residue
carboxylic acids and histidine by 1ꢀalkylꢀ
L
ꢀpGlu by a panel of hetero ringꢀcontaining
ꢀhistdines, in order to
L
gain insight into the essential structure features of the native
peptide (Fig. 1). The synthesized tripeptides were evaluated for
receptor binding assay, and cell signaling assay. Further, we
selected one CNS active TRHꢀlike peptide for the stability
analysis in rat blood plasma, effect on scopolamineꢀinduced
cognition impairment mice model and effect on cardiovascular
system and general behavior study using Functional
Observation Battery (FOB). The peptide exhibits superior CNS
activity resulting in its identification as a new TRHꢀlike peptide
possessing good cognitive enhancing activity in the
scopolamineꢀinduced memory impairment with safe
cardiovascular and CNSꢀbehavioral profile in the mouse model.
In TRH, the first residue LꢀpGlu is responsible for almost half of
peptide binding energy and higher sensitivity towards TRHꢀDE,
while modification on the histidine residue is known to increase the
lipophilicity.27ꢀ28 As indicated above, pGlu retains half of the peptide
binding energy, therefore, we explored its replacement with some
simple heteroꢀring containing residues to identify its most suitable
counterpart for improving stability, CNS activity and reducing
hormonal activity.29ꢀ31 The other objective of the replacement of the
CONH2
O
H
R1
N
N
LꢀpGlu residue at the Nꢀterminus was enhancing the hydrogen
bonding capability at the Nꢀterminus to note the effect on the
receptor sub type selectivity. The central His residue was modified
in such a way to substantially increase the hydrophobicity of the
designed peptides. The structural variations performed, include
modification of His with the incorporation of bulkier alkyl groups
such as methyl, ethyl, propyl, iꢀpropyl, and benzyl at the Cꢀ2
position of the ring, to investigate the effect of change in
hydrophobicity at the sideꢀchain of the involved amino acid.
O
N
N
R2
R1 = Heteroaromatic ring
R2 = CH3, C2H5, C3H7, CH(CH3)2, CH2C6H5
Fig. 1 General structure of the synthesized TRHꢀlike peptides
Results and discussion
Chemistry
The synthetic strategy adopted to synthesize the TRHꢀlike
Basis of this study is the earlier reported TRHꢀlike peptides in which
peptides was described in scheme 1. Firstly, the,
alkylꢀ ꢀhistidines (2aꢀe), precursors for TRHꢀlike peptides,
were synthesized using a earlier reported procedure in a single
NꢀαꢀBocꢀ1ꢀ
the Nꢀterminus
acid, including taltirelin (TAL), CGꢀ3703, YMꢀ14673 and RGHꢀ
2202. TAL ([1ꢀmethylꢀ(S)ꢀ4,5ꢀdihydroorotyl)]ꢀ ꢀHisꢀ ꢀProNH2, TAꢀ
LꢀpGlu residue is replaced with a nonꢀnatural amino
L
step from
N
ꢀαꢀBocꢀHisꢀOH (1
).32
L
L
0910) produces CNS effects at about 100 times lower doses than
TRH, and also showed eight times longer duration of antagonistic
action on PBꢀinduced sleep than TRH.14ꢀ15,18 It is the first centrally
acting TRHꢀlike peptide that received approval in Japan for use as a
drug for the treatment of adult spinal muscular atrophy under the
trade name Ceredist®.15 Previously, Matsuoka et al.19 showed that
TAL displayed higher activity in stimulating CNS effects than TRH
in rodents. The differences in the activities of TAL and TRH in the
CNS have been attributed to the higher stability of TAL in blood
against TRHꢀdegrading enzyme (TRHꢀDE), and its greater
lipophilicity than TRH that account for its increased penetration
across the bloodꢀbrainꢀbarrier.20ꢀ22 Thirunarayanan et al.23 recently
described the pharmacology of TAL at the human TRH receptor
CONH2
O
O
O
H
N
H
N
H
N
Boc
OH
Boc
OH
Boc
N
HN
i
ii
30ꢀ60%
CONH2
N
NH
N
N
N
N
R2
R2
3
4a-e
1
2a-e
iii
>90%
N
N
H3C
HO
N
N
N
NH2
CONH2
CONH2
O
O
N
H
R1
N
TFA.H2N
R1=
N
N
N
iv, v
20ꢀ55%
O
N
H
N
HO
N
N
N
N
N
R2
R2
6a-w
5a-e
Reagents and conditions: (i) NaH, R2X, DMF, ꢀ15 oC to ꢀ5 oC; (ii) DIC, HONB, DMF, 4 oC, 36h; (iii) 40% TFA, 0 oC, 30 min;
(iv) 7N NH3 in MeOH, 10 min; (v) R1CO2H, DIC, HOBt, DMF, 4 oC, 36h.
Scheme 1 Generalized scheme for peptide synthesis
expressed in heterologous cells at which TAL appeared to act as a
17
super agonist. Scalabrino et al
.
in a recent study have described
Further, intermediates 2aꢀe upon coupling reaction with
Lꢀ
compounds showing potent dualꢀaction and the lead compound,
ProNH2 ( ) in the presence N,Nꢀdiisopropylcarbodiimide (DIC)
3
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

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View ArticAle ROnTliInCeLE
DOI: 10.1039/C5RA06935A
and
the auxiliary nucleophile in DMF afforded
alkyl)ꢀ ꢀProNH2 4aꢀe). The removal of the Boc group in
dipeptides (4aꢀe) was accomplished by reaction with CF3CO2H
(40% solution in CH2Cl2) to produce dipeptide salts (5aꢀe). The
peptides 5aꢀe were neutralized with a solution of 7N NH3 in
N
ꢀhydroxyꢀ5ꢀnorborneneꢀ2,3ꢀdicarboximide (HONB) as
HEK TRHꢀR1, Receptor binding assay
N
ꢀαꢀBocꢀ ꢀHis(1ꢀ
L
5000
4000
3000
2000
1000
0
L
(
CH3OH for 10 min to obtain LꢀHis(1ꢀalkyl)ꢀLꢀProNH2, which
upon immediate coupling reaction with various hetero ringꢀ
containing carboxylic acids [pyrazineꢀ2ꢀcarboxylic acid (2ꢀ
Pyz), 5ꢀmethylpyrazineꢀ2ꢀcarboxylic acid (5ꢀMpyz), 3ꢀ
aminopyrazineꢀ2ꢀcarboxylic acid (3ꢀApyz), piperidineꢀ2ꢀ
carboxylic acid (2ꢀPip), 6ꢀhydroxypicolinic acid (6ꢀHpic) and
5ꢀhydroxypyrazineꢀ2ꢀcarboxylic acid (6ꢀHpyz)] in the presence
of DIC and 1ꢀhydroxybenzotriazole (HOBt) as the auxiliary
nucleophile at 4 oC in DMF for 36 h afforded the desired
TRH-like peptides
HEK TRHꢀR2, Receptor binding assay
15000
10000
5000
0
peptides 6aꢀw
.
In vitro pharmacology at TRHꢀR1 and TRHꢀR2
The primary screening through receptor binding assay of all
synthesized peptides were performed at a single dose in
duplicate in HEK293 EM cells stably expressing TRHꢀR1 or
TRHꢀR2 by incubating the cells with 10 µM dose of the test
peptides (Figure 2). Afterwards, all peptides were examined for
their functional signaling at multiple doses in HEK293 EM
cells stably expressing TRHꢀR1 or TRHꢀR2, for calculating
EC50. The extent of agonist behavior was then determined by
measuring signaling of calcium mobilization using FLIPR
assay, and the data are reported as EC50 values (ꢁM) in Table 1.
It was observed that none of peptides have effective
TRH-like peptides
Fig. 2 Receptor binding assay. Cells transfected with TRHꢀR1 or
TRHꢀR2 were incubated with 4 nM [3H]N(1)ꢀMeꢀHisꢀTRH in the
absence or presence of single dose 10 ꢁM of peptide at 0 oC. All
data represent the arithmetic mean of results obtained error bars
represent the ±SD of duplicate experiments.
A clear observation was made for the alkyl group placement at
the Nꢀ1 position of histidine residue. The increase in the size of
group results in increased selectivity toward TRHꢀR2 up to a
level, but further increase in the group size with groups such as
competition with [3H]MeTRH at 10 ꢁM (Figure
2), and some of
n
ꢀpropyl, iꢀpropyl and benzyl resulted in decreased selectivity.
the peptides exhibited weak signaling for TRHꢀR1 or TRHꢀR2,
but many of the peptides showed positive calcium signaling
towardss TRHꢀR2; however, in most cases potency was no
more than moderate when compared to that shown by TRH. For
example, peptide 6a (R1 = 2ꢀPyz, R2 = CH3) displayed 207ꢀfold
For example, 6i [R1 = 5ꢀMpyz, R2 = CH(CH3)2] and peptide 6j
(R1 = 5ꢀMpyz, R2 = CH2C6H5) displayed about 3ꢀfold and 2.6ꢀ
fold selectivity for TRHꢀR2. Peptide analogues 6kꢀl containing
a 3ꢀaminopyrazinyl moiety at the N
ꢀterminus (R1 = 3ꢀApyz, R2
= CH3/C2H5) exhibited equal selectivity but low signaling
potency toward TRHꢀR1 and TRHꢀR2 receptors with 21ꢀ30ꢀ
fold selectivity towards TRHꢀR2 receptor. Peptide 6m (R1 = 3ꢀ
Apyz, R2 = C3H7) exhibited 70ꢀfold selectivity toward TRHꢀR2
receptor, but moderate potency toward TRHꢀR2. Interestingly,
6n exhibited good signaling potency (EC50) of 0.007
0.001 M for TRHꢀR1 and TRHꢀR2, respectively. TRHꢀlike
selectivity towards TRHꢀR2 with potency (EC50 = 0.002 ꢀM),
while another peptide 6b (R1 = 2ꢀPyz, R2 = C2H5) showed 42ꢀ
fold selectivity toward TRHꢀR2 receptor. Peptide 6c (R1 = 2ꢀ
Pyz, R2 = C3H7) displayed promising signaling activity for
ꢀM and
TRHꢀR2, and lower for TRHꢀR1 (EC50 = 0.28 ꢀM and 50 ꢀM,
respectively), resulting in a 178ꢀfold relative selectivity toward
TRHꢀR2. Moreover, peptide 6d [R1 = 2ꢀPyz, R2 = CH(CH3)2]
ꢀ
peptides, wherein pGlu residue was replaced with 2ꢀpiperidinyl
ring also exhibited results along the same lines. For example,
peptide 6p (R1 = 2ꢀPip, R2 = CH3) exhibited 18ꢀfold selectivity
exhibited high potency to TRHꢀR1 (EC50 = 0.001
equipotent to the parent peptide in toward TRHꢀR1. Interesting
observations were made for peptide 6e (R1 = 2ꢀPyz, R2
CH2C6H5) that exhibited moderate potency (TRHꢀR1: EC50=
19.1 M; TRHꢀR2: EC50= 0.049 M) and produced 389ꢀfold
ꢀM) and was
toward TRHꢀR2 receptor, whereas 6q [R1 = 2ꢀPip, R2
=
CH(CH3)2] displayed signaling potencies (EC50) of 0.75
ꢀ
M
=
and 0.030
ꢀM to TRHꢀR1 and TRHꢀR2, respectively.
Similarly, peptides 6r (R1 = 5ꢀHpyz, R2 = CH3), 6s (R1 = 5ꢀ
Hpyz, R2 = C2H5) and 6t (R1 = 5ꢀHpyz, R2 = CH2C6H5)
exhibited moderate signaling for TRHꢀR1 and TRHꢀR2. The
ꢀ
ꢀ
selectivity in comparison to the parent peptide in signaling
potency toward TRHꢀR2. The peptide 6f, where pGlu was
replaced with a 5ꢀmethylpyrazinyl moiety (R1 = 5ꢀMpyz, R2 =
CH3) exhibited 22ꢀfold selectivity toward TRHꢀR2 receptor in
signaling potency, while 6g (R1 = 5ꢀMpyz, R2 = C2H5)
produced 75ꢀfold selectivity to TRHꢀR2 and displayed agonist
peptide 6r showed signiling potency (EC50) of 0.015
TRHꢀR2, which is 87ꢀfold selective toward TRHꢀR2. Peptide
6s exhibited an EC50 value of 0.072 M at TRHꢀR2, which is
44ꢀfold selective for TRHꢀR2. Finally, TRHꢀlike peptides 6uꢀ
, in which pGlu residue is replaced with 6ꢀhydroxypiperzinyl
group exhibited moderate activation potencies.
ꢀM at
ꢀ
w
activity at TRHꢀR2 with potency (EC50 = 0.037 ꢀM).
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

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DOI: 10.1039/C5RA06935A
Journal Name
RSC Publishing
Table 1 Results of in vitro receptor binding (IC50 in ꢀM) and receptor signaling potency (EC50 in ꢀM)
S. No.
R1
R2
EC50 (µM)b
FLIPR Assay
TRHꢀR1
IC50 (ꢀM)
TRHꢀR1
TRHꢀR2
TRHꢀR2
Fold
Selectivity
(R1/R2)
207
42.2
178.5
0.03
389.7
22.5
75.9
9.55
3.0
6a
2ꢀPyz
2ꢀPyz
2ꢀPyz
2ꢀPyz
CH3
C2H5
C3H7
CH(CH3)2
CH2C6H5
CH3
C2H5
C3H7
CH(CH3)2
CH2C6H5
CH3
C2H5
C3H7
CH(CH3)2
CH2C6H5
CH3
CH(CH3)2
CH3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ꢀ
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ꢀ
0.414
10.48
50
0.002
0.248
0.28
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6t
6u
6v
6w
TRH
0.001
19.10
0.27
2.81
4.46
0.012
0.085
1.31
2.87
0.78
0.007
0.058
3.55
0.75
1.31
3.18
0.12
4.04
ꢀ
0.030
0.049
0.012
0.037
0.467
0.004
0.032
0.061
0.095
0.011
0.001
0.019
0.195
0.030
0.015
0.072
0.437
0.172
ꢀ
2ꢀPyz
5ꢀMpyz
5ꢀMpyz
5ꢀMpyz
5ꢀMpyz
5ꢀMpyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
2ꢀPip
2.6
21.4
30.2
70.9
7
3.05
18.2
25
87.3
44.1
0.27
23.4
ꢀ
2ꢀPip
5ꢀHpic
5ꢀHpic
5ꢀHpic
5ꢀHpic
6ꢀHpyz
6ꢀHpyz
6ꢀHpyz
pGlu
C2H5
C3H7
CH2C6H5
CH3
C2H5
>50
>50
0.026
>50
>50
.007
ꢀ
2.228
0.436
0.00052
2.22
0.25
1.9
CH2C6H5
H
0.11
0.001
For binding assays, cells transfected with TRHꢀR1 or TRHꢀR2 were incubated with 4 nM [3H]MeTRH in the absence or presence of various doses
of unlabeled TRH analogues for 4 h at 0 oC. For signaling, transient changes in [Ca2+] induced by ligands were measured using the
FLIPRTETRA® system at multiple doses. All experiments were performed three times in duplicate or triplicate. “ꢀ“ Not tested.
The sleeping time was recorded as the time elapsed from the
The results of the in vitro data showed that none of the peptide
onset of loss of righting reflex until it returned (Table 2). Some
tested appear to bind to known TRH receptors expressed in
of peptides showed a significant decrease in sleeping time as
HEK cells, while they show selectivity for TRHꢀR2 over TRHꢀ
compared with the control group. The observed potentiation of
R1 in inhibiting calciumꢀdependent fluoresence response in the
pentobarbitalꢀinduced sleep activity may be caused by
alterations in receptor interactions due to the specific properties
of the substitution on the His residue of the TRHꢀlike peptides.
Most of the tested peptides showed positive results when
evaluated in vivo for antagonism of pentobarbitalꢀinduced
sleeping time in mice.
FLIPR assay. With the exception of 6d, all of the compounds
exhibited EC50 values greater than TRH. These receptorꢀbased
results are consistent as found earlier, when analogue where
ring NH of pGlu was replaced by a methylene group did not
show binding affinity, confirming that the ring NH group of
pGlu is necessary for the binding of the peptide to TRH
receptors, while the modulation of the His residue at the
Nꢀ1
position imparts selectivity toward TRHꢀR2.14aꢀb
In vivo reversal of pentobarbitalꢀinduced sleeping time
assay of the peptides
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Journal Name
Table 2 Results of reversal of pentobarbitalꢀinduced sleeping time assay
RSC Advances
View ArticAle ROnTliInCeLE
DOI: 10.1039/C5RA06935A
S. No.
R
R1
Sleeping duration in min (%) Reduction in time (Mean±
(Mean± SEM)
SEM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
Control
TRH
CH3
C2H5
C3H8
CH(CH3)2
CH2C6H5
CH3
C2H5
C3H7
2ꢀPyz
2ꢀPyz
2ꢀPyz
2ꢀPyz
82.7±4.2
81.1±7.1
39.0±7.1
57.1±0.7
93.5±4.4
65.6±4.9
51.6±7.7
65.5±4.5
19.1±2.2
51.1±8.5
31.5±6.7
47.0±11.0
85.1±5.5
67.6±6.1
93.8±3.5
53.9±3.7
11.8
13.5
58.5
39.1
0.32
30.0
44.9
30.1
79.6
45.5
54.6
49.8
9.2
2ꢀPyz
5ꢀMpyz
5ꢀMpyz
5ꢀMpyz
5ꢀMpyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
3ꢀApyz
–
CH(CH3)2
CH3
C2H5
C3H7
CH(CH3)2
CH2C6H5
–
27.93
0.0
42.5
H
pGlu
Pentobarbital was injected (50 mgkgꢀ1 i.p.) 10 min after i.v. Injection of the TRH/TRH analogues. Sleep was recorded as the time elapsed
from the onset of loss to regaining of the righting reflex. Six to eight Swiss albino mice (body weight 20–30 g) were used in each group.
Values are expressed as the mean SEM. p< 0.05 relative to control using oneꢀway ANOVA followed by post hoc Tukey test. p<0.05
relative to TRH using oneꢀway ANOVA followed by post hoc Tukey test.
The sleeping time after administration of peptides 6c and 6i hydrophobicity of TRH and 6c was predicted by calculating C
were 39.0±7, and 19.1±2.2 min, respectively. The decrease in log P values using Chemoffice 6.0 software. The predicted C
sleeping time caused by these peptides was significantly better log P value of 6c is ꢀ0.6425 and that of TRH is ꢀ2.791, which
from control (93.8±3.5 min) and TRHꢀtreated group (53.9±3.7 confirms more lipid solubility of 6c compare to TRH. Further
min). Analogues 6c and 6i were found to be the most potent the stability studies of 6c have indicated it to be more stable in
analogues, effecting decreases in sleep time by 58.5% and plasma as compared to TRH, which represents its good stability
79.6%, relative to control. In continuation, we also observed profile and lower tendency for early degradation by degrading
interesting result with peptide 6k (31.5±6.7 min) showing enzymes.
54.6% reduction in sleep time. The outcome of these results
show that TRHꢀlike peptides 6c and 6i are more potent than
TRH in their ability to decrease pentobarbitalꢀinduced sleeping
150
125
time in mice. The promising analeptic potential, along with the
ease in the synthesis, and specificity to TRHꢀR2 receptor in the
100
75
50
25
0
TRH
6c
singaling assay prompted us to undertake further biological
evaluation of 6c. It may be noted that earlier studies indicated
the possibility that novel TRH receptors may mediate the CNS
actions of TRHꢀlike peptides, and it can be postulated that the
CNS effects of the peptides reported herein may be mediated
through such a novel TRH receptor subtype.23ꢀ25
Time in (hour) after incubation
Stability study of TRH and TRH analogue 6c in plasma
Fig. 3 Exꢀvivo stability study of 6c and TRH in plasma sample of rat at
different time intervals (values represent the % remained in plasma).
To check the stability profile of 6c over TRH, we have studied its
stability in plasma using HPLC.4 The peptide 6c was found to be
more stable in plasma than TRH; TRH degraded by 80% within 3 h
whereas 6c remained 60% intact up to 18 h of incubation in the
plasma at 37 °C, and therefore is considered more stable compared
to TRH in the blood plasma (Figure 3).
Behavioral experiments
Scopolamineꢀinduced memory impairment experiments were
with the Passive avoidance and Morris water maze test in which
studies were conducted with five groups, namely, control
(saline), positive control (rivastigmine), scopolamine, TRH and
Permeability prediction
The hydrophobicity of a drug molecule can be used to predict
its cellular permeability. More the hydrophobicity of the
molecule more will be the accessibility in the brain. The
investigational peptide 6c
Effect of 6c on scopolamineꢀinduce memory impairment
.
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5RA06935A
a) Effect of peptide 6c at different dose on reference The efficacy of the TRHꢀlike peptide 6c (3.9 mg/kg) was
memory investigated in scopolamineꢀinduced memory impairment
Peptide 6c in Morris water maze learning showed decrease in model through Morris water maze test. It was compared with
latency time at dose level of 1.9 and 3.9 mg/kg, but at the dose TRH and the positive control, rivastigmine. Control mice
level of 7.8 mg/kg it failed to do so (Figure 4). In the amnesic rapidly learned the platform location but scopolamineꢀtreated
mice treated with 6c (1.9 and 3.9 mg/kg), the mean escape mice fail to find the platform. Scopolamineꢀtreated mice took
latency of 111.2±4.7 sec and 114.8±5.2 sec was observed on significantly longer time to find the platform from day 2
day 1, which fell to 72.6±9.6 sec and 53.1±6.8 sec by day 4, onwards, in which escape latency (106.8±4.9 sec) on day 1 was
respectively. Peptide 6c at a dose of 3.9 mg/kg showed reduced to (66.2±3.8 sec) on day 4, as compared to saline
significant reduction of the escape latency in scopolamineꢀ (91.8±4.1 sec) on day 1 reduced to (31.4±2.0 sec) on day 4. In
treated mice on day 2, 3 and 4, and at 1.9 mg/kg on day 2 and 3 the amnesic mice treated with 6c (3.9 mg/kg), the mean escape
but not on day 4. On the other hand 6c (7.8 mg/kg) failed to latency of (102.9±3.2 sec) observed on day 1 fell to (34.2±2.3
improve learning at all stages. The area under curve of the sec) by day 4. The peptide 6c showed significant reduction of
escape latencies in fourꢀday trial of 6c at 1.9 mg/kg was the escape latency in scopolamine treated mice on day 2, 3 and
261.3±25.6 sec, and at 3.9 mg/kg was 217.8±20.5 sec, which 4. On the other hand, TRH significantly reduce the escape
was significantly different from scopolamine (337.5±12.2 sec) latency on day 3 and 4 only in which the escape latency on day
treated group (Figure 5).
1 (102.9±3.9 sec) fell to (46.2±1.0 sec) on day 4. Rivastigmine,
a cholinesterase inhibitor at a dose of 1.5 mg/kg, also reverse
the scopolamineꢀinduced amnesia in which the latency time of
101.7±5.5 sec on day 1 was reduced to 36.2±3.4 sec on day 4
(Figure 6). The mean and maximum swimming speed of the
animal in the water maze did not differ significantly between
groups (Figure 7). The area under curve of the escape latencies,
which shows the overall comparison was calculated for all
groups in a four days trials, in which 6c (148.3±7.0 sec) and
rivastigmine (170.1±9.1 sec) significantly differ from
scopolamine (246±12.5 sec) treated group (Figure 8).
150
Saline
###
###
Scopalamine
6c (1.9 mg/kg)
6c (3.9 mg/kg)
6c (7.8 mg/kg)
###
***
100
50
0
***
***
***
***
***
140
120
100
80
SALINE
SCP
Fig. 4 Antagonism of scopolamineꢀinduced memory impairment by
peptide 6c at different doses in Morris water maze on latency time to
reach the platform. Values represent mean± SEM of total escape
latencies for daily trials; n = 8ꢀ10,***p< 0.001. 6c (1.9, 3.9 and 7.8
mg/kg equivalent to 5, 10 and 20 ꢀmol/kg) vs Scopolamine;
###p<0.001 scopolamine vs saline, One way ANOVA followed by
Dunnet test).
###
SCP +TRH
SCP + RIVA
SCP + 6c (3.9 mg/kg)
***
***
###
***
###
***
***
***
***
60
***
***
40
20
0
Day 1
Day 2
Day 3
Day 4
400
###
300
200
100
0
***
Fig. 6 Antagonism of scopolamineꢀinduced memory impairment by TRH
and 6c in Morris water maze.Values represent mean ± SEM. *p< 0.05
vs. scopolamine; Values represent mean ±SEM of total escape
latencies for daily trials; n = 8ꢀ10, *p< 0.05, ***p< 0.001, 6c and TRH vs
Scopolamine; ###p< 0.001, #p<0.05 scopolamine vs Saline, One way
ANOVA Followed by Dunnet test.
***
SALINE
SALINE
0.40
0.35
0.30
0.65
0.60
0.55
0.50
0.45
SCP
SCP
TRH (3.6 mg/kg)
TRH (3.6mg/kg)
Riva
6c
Riva
6c
0.25
0.20
Day 1
Day 2
Day 3
Day 4
Day 1
Day 2
Day 3
Day 4
Fig. 5 Area under curve of the escape latencies in four day study of
peptide 6c at different doses in daily trials in Morris water maze. Values
Fig. 7 Antagonism of scopolamineꢀinduced memory impairment by TRH
and 6c in Morris water maze. a) Mean speed of mice during test. b)
Maximum speed of mice during test and values represent mean+ SEM
of average speed and maximum speed for daily trials; n = 8ꢀ10. Values
were analyzed by one way ANOVA Followed by Dunnet test.
represent mean
± SEM. ###p< 0.001 scopolamine vs. Saline
***p<0.001 vs. scopolamine.
b) Effect of peptide 6c on reference memory
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA06935A
(235.1±27.6 sec), was significantly (p<0.001) greater than for
scopolamine treated mice. The peptide 6c (7.8 mg/kg) did not
show (68.3±32.0 sec) any significant increase in retention
latency as compared to scopolamine (Figure 10).
300
250
200
150
100
50
#
SALINE
Scopolamine (1 mg/kg)
TRH (3.6 mg/kg)
*
*
*
6c (3.9 mg/kg)
RIVA (1.5 mg)
Saline
Scopolamine
150
100
50
150
100
50
Trial 1
Trial 2
Trial 1
Trial 2
0
***
**
**
0
0
Scopolamine + 6c
Scopolamine + TRH
150
150
100
50
Trial 1
Trial 2
Trial 1
Trial 2
Fig. 8 Antagonism of scopolamineꢀinduced memory impairment by
TRH and 6c in area under curve of the escape latencies in four day,
100
50
0
*
**
**
*
daily trials and values represent mean
± SEM. *p< 0.05 vs.
**
***
*
scopolamine; Values represent mean± SEM of total escape latencies
for daily trials; n = 8ꢀ10, *p< 0.05, ***p< 0.001, 6c and TRH vs
Scopolamine; ###p< 0.001, #p<0.05 scopolamine vs Saline, One way
ANOVA Followed by Dunnet test.
0
Scopolamine + Riva
Riva + 6c
150
100
50
150
c) Effect of 6c on working memory
Trial 1
Trial 2
Trial 1
Trial 2
100
50
0
*
The Morris water mazeꢀlearning task is used to assess the
hippocampal dependent spatial learning ability. The reductions of
latency time (time to reach the platform) from day to day reflect
learning with respect to reference or longꢀterm memory, which is not
the case with scopolamine. Effect of peptide 6c on working memory
was analyzed by comparing the escape latencies in trial 1 and trial 2,
on each day for four days. Scopolamineꢀtreated amnesic group did
*
***
*
***
***
**
0
Fig. 9 The effect of TRH and 6c on trial 1 and 2 in scopolamine (1
not show any significant difference between two trials. On the other mg/kg) induced amnesia in Morris water maze. Values represent
mean ±SEM. ***p<0.001, **p<0.01, *P<0.05 significantly differ from
values in trial 1.
hand control mice on day 2 and 3 showed significant difference on
day 1, 2 and 3. In the TRH treated mice, the escape latencies
significantly decrease in trial 2 compared to trial 1 on all four days
(Figure 9). Escape latencies in trial 1 and 2 in mice treated with
rivastigmine showed significant effect on day 1, 2 and 3. The escape
latencies in trial 1 and 2 with 6c (3.9 mg/kg) showed significant
effect on day 1, 2 and 4. The treated mice of rivastigmine and 6c
showed significantly reduction in the latency time on day 1, 2, 3 and
4. In this study 6c was found to ameliorate the scopolamineꢀinduced
memory impairment similar to rivastigmine.
Retention trial
Acquisition trial
***
300
250
200
150
100
50
***
***
Passive avoidance task
###
The effect of TRH and 6c on the scopolamineꢀinduced memory
impairment was evaluated using passive avoidance test. In this
task, no significant difference was found in the mean initial trial
latency between control, scopolamine, TRH, 6c (1.8, 3.9 and
7.8 mg/kg) and rivastigmine groups (Figure 10). Scopolamine
treated group showed significant difference in retention trial
(39.9±7.8 sec) as compared to control group (252.4±27.3 sec).
A significant group effect was observed during retention trial
for 6c at 1.8 and 3.9 mg/kg (195.5±37.6 sec) and (148.8±27.9
sec) as compared to scopolamine. The step through latency of
scopolamine treated mice was significantly shorter than that of
saline treated control mice. In rivastigmine (positive control)
plus scopolamine treated mice, step through latency,
0
Fig. 10 Effect of TRH and 6c (1.9, 3.9 and 7.8 mg/kg) on scopolamine
induced memory impairment in passive avoidance task. Memory was
impaired by scopolamine (1 mg/kg). Data represents mean±SEM, n =
8ꢀ13, ***p< 0.001 vs scopolamine, ### p<0.001 vs Saline, by One way
ANOVA followed by Dunnet test.
This journal is © The Royal Society of Chemistry 2012
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ARTICLE
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Table 3 Effect of 6c and TRH on Cardiovascular System
S. No.
Treatment
∆ MAP
(mm Hg)
∆ mean
Systolic
BP (mm Hg)
∆ mean
Diastolic
BP (mm Hg)
∆ Heart
Rate
(BPM)
1
2
3
4
5
6
Saline
2.1±0.8
2.3±1.2
2.4±0.6
37.2±3.2*
2.6±1.1
12.6±2*
2.6±0.5
2.3±0.7
10.5±2
56.6±9*
20±4
Noradrenaline
TRH (3.6 mg/kg)
TRH (7.2 mg/kg)
6c (3.9 mg/kg)
6c (7.8 mg/kg)
42.8±2.2*
3.8±1.4
54.1±1.7*
5.5±2.6
17.8±1.2*
2.6±0.9
19.6±2.4*
3.8±1.4
95±6*
11±2
2.8±0.9
3.0±0.5
26±8
Mice (4ꢀ5) were used to study the effect of TRH and 6c on CVS. Values are expressed as Mean ± SEM, n=4ꢀ5, *p<0.05 compared
with saline (One way ANOVA Followed by Dunnet Test). Basal recordings (Blood Pressure: ꢀ Sys BP 100ꢀ105; Mean BP 82ꢀ85; Dia
BP 74ꢀ78; Heart rate:ꢀ 500ꢀ600 BPM)
Effect of 6c on cerebral blood flow
1a. Spontaneous activity
TRH and 6c were found to improve cerebral blood flow in The spontaneous activity 6c treated mice at all dose levels do
anesthetized mice (n=4ꢀ5). TRH and 6c (3.9 mg/kg) have not significantly change as compared to saline and TRH.
significantly increased the cerebral blood flow (93.4±2.9%) and 1b. Respiration
(42.7±12.6%), respectively as compared with saline It is found that TRH and peptide 6c do not affect normal
(5.7±1.3%), p<0.001 (Figure 11).
respiration after administration. The breathing pattern of control
and treated mice remained normal throughout the study period.
1c. Posture
120
The normal posture was observed in saline, TRH and 6c treated
animals right from the beginning.
***
90
2. Home cage removal and excitability
2a. Excitability
***
60
30
0
Measures of reactivity (excitability) assess the animal’s level of
responsiveness to nonspecific stimuli such as handling or being
placed in an open field. Peptide 6c do not show increase
excitability as compare to saline and TRH of animal to
auditory, visual, olfactory and prick response was noted down.
2b. Other parameters
Saline
TRH
6c
Other parameters such as, salivation and lacrimation was
normal in all groups at all time intervals. Piloerection, ptosis
and exopthalmia were absent.
Fig. 11 Effect of TRH and 6c on cerebral blood flow in anesthetized
mice n = 4ꢀ5 per group. Data represents the mean ±SEM of the %
increase in cerebral blood flow, ***p< 0.001 vs saline. Baseline values
for cerebral blood flow in mice were 70 + 10 Perfusion units
3. Open fields Activity
3a. Spontaneous activity in open field
Effect of 6c on mean arterial blood pressure
The TRH and 6c do not increase spontaneous activity in open
field as compared to saline.
3b. Gait and Posture
Changes in posture and movements reflect alterations in the
central and peripheral nervous systems and as such are sensitive
indicators of neurological dysfunction. Gait descriptions refer
to movements of the limbs as the rat ambulates. Postural
descriptions refer to the placement of the body and spinal
curvature. The gait and posture of the TRH and 6c and vehicle
treated animals was normal till three hours.
TRH has shown dose dependently increase in mean arterial
pressure (MAP) at the dose of 7.2 mg/kg and also significant
(p<0.05) increase in MAP (17.8±1.2) compared with saline
(2.1±0.8) whereas peptide 6c did not cause any change
(2.8±0.9) in cardiovascular parameters at ta dose of 7.8 mg/kg
(Table 3).
Effect of 6c on functional observation battery (FOB)
1. Home cage observations
Home cage observations are made while the animal is
undisturbed in the cage.
3c. Arousal
8 | J. Name., 2012, 00, 1-3
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enhancing activity in the scopolamineꢀinduced memory impairment
mice model similar to rivastigmine.
Arousal or alertness is inferred from watching the animal in the
open field. It can be thought of as the attentiveness or vigilance
of the animal. Observing the locomotion, rearing and sniffing
behaviors of the animal can infer arousal; the animal treated
with TRH and 6c shows moderate arousal. It did not differ with
control animals.
Experimental sections
Material and methods
All Amino acids, and coupling reagents were purchased from
either ChemꢀImpex International or Nova Biochem (Merck
Ltd.). All solvents used for synthesis were of analytical grade.
Sensory responses
Sensory reactivity tests measure the responses to stimuli of
different sensory modalities. In these parameters the
responsiveness. A scale of 1 to 5 was used to determine the
response of animals to these stimuli. The scoring was done as, 1
= no reaction or response; 2 = slight or sluggish reaction (flinch
or startle as evidence of perception); 3 = obvious reaction
(locomotor orientation as evidence of perception); 4 = clear
reaction or response (more intense startle or locomotion); 5 =
exaggerated reaction (may jump, bite, or attack). Mice treated
with 6c showed increase in somatosensory response at all dose
level at some time point. Peptide 6c (1.9 mg/kg) showed
significant increase in somatosensory response. It did not show
that much significant increase in auditory, visual and olfactory
response. Other sensory responses including pinna reflex,
extensor thrust reflex, palpebral reflex, visual placing, surface
righting, aerial righting, tail pinch response and pupil reaction
was present in all groups at all time points.
The peptides were synthesized using the
synthesis protocol.
tꢀBoc solution phase
Synthesis of peptides and purification
General method for the synthesis of
OH (2aꢀe)
NꢀαꢀBocꢀ1ꢀalkylꢀLꢀHisꢀ
N
ꢀαꢀBocꢀ1ꢀalkylꢀ
earlier reported procedure in a single step from
OH (
).32
General method for the synthesis of
ꢀProNH2 (4aꢀe
To a solution of respective
mmol) and ꢀProNH2 (1 mmol) in DMF was added HONB (1.1
L
ꢀhistidines (2aꢀe) were synthesized using a
NꢀαꢀBocꢀHisꢀ
1
N
ꢀαꢀBocꢀLꢀHis(1ꢀalkyl)ꢀ
L
)
Nꢀαꢀ1ꢀalkylꢀLꢀhistidine (2aꢀe, 1
L
mmol) and DIC (1.1 mmol). The resulting mixture was stirred
o
at 4 C for 36 h. After the completion of reaction, the solvent
was removed under reduced pressure, and the residue thus
obtained was purified by flash column chromatography using a
stationary phase of neutral alumina and a mobile phase of 6%
Neuromuscular response
MeOH in CH2Cl2 to afford 4aꢀe 33
General method for the synthesis of R1ꢀ
ProNH2 (6aꢀw
.
This parameter was recorded by measuring landing hind limb
foot splay. The animal was dropped from a height of about 30 cm
thrice. A dye was applied underneath the hind foot, which serves
to mark the distance between the hind limbs when animal lands
over a piece of paper. The stretching limits of the normal control
animals do not differ significantly throughout the study period.
Peptide 6c did not shows any difference in hind limb foot splay
distance as compare to control. Table 4ꢀ5 available in electronic
Supplementary Information (ESI) provides details of FOB.
LꢀHis(1ꢀalkyl)ꢀLꢀ
)
The respective dipeptides (4aꢀe) were dissolved in 40%
o
CF3CO2H in CH2Cl2 (5 mL) and stirred for 30 min at 0 C. The
resulting dipeptide salt (5aꢀe) were neutralized by added a
solution of 7N methanolic ammonia solution (5 mL), and
stirring the mixture for 10 min at ambient temperature. The
solvent was evaporated under reduced pressure to afford free
dipeptide, which was used in the next coupling step without
isolation. The free dipeptide (1 mmol) was dissolved in DMF (5
Conclusion
A new class of TRHꢀlike peptides has been synthesized by the
solution phase peptide synthesis protocol. We have observed
interesting receptor binding and signaling assay results for some of mL) and cooled to 4
°C. The requisite hetero ringꢀcontaining
the tested peptide. For example, peptides 6aꢀc 6e, 6g, 6m and 6s
have displayed high selectivity towards TRHꢀR2 in the FLIPR assay.
In the pentobarbitalꢀinduced sleeping time model, twentyꢀfour
peptides were investigated for their CNS activity. One of the
promising TRHꢀlike peptide 6c was selected for further
pharmacological evaluation. The ClogP calculation of 6c predicts
more lipid solubility for the peptide, when compare to TRH. The
plasma stability studies of 6c confirmed its enhanced stability in
plasma. Since previous research indicates that an Nꢀterminus pGlu
appears to be essential for the catalytic activity of TRHꢀDE and
Ceredist is not metabolized by TRHꢀDE, thus 6c is unlikely to be
metabolized by this enzyme. Peptide 6c at the doses of 1.9 mg/kg
and 3.9 mg/kg was found effective in scopolamineꢀinduced memory
impairment model, using Morris water maze and Passive avoidance
test. It is pertinent to mention that it can also be speculated that
peptide 6c exhibits CNS activity by binding to a novel TRH receptor
subtype, and is the topic of further investigations. Peptide 6c was
also tested for cardiovascular activity and was found to be safe in the
absence of any effect on the CVS. The peptide 6c was also found to
have CNS safety profile, as it does not show any abnormal behavior
in Functional Observation Battery (FOB). This study suggests that
6c is a CNS active TRHꢀlike peptide that possesses cognitiveꢀ
carboxylic acid (1 mmol), DIC (1.1 mmol) and 1ꢀ
hydroxybenzotriazole HOBt (1.1 mmol) was added and the
resulting mixture stirred at 4 oC for 36 h. The solvent was
removed under reduced pressure and residue was purified by
flash column chromatography using a stationary phase of
neutral alumina and a mobile phase of 0ꢀ7% CH3OH in CH2Cl2
to afford 6aꢀw
.
In vitro molecular pharmacolgy
Material and methods
Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum were purchased from Biosource (Rockville, MD,
USA). TRH (pGluꢀHisꢀProNH2) and MeTRH [pGluꢀHis(1ꢀ(τ)ꢀ
methyl)ꢀProNH2] were purchased from Sigma (St. Louis, MO,
USA). [3H]MeTRH (0.25 mCi) was purchased from Perkin
Elmer (Waltham, MA, USA).
Cell culture and transfection
The generation of HEKꢀEM 293 (human embryonic kidney)
cells stably expressing murine TRHꢀR1 or TRHꢀR2 was
described earlier by Engel et al.24 HEKꢀEM cells stably
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expressing all TRH receptors were grown in DMEM containing Sunnyvale, CA). The agonistic responses of ligands were
10% fetal bovine serum, 100 units/mL penicillin, 10 mg/ml assessed immediately upon their addition in a concentration
streptomycin and 200 mg/mL hygromycin B (Invitrogen, range from 0.0001 ꢁM to 30 ꢁM. Responses were measured as
Carlsbad, CA) at 37
Receptor binding assay
°
C in a humidified 5% CO2 incubator.
peak fluorescent intensity minus basal fluorescent intensity at
each peptide concentration and are presented as % of the
maximum response. Data are reported as EC50 (µM) values.
In vivo reversal of pentobarbitalꢀinduced sleeping time
assay
All peptides were examined for both their affinity for mTRHꢀ
R1 and mTRHꢀR2 and their ability to serve as agonists and
their selectivity for the receptors. Twentyꢀfour hour before the
experiment (human embryonic kidney cells 293) HEK293 cells
stably expressing mTRHꢀR1 or mTRHꢀR2, were plated in
appropriate cell culture plate 24 well plates. 300,000 Cells per
well were seeded and incubated overnight at 37 °C. The
following day, the cell monolayer was washed 3 times and the
plate and buffer were placed on ice for at least 45 min prior the
Animals
Male Swiss albino mice (20–30 g) were procured from the
Central Animal Facility of the Institute. Animals were fed a
normal pellet diet (Ashirwad Industries, Chandigarh, India) and
purified water ad libitum and maintained at 22 ± 2 °C and 50 ±
5% relative humidity with a 12ꢀhour dark/12ꢀhour light cycle
(lights on 06:00ꢀ18:00 hours). The Institutional Animal Ethics
Committee NIPER approved all animal experimental protocols
and experiments were conducted in accordance with the
guidelines of CPCSEA, Government of India, for animal care
and experimentation.
o
experiment. The plates were kept at 0ꢀ4 C and washed once
with 1 mL/well of ice cold Hank's Balanced Salt Solution
(HBSS)
and
with
10
mM
4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀ
piperazineethanesulfonic acid (HEPES), without disturbing the
cell monolayer. The compounds were added and mixed with
[3H]MeTRH (100 µL), TRH analogues (250 µL) at 4 ºC before
adding in plate (cold), all threeꢀfold dilution in a single well
totaling 350 µL (in duplicate). The plates were kept in the cold
room in an ice tray up to 4 h and then the supernatant was
aspirated and cells were carefully washed three times by adding
1 mL ice cold buffer per well (without disturbing monolayer of
cells). Later, 0.4 N NaOH solution (1 mL) was added to it, and
shaken for 30 min on the shaking platform for cell lysis. In
small scintillation vial, 4 mL of scintillation fluids was taken
and dispensed with 0.7 mL of lysate. It was shaken using a
vortex mixer for 5 sec, and placed in the scintillation counter.
Affinities, reported as IC50 (µM), were determined as described
earlier; briefly by measuring the concentration of the peptides
required to displace 50% of 4nM [3H]ꢀ1ꢀMeTRH from the
mTRH receptor. 31
Fluorescent imaging plate reader (FLIPR) (Ca+2) assay
HEK293 cells stably expressing mTRHꢀR1 or mTRHꢀR2 were
seeded in blackꢀwalled, clearꢀbottomed 96ꢀwell plates
(Corning, NY) at a density of 6×104 cells/well in DMEM (10%
FBS, 1% penicillinꢀstreptomycin, 0.1% hygromycin) media and
incubated for 24 h at 37 oC and 6% CO2. On the following day,
the culture media was replaced with 100 ꢁL of HBSS
supplemented with 20 mM HEPES, pH 7.5 and the cells were
loaded with 100 ꢁL of calcium 3 fluorescent dye (Molecular
Devices, Sunnyvale, CA) for 1 h at room temperature before
addition of compounds. Transient changes in intracellular
(Ca2+) induced by ligands were measured. Changes in
fluorescence were detected at the emission wavelength of 515 –
575 nm using FLIPR calcium assay kit according to the
manufacturer’s recommendation.34 This kit includes a calcium
sensitive dye that is taken into the cytoplasm of the cell during
incubation. The kit masking technology remains outside the cell
and blocks background fluorescence. Upon ligand binding to
the receptor, calcium released into the cytoplasm of the cell, the
dye binds to the intracellular calcium and becomes fluorescent;
fluorescence is measured by the FLIPRTETRA® high
throughput cellular screening system (Molecular Devices,
Drugs and Solutions preparation
Pentobarbital sodium was procured from Sigma, (USA),
scopolamine hydrobromide from Tocris Biosciences (USA) and
rivastigmine was a gift sample from Dr. Reddy’s Lab, India.
All compounds were dissolved in saline.
Analeptic activity
Analeptic activity of TRHꢀlike peptides was studied using the
antagonism of the pentobarbital (PB)ꢀinduced sleeping time
method in albino Swiss mice as described elsewhere as it is a
well known model to study the effectiveness of TRH and
analogues on central nervous system.35ꢀ38 Vehicle (5 mL/kg),
TRH or TRH likeꢀpeptides (10 ꢁmol/kg equivalent to 3.6
mg/kg of TRH) were injected into mice (n = 8–10/group) via
the tail vein. Ten minutes after injection, each mouse received
PB (50 mg/kg, i.p.). Sleep duration was recorded as the time
elapsed from the onset of loss to regain of the righting reflex.
Based on this study, the peptide, which showed the maximum
analeptic effect, was selected for further studies. (Tableꢀ2).
Analeptic activity = (1ꢀ S/C) x 100
(S = Sleeping time in compound treated, C= Sleeping time in
Vehicle treated group)
Stability assay (exꢀvivo in plasma)
Animals were sacrificed and truncated blood was collected in
freeze heparin containing centrifuge tube, immediately
centrifuge at 10000 rpm for 5 min for plasma. Then 1 mL of
plasma was incubated with 500 nmol of TRH or analogue and
kept at 37 oC in water bath, 50 ꢁL aliquots were taken at
different intervals i.e. at 0, 15, 30, 60, 90 and 180 min and
immediately mixed with 200 ꢁL of acetonitrile and centrifuged
at 10000 rpm for 5 min. Supernatant were collected and run in
gradient mobile phase containing 0.1% TFA in water (A) and
0.08% TFA in acetonitrile (B). Gradient flow containing A and
B in ratio of 98:2 for 3 min, reduced to 50:50 in 12 min, further
increased to 98:2 in 14 min and remained same for 20 min for
6c and for TRH 99:1 for 4 min, reduced to 90:10 in 10 min,
further reduced to 50: 50 in 16 min, then increased to 99:1 in 20
min and remained same for 24 min, was used. Samples were
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DOI: 10.1039/C5RA06935A
injected using Reno dyne manual injector at the volume of 20 was kept same in between the entire trials. In the water maze
ꢁL. Compounds were analyzed using UVꢀVIS detector (Waters experiments, the day prior to the experiment was dedicated to
2489) at the 210 nm and TRH at 254 nm.
Passive avoidance test
swim training for 60 sec, in the absence of the platform. In the
days following, the mice were given two trial sessions each day
Training and testing of passive avoidance performance were for four consecutive days. Mice climb this platform to escape
carried out with an apparatus (Ugo Basile, Typeꢀ7551, Italy) the necessity of swimming. On four consecutive days, mice
consisted of a rectangular box (52cm x 30cm x 35cm) divided were given two acquisition trials per day with an interꢀtrial
into two compartments, a light chamber illuminated with a 40 interval of 20 min. Mice were allowed to locate the platform for
W bulb and a dark chamber, separated by an automatically a maximum of 120 sec and those fail will be drifted towards it
operated sliding door. Amnesia was induced by scopolamine (1 and then permitted to stay on it for a maximum of 10 sec.
mg/kg, ip). TRH and analogues were given 20 min after Scopolamine (1 mg/kg, i.p.) was given 30 min prior to test and
scopolamine treatment.39 Mice were initially placed in the light TRH and test analogue just 10 min prior the test. Rivastigmine
chamber and 10 sec later the door between compartments was was given 5 min after scopolamine treatment.42ꢀ45 During the
opened. When mice entered the dark compartment, the door experiments, the pool was videotaped and scores for the escape
automatically closed and an electrical foot shock 0.5 mA lasting latency (time taken to find the hidden platform), mean
for 5 sec was delivered through the stainless steel rods swimming speed and the maximum swimming speed were
(Acquisition trial). 8ꢀ10 Mice were used per treatment. The recorded by AnyꢀMaze software (Stoelting, USA).
time taken by the mice to step into the dark compartment is Functional Observational Battery
recorded as initial trial latency (ITL). Mice, which were failed Functional Observational Battery is
a systematic study
to enter the dark compartment in 90 sec, are not considered for performed to determine the effect of any disease or drug
further experiments. After twentyꢀfour hours, retention trial is treatment on different functions of central nervous
performed and latency to step into the dark compartment is system/functions. This consists of measurement of various
recorded as retention trial latency (RTL) to a maximum of 300 behavioral parameters in the home cage, hand held and open
sec.
field cages.40 The scoring patterns are a modified form of
Irwin’s scoring.40 Various functional parameters, which were
Cerebral Blood Flow
Mice (n=4ꢀ5/group) were anaesthetized with urethane (1.5 g/kg, studied, result are represent into table 4ꢀ5.
i.p.). Jugular vein was cannulated for administration of saline, Statistical analysis
TRH and 6c (3.9 mg/kg). Cerebral blood flow (CBF) was All the values are expressed as mean±S.E.M. Statistical
measured using a laserꢀDoppler blood flow meter (Periflux analysis was performed using Sigma Stat 2.0 statistical
4001, Perimed, Sweden). Laser Doppler probe was positioned software. Statistical significance for multi group was assessed
(1 mm posterior and 2 mm lateral to bregma), without by using oneꢀway ANOVA followed by Dunnet test. Median
damaging duramater. CBF was calculated as averaged values in was determined in FOB and KruskalꢀWallis One Way Analysis
perfusion units (PU) 10 min before and after TRH and 6c of Variance on Ranks followed by Dunnet test assessed
administration. Baseline values for cerebral blood flow in mice statistical significance. P<0.05 is considered as statistically
were 70±10 Perfusion units. Basal value was taken as 100 % significant.
and results are presented as % changes from the basal value.40ꢀ
41
ABBREVIATIONS
TRH
pGlu
Thyrotropin releasing hormone
Pyroglutamic acid
Cardiovascular parameters
Mean arterial blood pressure (MABP) was recorded using the
Power Lab System (AD Instruments, Australia). Briefly, mice
(n = 4 or 5/group) were anesthetized with urethane (1.5 g/kg,
i.p.), TRH and 6c, and the carotid artery was cannulated for the
recording of blood pressure. The effect of TRH and 6c (3.9
mg/kg) on MABP was recorded.
His
Histidine
ProNH2 Prolinamide
HOBt
DIC
1ꢀHydroxybenzotriazole
N,N’ꢀDiisopropylcarbodiimide
HPLC
Highꢀpressure liquid chromatography
HONB N-Hydroxyꢀ5ꢀnorborneneꢀ2,3ꢀdicarboximide
Behavioral experiments
IC50
Inhibitory concentration that affords 50% of competitive
inhibition binding
Scopolamineꢀinduced memory impairment was studied with the
Passive avoidance and Morris water maze test in which studies
were conducted with five groups, namely, control (saline),
EC50
Therapeutic effective concentration that affords 50% response
HEK293 Human embryonic kidney 293 cells
positive control (rivastigmine), scopolamine, TRH and 6c
.
TRHꢀRs Thyrotropin releasing hormone receptors
Morris water maze (cued test version)
FLIPR
IPꢀ1
Fluorescent imaging plate reader
Inositolꢀ1ꢀphosphate
A spatial memory test was performed by the method of Morris
with minor modifications. Water maze consists of a large
circular pool (80 cm in diameter, 30 cm in height and 25 cm
deep, filled with water) painted black from inside and a
submerged platform, painted black, was placed inside this pool
1 cm below the surface of water. The position of the platform
HPT
TSH
CBF
PKC
Hypothalamicꢀpituitaryꢀthyroid
Thyrotropinꢀstimulating hormone
Cerebral Blood Flow
Protein kinase C
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DOI: 10.1039/C5RA06935A
Biochemistry 1997, 36, 15670; (
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b) R. Jain, J. Singh, J. H.
GPCR
Gꢀprotein coupled receptor
BLAST Basic Local Alignment Search Tool
Erap1 Endoplasmic reticulum aminopeptidase 1
c
d
Acknowledgements
e
* Corresponding author. Tel. +91 (172) 2292024; Fax: +91 (172)
2214692; Email: rahuljain@niper.ac.in
6
f
Dr. Chhuttan L. Meena acknowledges the support provided by Dr.
Marvin C. Gershengorn NIDDK, NIH, USA for this study. This study
was supported by Rajiv Gandhi National Fellowship Award (RGNF)
2006, University grant commission (UGC), New Delhi, India and
Fulbright Nehru Doctrol and Professoinal Research Fellowship (FNꢀ
DPR), United State India Education Foundation (USIEF,
1493/DPR/2011ꢀ2012).
aDepartment of Medicinal Chemistry, National Institute of
Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, 160
062, Punjab, India
,
g
h
Nandekar, B. Wahlang, N. Shaikh, Y. B. Pawar, C. L. Meena, A.
T. Sangamwar, R. Jain, K. B. Tikoo, and A. K. Bansal, Mol.
Pharmaceutics 2012, 9, 2458; (i) P. Bagul, K. S. Khomane, S. S.
Kesharwani, P. Pragyan, P. P. Nandekar, C. L. Meena, A. K.
Bansal, R. Jain, K. B. Tikoo, and A. T. Sangamwar, J. Mol.
Recognit. 2014, 27, 609.
bDepartment of Pharmacology and Toxicology, National Institute of
Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, 160
062, Punjab, India
15.
16.
17.
K. S. Khomane, C. L. Meena, R. Jain and A. K. Bansal,
Expert Opin. Ther. Pat., 2011, 21, 1673.
J. A. Kelly, G. R. Slator, K. F. Tipton, C. H. Williams and
K. Bauer, J. Biol. Chem., 2000, 275, 16746.
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M. Fitchett, S. M. Draper, G. W Bennett, P.M. Hinkle, K.
Bauer, C. H. Williams, K. F. Tipton, and J. A. Kelly,
cDepartment of
Pharmacoinformatics, National Institute
of
Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, 160
062, Punjab, India
Electronic Supplementary Information (ESI) available: Complete spectral
1
data, H, 13C, HPLC of all peptides and tables depicting effect of 6c on
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