Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 1961-1969 © The Author(s) 2024. This article is an open access publication.

1961

Low-Molecular Neurotrophin-3 Mimetics with Different

Patterns of Postreceptor Signaling Activation

Attenuate Differentially Morphine Withdrawal in Rats

Larisa G. Kolik

1,a

*, Mark A. Konstantinipolsky

, Sergey V. Nikolaev

Ilya O. Logvinov

, Tatyana A. Antipova

, and Tatiana A. Gudasheva

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies,

125315 Moscow, Russia

e-mail: kolik_lg@academpharm.ru

Received June 4, 2024

Revised July 17, 2024

Accepted July 18, 2024

Abstract— The accumulated evidence suggests that varying levels of tyrosine kinase receptor signaling path-

way activity may regulate opiate-associated neuroadaptation of noradrenergic system. Neurotrophin-3 (NT-3)

interacts with tropomyosin receptor kinases (TRKs), binding mainly to TRKC receptors, which are expressed

within noradrenergic neurons in the blue spot (locus coeruleus, LC). Considering the difficulties in delivering

full-length neurotrophins to the CNS after systemic administration, low-molecular mimetics of loop4 in NT-3,

hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), and hexamethylenediamide

bis-(N-γ-oxybutyryl-L-glutamyl-L-asparagine) (GTS-302), activating TRKC and TRKB receptors, were synthesized.

The aim of the study is comparative examination of the effects of NT-3 dipeptide mimetics on the signs of

morphine withdrawal in outbred white rats with opiate dependence, as well as investigation of activation of

postreceptor signaling pathways by the mimetics. Dipeptides GTS-301 and GTS-302 after acute administration

at doses of 0.1, 1.0, and 10.0mg/kg (i.p., intraperitoneal) had a dose-dependent effect on the specific morphine

withdrawal symptoms with the most effective dose being 1.0  mg/kg. Maximum decrease in the total index of

morphine withdrawal syndrome for GTS-301 was 31.3% and for GTS-302 – 41.4%. Unlike GTS-301, GTS-302

weakened mechanical allodynia induced by morphine withdrawal, reducing tactile sensitivity. When studying

activation of the postreceptor signaling pathways by the NT-3 mimetics in the HT-22 hippocampal cell culture,

a different pattern of postreceptor signaling was shown: GTS-302 (10

−6

  M), similar to NT-3, activates all three

MAPK/ERK, PI3K/AKT/mTOR, and PLCγ1 pathways, while GTS-301 (10

−6

  M) triggers only MAPK/ERK and PLCγ1

pathways. Thus, the identified features of attenuation of the morphine withdrawal syndrome in the rats under

GTS-301 and GTS-302 effects could be associated with different activation pattern of the postreceptor pathways.

DOI: 10.1134/S0006297924110105

Keywords: neurotrophin-3 mimetics, withdrawal syndrome, morphine, rats, MAPK/ERK, PI3K/AKT, PLCγ1, HT-22

Abbreviations: AKT, protein kinase B; ERKs, extracellular signal-regulated kinases; LC (Lat. locus coeruleus), blue spot;

MAPK, mitogen-activated protein kinase; mTOR, rapamycin complex 1; NT-3, neurotrophin-3; PI3K, phosphatidylinositol

3-kinase; PLC, phospholipase C; TRKA, TRKB, TRKC, tropomyosin receptor protein kinases of A, B, and C types, respec-

tively; WS, withdrawal syndrome.

* To whom correspondence should be addressed.

INTRODUCTION

Drug dependence is often associated with pain-

ful withdrawal symptoms experienced by the patients

when they stop abruptly or reduce rapidly the dose of

analgesics. Somatic component of drug dependence is

assumed to be associated to a greater degree with meso-

limbic areas of the brain, blue spot (locus coeruleus,LC)

KOLIK et al.1962

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

and periaqueductal gray area [1, 2]. Hyperactivity and

disinhibition of brainstem noradrenergic neurons are

considered to be the key mechanisms of aversive state

and many somatic symptoms that occur during the

acute opioid withdrawal [3].

Neurotrophin-3 (NT-3) belongs to the family of

neurotrophins and interacts with the tropomyosin-

associated protein kinases of A, B, and C types (TRKA,

TRKB, and TRKC, respectively), binding preferential-

ly to the TRKC receptors, which are expressed in the

noradrenergic LC neurons [4]. It is known that LC ex-

presses high levels of NT-3 and TRKC [5, 6], and NT-3

also increases survival of the LC neurons in  vitro [7].

Involvement of the TRKC signaling pathways in the

opiate-induced adaptation of the catecholaminergic

system has been shown in in  vitro  [7] and in  vivo ex-

periments  [8]. Transgenic mice overexpressing TRKC

receptors (TgNTRK3) showed an altered frequency of

spontaneous excitation of the LC neurons and of the

noradrenergic system response to chronic exposure to

opiates, which is probably associated with the shifts in

regulation of neurotrophins [9]. The accumulated evi-

dence suggests that changing combinations and activi-

ty levels of the TRK receptor signaling pathways in the

neuronal circuits, which are interconnected with nor-

adrenergic neurons, could regulate and fine-tune the

opiate-induced adaptations of noradrenergic system.

Considering the difficulties in delivering full-length

neurotrophins to the CNS during systemic adminis-

tration, the Federal Research Center for Innovator

and Emerging Biomedical and Pharmaceutical Tech-

nologies of the Federal State Budgetary Scientific In-

stitution is developing small-molecule NT-3 mimetics,

which were designed using the proprietary technology

for constructing dipeptide neurotrophin mimetics [10].

The most exposed part in the NT-3 structure is loop4,

a fragment of which (-Ser91-Glu92-Asn93-Asn94-Lys95-

Leu96-), presumably occupies geometrically the most

advantageous position for interaction with the recep-

tor. When constructing the GTS-301 mimetic, the di-

peptide fragment (-Asn93-Asn94-) of the beta-turn-like

region was retained and the preceding Glu92 amino

acid residue was replaced with the succinic acid res-

idue. Dimeric structure of neurotrophin was repro-

duced using the hexamethylenediamine spacer at the

C-terminus (hexamethylenediamide bis-(N-monosuc-

cinyl-L-asparaginyl-L-asparagine) [11]. Another NT-3

mimetic, GTS-302, was constructed from the same

fragment of the loop  4, but possessed one amino acid

residue shifted to the left relative to (-Asn93-Asn94-),

with the dipeptide region (-Glu92-Asn93-) retained

and the preceding Ser91 amino acid residue replaced

with gamma-hydroxybutyric acid; dimerization was

also carried out with the hexamethylenediamine

(hexamethylenediamide bis-(N-γ-oxybutyryl-L-glutam-

yl-L-asparagine)) spacer  [12]. The aim of the study is

comparative examination of the effects of NT-3 loop  4

mimetic on morphine withdrawal symptoms in the

animals with formed opiate dependence, as well as

investigation of activation of the postreceptor signal-

ing pathways by mimetics.

MATERIALS AND METHODS

In  vivo experiments. The experiments were per-

formed with outbred white male rats weighing 230-

250  g (n  =  112) (FSBSI, Scientific Center of Biomedical

Technologies of the Federal Medical Biological Agency,

Stolbovaya Branch). Animals were kept in groups of 8

individuals in a cage in the vivarium of the Federal

Research Center for Innovator and Emerging Biomed-

ical and Pharmaceutical Technologies (FSBSI) at tem-

perature of 21-23°C and relative humidity of 40-60%

with natural illumination and free access to water and

briquetted feed for 10 days before testing.

Drugs. Morphine hydrochloride (Minmedbio-

prom, Chimkentbiopharm, substance) was dissolved

in distilled water for injection and administered in-

traperitoneally (i.p.) at the rate of 0.1  ml/100  g of rat

body weight. GTS-301 [melting point was 214-229°C

(with decomposition); [α]

  –  20.2° (c  =  1, DMSO)] and

GTS-302 [melting point was 173-178°C (with decom-

position); [α]

  –  7.76° (c  =  1, DMSO)]  [11] were syn-

thesized in the chemistry department of the Federal

Research Center of Original and Promising Biomedical

and Pharmaceutical Technologies of the Federal State

Budgetary Scientific Institution and administered i.p.

as a suspension in 1% aqueous Tween  80 at a rate of

0.1  ml/100  g of animal weight.

Method of opiate dependence formation and

assessment of somatic symptoms of morphine with-

drawal syndrome. Morphine dependence was induced

in rats and behavior was evaluated for specific signs

of withdrawal syndrome(WS) according to the scheme

described earlier [13]. To obtain morphine-dependent

animals, the drug was administered to animals in in-

creasing doses (10-20  mg/kg) 2 times a day with an

interval of 8  h for 5 days: 1  day – 10 and 10  mg/kg;

2nd  day– 10 and 20  mg/kg; 3rd  day– 20 and 20  mg/kg;

4th  day– 20 and 20  mg/kg; 5th  day– 20  mg/kg. On the

5th day of the experiment, 5  h after the last morphine

injection, GTS-301 and GTS-302 were administered at

doses of 0.1, 1.0, and 10.0  mg/kg (i.p.), and control ani-

mals (groups 0.0) were injected with water containing

1%  (v/v) Tween  80 in an equivalent volume 60  min be-

fore testing. Animals were tested for specific signs of

morphine WS for 5  min in an open field (illuminated

round arena with a diameter of 80  cm) 15  min after

administration of naloxone, opiate receptor antagonist

(Du Pont De Nemours Int. Switzerland) at a dose of

1.0  mg/kg (i.p.). Specific symptoms of morphine WS

NT-3 MIMETICS ATTENUATE MORPHINE WITHDRAWAL 1963

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Table 1. Specific symptoms of morphine withdrawal

syndrome in rats

Withdrawal

signs

Assessment

Withdrawal

signs

Assessment

Diarrhea points 1-5 spasms +/–

Teeth

chattering

number

of episodes

stereotypy +/–

“Wet-dog”

shakes

number

of episodes

vocalization +/–

Escape

attempts

+/– chewing +/–

Urination +/– nosebleed +/–

Postural

disturbance

+/– rhinorrhea +/–

Piloerection +/– head shakes +/–

Dyspnea +/– paw shakes +/–

Ptosis +/– convulsions +/–

Note. The mean level of WS severity in the control group

(group 0.0) was taken as 100%.

were recorded for all groups. Total index (TI) of the

WS severity for each animal and average values for

the experimental and control groups were calculat-

ed based on alternative symptoms (sign presence –

1  point, sign absence– 0  points), a total of 18WS signs

were recorded, each animal had its own set of signs

(Table 1).

Changes in tactile sensitivity in rats were as-

sessed using a standard set of 20 nylon monofilaments

(von Frey filaments set; Ugo Basile, Italy). During test-

ing, a single filament was applied to the surface of

the hind paws of the rat for 1-2 seconds through the

mesh surface of the platform, withdrawal or displace-

ment of a paw was considered as a positive reaction.

Threshold level of the tactile stimulus was determined

by its lowest response value in rats, according to the

gram-based scale indicated on each filament. During

the study, the testing procedure was carried out twice:

on the day preceding the first day of morphine ad-

ministration and 24  h after morphine withdrawal [13].

In  vitro experiments. Cell cultivation. Experiments

were carried out with HT-22 mouse hippocampus cells

(Cell Bank of Utrecht University, Holland). All exper-

iments were performed under strictly sterile condi-

tions. Cells were cultured at 37°C under 5%  CO

in a

DMEM medium (Dulbecco’s modified Eagle’s medium;

HyClone, USA)  [14] containing 5%  FBS (fetal bovine

serum; Gibco, USA) and 2  mM L-glutamine (ICN Phar-

maceuticals, USA). The culture medium was changed

24  h after seeding and every 2-3 days after that. Re-

seeding into 75-cm

culture flasks (TPP, Switzerland)

was performed 3 times a week.

Western blot analysis. Samples were lysed in

a 50  mM Tris-HCl (pH  7.5) buffer containing 5  mM

EDTA, 1  mM dithiothreitol, 1%  (w/v) Triton X-100 at 5,

15, 30, 60, and 180min after adding NT-3 (100ng/mL)

(Sigma, USA), or GTS-301 (L,L) (10

−6

  M), or GTS-302

(L,L) (10

−6

  M). NT-3 (100  ng/mL) was used as a positive

control. Non-phosphorylated protein kinase B (AKT),

extracellular signal-regulated kinases (ERK1/ERK2),

and phospholipase  C (PLCγ1) were used as loading

controls. Protein level was examined in the cytoso-

lic fraction. Protein concentration in the samples was

measured using Lowry method  [15]. 75  μg of protein

was loaded into run in the gel. Proteins were sepa-

rated using electrophoresis in a 10% polyacrylamide

gel with a Mini-Protean Tetra Cell electrophoretic sys-

tem (Bio-Rad, USA)  [16]. The proteins were then trans-

ferred to a PVDF membrane (Santa Cruz, USA) in a

Trans-Blot Turbo transfer system (Bio-Rad). All West-

ern blots were preincubated in TBS-T buffer (20  mM

Tris-HCl, 137  mM NaCl, 1%  (v/v) Tween-20; pH  =  7.5)

containing 3%  (w/v) BSA (Bio-Rad) for 1  h using pri-

mary anti-p-AKT1/2/3 antibodies (#  PA5-104445; Invi-

trogen, Thermo Fisher Scientific, USA), anti-p-ERK1/2

(#  PA5-37828; Invitrogen, Thermo Fisher Scientific),

anti-p-PLCγ1 (#2821S; Cell Signaling Technology, USA)

against AKT (#  PA5-77855; Invitrogen, Thermo Fisher

Scientific), ERK1/ERK2 (#61-7400; Invitrogen, Thermo

Fisher Scientific), and PLCγ1 (#2822S; Cell Signaling

Technology) at a dilution of 1  :  1000 overnight at 4°C.

Next, after washing in a TBS-T buffer containing

0.05%  (w/v) BSA, the membranes were incubated in

the presence of secondary goat anti-rabbit IgG anti-

bodies (#  31460; Thermo Fisher Scientific) conjugat-

ed with horseradish peroxidase (at 1  :  1000 dilution)

for 1  h. Detection of proteins was performed after

washing from secondary antibodies in a buffer with

ECL reagents (#  32106; Pierce ECL WB Substrate (Ther-

mo Fisher Scientific)) using Alliance 4.7 gel documen-

tation system (UVITEC, UK). Densitometry of the imag-

es was performed using the GIMP2 software.

Statistical analysis of the results of in  vivo exper-

iments was performed by descriptive statistics, one-

way analysis of variance (ANOVA), repeated measures

ANOVA for related groups; the results of in  vitro ex-

periments were analyzed using Mann–Whitney U test

and Statistica 10 software.

RESULTS

The obtained results show that GTS-301 and

GTS-302 (Fig. 1) exhibit a dose-dependent effect on

the specific signs of morphine withdrawal in rats.

KOLIK et al.1964

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Table 2. Effect of GTS-301 on some symptoms of morphine withdrawal

Behavioral signs

GTS-301, doses, mg/kg

0.0 0.1 1.0 10.0

Diarrhea 4.13 ± 0.30 2.50 ± 0.60* 2.50 ± 0.70* 1.87 ± 0.77*

Postural disturbance 0.87 ± 0.00 1.00 ± 0.00 0.75 ± 0.16 0.38 ± 0.18*

Ptosis 0.38 ± 0.18 0.00 ± 0.00* 0.25 ± 0.16 0.25 ± 0.16

Teeth chattering 1.13 ± 0.78 1.75 ± 0.88 0.13 ± 0.12* 0.50 ± 0.50

“Wet-dog” shakes 2.50 ± 1.12 1.00 ± 0.42 1.00 ± 0.53 0.75 ± 0.52

Vocalization 0.50 ± 0.19 0.62 ± 0.18 0.13 ± 0.12* 0.75 ± 0.16

Head shakes 0.50 ± 0.19 0.38 ± 0.18 0.25 ± 0.16 0.25 ± 0.16

Paw shakes 0.75 ± 0.16 0.25 ± 0.16* 0.38 ± 0.18 0.13 ± 0.13**

Rhinorrhea 1.00 ± 0.00 0.88 ± 0.12 0.38 ± 0.18** 0.38 ± 0.18**

Note. *  p  <  0.05; **  p  <  0.01; ***  p  <  0.001 – significant difference from the control (group “0.0”); each group consists of 8 an-

imals; the data are presented as a mean ± standard error of the mean (M ± SEM).

Table 3. Effect of GTS-302 on some symptoms of morphine withdrawal

Behavioral signs

GTS-302, doses, mg/kg

0.0 0.1 1.0 10.0

Diarrhea 3.10 ± 0.71 3.9 ± 0.22 1.60 ± 0.70 1.50 ± 0.59

Postural disturbance 1.00 ± 0.00 0.25 ± 0.16*** 0.25 ± 0.16*** 0.38 ± 0.18**

Ptosis 0.38 ± 0.18 0.37 ± 0.18 0.25 ± 0.16 0.13 ± 0.11

Teeth chattering 1.88 ± 0.81 0.25 ± 0.25* 0.50 ± 0.50 0.13 ± 0.13*

“Wet-dog” shakes 0.70 ± 0.15 0.25 ± 0.25 0.0 ± 0.0* 0.0 ± 0.0*

Vocalization 0.65 ± 0.17 0.63 ± 0.18 0.13 ± 0.11* 0.49 ± 0.17

Head shakes 0.75 ± 0.16 0.61 ± 0.18 0.13 ± 0.13** 0.50 ± 0.19

Paw shakes 0.75 ± 0.16 0.25 ± 0.16* 0.38 ± 0.18 0.25 ± 0.16*

Rhinorrhea 0.62 ± 0.18 0.38 ± 0.18 0.50 ± 0.19 0.88 ± 0.12

Note. *  p  <  0.05; **  p  <  0.01; ***  p  <  0.001 – significant difference from the control (group “0.0”); each group consists of 8 an-

imals; the data are presented as a mean ± standard error of the mean (M ± SEM).

Decreases in the WS total index (TI) in the group

“GTS-301; 0.1  mg/kg” was 12% (F

(1.14)

  =  4.66; p  <  0.05);

in the group “GTS-301; 1.0  mg/kg” it amounted to

31.1% (F

(1.14)

  =  9.14; p  <  0.01); in the group “GTS-301;

10.0  mg/kg” – 26.5% (F

(1.14)

  =  21.44; p  <  0.001), com-

pared to the 0.0 control group. WS TI drop in the

group “GTS-302; 0.1  mg/kg” was 26.9% (F

(1.14)

  =  10.19;

p  <  0.01); in the group “GTS-302; 1.0  mg/kg” it

amounted to 41.4% (F

(1.14)

  =  12.49; p  <  0.01), in the

group “GTS-302; 10.0  mg/kg” – 36.5% (F

(1.14)

  =  25.64;

p  <  0.001). The most pronounced effect of NT-3 mi-

metics was demonstrated at the dose of 1.0  mg/kg.

When evaluating correction of the individual signs

of morphine WS, it is necessary to note that reduc-

tion in the diarrhea symptoms, as well as elimina-

tion of the postural disturbance, ptosis, paw shaking,

NT-3 MIMETICS ATTENUATE MORPHINE WITHDRAWAL 1965

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig. 1. Effect of the NT-3 loop 4 mimetics on the total index

of naloxone-induced morphine withdrawal syndrome in the

outbred male rats (as % of control). * p < 0.05; ** p < 0.01;

***p<0.001– significant difference from the control (group

“0.0”); each group consists of 8 animals; the data are pre-

sented as a mean ± standard error of the mean (M ± SEM).

vocalization, and rhinorrhea were observed with

GTS-301 administered at all doses examined (Table2).

For GTS-302, decrease in the severity or complete

elimination of the signs, such as diarrhea, postural

disturbance, teeth chattering, “wet-dog” shakes, vocal-

ization, head, and paw shakes was recorded (Table3).

In total, GTS-301 and GTS-302 attenuated 7 of the

18 analyzed signs of morphine WS, along with the

greatest decrease in the total WS index (41.1%) ob-

served in the GTS-302 group at a dose of 1.0  mg/kg

(Table 3).

The main clinical symptoms of opioid withdrawal

syndrome, in addition to vegetative disorders (yawn-

ing, rhinorrhea, watery eyes, diarrhea, pupillary di-

lation, piloerection, etc.), are often accompanied by

the pain syndrome and hyperesthesia. The last ones

are considered to be important diagnostic symptoms

belonging to the category of disorders most severely

experienced by patients. In additional series of ex-

periments the effect of GTS-301 and GTS-302 on the

thresholds of tactile sensitivity under conditions of

morphine withdrawal in the dependent rats was ex-

amined. Pre-testing of the animals using von Frey fil-

aments showed that the individual rats did not differ

statistically significantly in the initial level of sensitiv-

ity to the mechanical tactile action: for GTS-301 it was

(3.28)

  =  0.957, p  =  0.426; for GTS-302 – F

(3.28)

  =  1.159,

p  =  0.342. During the morphine withdrawal model-

ing with GTS-301 a significant decrease in the sen-

sitivity threshold was observed in the control and

experimental groups compared to the initial values

(3.28)

  =  4.078; p  =  0.0002), which is consistent with the

data published previously  [13]. However, GTS-301 at

the studied single-administration doses did not affect

mechanical allodynia formed during the morphine

withdrawal. When evaluating efficacy of GTS-302, it

was shown that, with the morphine withdrawal in the

control and experimental groups, there was a signifi-

cant decrease in the sensitivity threshold compared to

the baseline level (pre-test) (F

(3.28)

  =  5.125; p  =  0.0059).

In contrast to GTS-301, GTS-302 increased dose-de-

pendently the threshold of response to acute admin-

istration at all studied doses with the maximum effect

observed for the dose of 1.0  mg/kg, at which there was

a 3-fold increase in the recorded parameter compared

to the control group (F

(1.14)

  =  17.42; p  =  0.009). Never-

theless, in all experimental groups the level of tactile

sensitivity remained lower than during the prelimi-

nary testing (Table 4).

Pharmacological advantages of GTS-302 over

GTS-301 revealed by the in  vivo modeling of opioid

dependence may be due to peculiarities of activation

of the postreceptor signaling pathways. Therefore, ac-

tivation of the signaling ways of PI3K/AKT, MAPK/ERK,

PLCγ1 by GTS-301 and GTS-302 mimetics in the HT-22

hippocampal cell culture was investigated. GTS-301

(10

–6

  M) and GTS-302 (10

–6

  M) were added to the cul-

ture medium as a sterile solution at effective concen-

trations established in the experiments on neuropro-

tective effect of these compounds on the HT-22 culture

line of hippocampal cells [12, 17]. NT-3 (10

–9

  M) was

used as a positive control. Cells were lysed at 5, 15,

30, 60, and 180 min after peptide adding.

Despite the fact that we have previously shown

activation of the TRKB and TRKC receptors by both

mimetics, a different pattern of postreceptor signal-

ing in them was revealed: GTS-302 (10

−6

  M), similar to

NT-3, activates all three MAPK/ERK, PI3K/AKT/mTOR,

and PLCγ1 pathways, while GTS-301 (10

−6

  M) triggers

only the MAPK/ERK and PLCγ1 signaling pathways.

As can be seen from Figs.2 and3, a significant increase

Table 4. Effect of GTS-302 on thresholds of tactile sensitivity during the foot shock-induced stress in rats

Groups

Doses, mg/kg

0.0 0.1 1.0 10.0

Pre-test 1.35 ± 0.15 1.23 ± 0.14 1.05 ± 0.14 1.05 ± 0.09

Test 0.040 ± 0.005*** 0.060 ± 0.005***

0.12 ± 0.02***

,###

0.08 ± 0.01***

,##

Note. Each group included 8 animals; ***p<0.001 is a significant difference from the Pre-test group; #p<0.05, ##p<0.01,

### p < 0.001 are significant differences from the control group. Data are presented in grams as a mean ± standard error

of the mean (M ± SEM).

KOLIK et al.1966

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig. 2. Phosphorylation of AKT1/2/3 (a), ERK1/2 (b), and

PLC-γ1 (c) after NT-3 and GTS-301 (10

−6

  M) adding of the

HT-22 mouse hippocampal cell culture (original Western blots

and densitometry results). Lanes: 1, 6)  Control; 2)  GTS-301,

5  min; 3)  NT-3, 5  min; 4)  GTS-301, 15  min; 5)  NT-3, 15  min;

7)  GTS-301, 30  min; 8)  NT-3, 30  min; 9)  GTS-301, 60  min;

10)  NT-3, 60  min; 11)  GTS-301, 180  min; 12)  NT-3, 180  min.

Data are presented as a mean ±standard deviation (M ± SD)

of three independent experiments; *  p  <  0.05 is a significant

difference from the control.

Fig. 3. Phosphorylation of AKT1/2/3(a), ERK1/2(b), PLC-γ1(c)

after NT-3 and GTS-302 (10

−6

  M) adding of the HT-22

mouse hippocampal cell culture (original Western blots

and densitometry results). Lanes: 1, 6) Control; 2) GTS-302,

5  min; 3)  NT-3,  5 min; 4)  GTS-302, 15  min; 5)  NT-3, 15  min;

7)  GTS-302, 30  min; 8)  NT-3, 30  min; 9)  GTS-302, 60  min;

10)  NT-3, 60  min; 11)  GTS-302, 180  min; 12)  NT-3, 180  min.

Data are presented as a mean ±standard deviation (M ± SD)

of three independent experiments; *  p  <  0.05 is a significant

difference from the control.

in phosphorylation of AKT kinases was observed at 5,

15, 60, and 180min after the GTS-302 (10

−6

  M) adding,

as well as at 5, 15, 30, and 60min after NT-3 adding; a

significant increase in phosphorylation of Erk kinases

was observed 5, 60, and 180 min after the GTS-302

adding; and 30, 60, and 180 min after the GTS-301

NT-3 MIMETICS ATTENUATE MORPHINE WITHDRAWAL 1967

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

adding. The PLCγ1 signaling pathway was activated 15

and 30min after adding of both GTS-302 and GTS-301.

DISCUSSION

When modeling morphine dependence in rats,

naloxone at low doses triggers general negative af-

fective state (motivational component), while at high

doses naloxone induces somatic signs of withdrawal

[18], and that was demonstrated in our work: devel-

opment of the somatic-vegetative disturbances typical

to opioid abstinence syndrome was observed in the

rats 15  min after naloxone administration at a dose of

mg/kg. Previously, the in  vitro experiments showed

that treatment of the LC neurons with morphine led

to 20%-decrease in the norepinephrine uptake and

a 12%-decrease in the number of tyrosine hydroxy-

lase-immunoreactive (TH+) cells. At the same time, in-

crease in the norepinephrine uptake and number of

the TH+ cells was noted in the cell culture supplement-

ed with NT-3 [7]. In the ex  vivo experiments, infusion

of NT-3 directly into the ventral tegmental area in the

rats prevented biochemical changes in the mesolimbic

dopaminergic system that occur during prolonged ac-

tion of morphine

  [19]. Despite the change in the NT-3

and TRKC mRNA levels in several brain areas during

opiate withdrawal, there is very little evidence for the

role of NT-3 in the development of opioid withdraw-

al in

vivo. In mice with conditional ablation of NT-3

in the brain, somatic symptoms of opiate withdrawal

were attenuated, which was accompanied with the

reduced expression of tyrosine hydroxylase in the LC

[20]. Opiate withdrawal led to the significant decrease

in the levels of NT-3 mRNA in the LC 20 and 70

  h after

morphine withdrawal, while the TRKC mRNA levels

were also below the control values  [4]. It cannot be

ruled out that the NT-3 mimetics could have certain

advantages, when used as agents for rapid pharmaco-

therapy of the opioid withdrawal syndrome.

In our work for the first time it was shown invivo

that the NT-3 dipeptide mimetics after acute systemic

administration attenuate dramatically somatic signs of

the naloxone-induced morphine withdrawal in rats,

which is consistent with the idea of participation of

neurotrophins in the mechanisms associated with for-

mation of opiate dependence

  [21]. It was first shown

in 1995 by in  situ hybridization that the individual

CNS neurons can express mRNA for high-affinity NT-3,

TRKB, and TRKC receptors [22]. The ability of GTS-301

and GTS-302 to activate, same as full-length neurotro-

phin, two types of receptors (TRKB and TRKC) in the

HT-22 hippocampal cells during oxidative stress mod-

eling [17, 12] suggests high level of regulatory activity

of the small-molecule NT-3 mimetics under patholog-

ical conditions that occur during withdrawal of psy-

choactive substances, including analgesics. GTS-301

and GTS-302 compounds attenuated significantly 7 of

18 signs of the morphine withdrawal. However, with

acute systemic administration, GTS-302 caused not

only the greatest decrease in the total index of with-

drawal syndrome, but also weakened in a dose-depen-

dent manner mechanical allodynia in the rats with

drug dependence, which does not rule out existence

of the structure-property relationship.

In this work, for the first time, differences in the

activation patterns of postreceptor pathways by the

NT-3 mimetics during interaction with TRK receptors

have been demonstrated. One of the peculiarities of

the GTS-302 action, similar to NT-3, is the ability to ac-

tivate three intracellular cascades, while GTS-301 did

not show the ability to activate the PI3K/AKT/mTOR

signaling pathway. It is known that PI3K/AKT/mTOR

inhibits the rapamycin complex

1 (mTOR), change in

functioning of which has been established in sub-

stance abuse  [23]. In the work of Zhu et  al.  [24] pub-

lished in 2021, a possible role of the PI3K/AKT/mTOR

signaling pathway in heroin addiction was revealed,

since the authors showed involvement of the PI3K/

AKT/mTOR phosphorylation in the nucleus accumbens

in formation of the heroin-seeking behavior in rats.

Gene expression analysis demonstrated statistically

significant increase in the mTOR expression with the

decreased levels of AKT as well as PI3K (phospha-

tidylinositol 3-kinase) expressions among the hero-

in-dependent patients compared to the control group

(p  <  0.001), with insufficient levels of PI3K and AKT

phosphorylation and, in some cases, overexpression

of mTOR (p  <  0.001) [25].

It should be noted that neuroprotective prop-

erties aimed at attenuation of mechanical allodynia

in hyperglycemia can be performed, among other

things, through phosphorylation of AKT, as well as of

its downstream target factor mTOR, that is, increase

in viability of the Schwann cells seems to be medi-

ated through the PI3K/AKT/mTOR signaling pathway

[26]. However, in other work, the nociceptin opioid

peptide receptor (NOP) antagonist, also known as

FQ nociceptin/orphanin receptor (N/OFQ), alleviated

mechanical allodynia associated with the chemother-

apy-induced neuropathic pain by inhibiting pacli-

taxel-enhanced expression of PI3K and phospho-AKT

in the dorsal root ganglion  [27] Overall, the role of

PI3K, in formation of individual symptoms of opiate

dependence manifested as central sensitization af-

ter exposure to harmful stimuli remains completely

unclear. However, the ability of GTS-302 to not only

stop main manifestations of the morphine withdrawal

syndrome, but also to weaken noticeably mechanical

allodynia in the rats with formed drug dependence

suggests that these effects of NT-3 mimetic may be

due to involvement of the PI3K/AKT/mTOR signaling

KOLIK et al.1968

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

pathway activation in the processes, including in the

processes of central sensitization. This fact requires

further investigation.

CONCLUSION

For the first time, the ability of small-molecule

neurotrophin-3 mimetics to reduce morphine with-

drawal syndrome in the opiate-dependent rats was

experimentally proven, while the pharmacologically

more significant effect of mimetic was observed that

involves activation of all three main postreceptor sig-

naling cascades (PI3K/AKT, MAPK/ERK, and PLCγ1)

same as in the case of full-length neurotrophin.

Contributions. L.G.K. supervised the study, dis-

cussed the results of the study, prepared the manu-

script; M.A.K. conducted the invivo experiments, per-

formed statistical analysis of the data; S.V.N. conducted

the in vitro experiments, edited the manuscript and

images; I.O.L. conducted the invitro experiments, bio-

chemical analysis; T.A.A. performed statistical analysis

of the data and discussed the obtained results, pre-

pared the manuscript; T.A.G. developed the concept

and discussed results of the study.

Funding. This work was financially supported by

the Ministry of Science and Higher Education of the

Russian Federation (topic no.FGFG-2022-0002 Preclin-

ical evaluation of drug safety and experimental devel-

opment of tools for health-saving and prevention of

diseases caused by the interaction of the body with

adverse environmental factors).

Ethics declarations. All applicable international,

national, and/or institutional guidelines for the care

and use of animals were followed and were approved

by the Biomedical Ethics Committee of the V.

 V.  Zakusov

Research Institute of Pharmacology of FSBSI (Protocol

No. 01 of February2, 2023). The authors of this work

declare that they have no conflict of interests.

Open access. This article is licensed under a Cre-

ative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s)

and the source, provide a link to the Creative Com-

mons license, and indicate if changes were made.

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Commons license and your intended use is not permit-

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use, you will need to obtain permission directly from

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