Regulation of the Melanocortin-Sensitive Adenylate Cyclase System by N-Acylated Peptide 71-82 of Type 4 Melanocortin Receptor.

The peptides structurally corresponding in to cytoplasmic loops of G protein-coupled receptors (GPCR) are able to control functional activity of homologous receptors and the corresponding signaling pathways. Modification of these peptides with hydrophobic radicals enhances their biological activity due to penetration of lipophilic derivatives through the membrane and anchoring near their targets, GPCR. We synthesized an N-palmitoylated peptide Palm-Val-[Lys-Asn-Lys-Asn-Leu-His-Ser-Pro-(Nle)-Tyr-Phe-Phe71-82]-amide-Palm-Val-(71-82) structurally corresponding to cytoplasmic loop 1 of melanocortin 4 receptor (M4R). We found that in micromolar concentrations it very effectively suppresses stimulation of basal adenylate cyclase activity and basal level of GppNHp binding of heterotrimeric G proteins produced by THIQ and α-melanocyte stimulating hormone (α-MSH), agonists of M4R homologous to the peptide, in synaptosomal membranes of rat brain. The peptide Palm-Val-(71-82) also reduced, albeit to a significantly less extent, stimulation of adenylate cyclase and G-proteins by M3R agonist of γ-MSH, due to high homology of the peptide primary structure to M3R cytoplasmic loop 1. The synthesized peptide with activity of M4R/M3R antagonist can be used for the development of regulators of M4R and M3R and the corresponding biochemical and physiological processes.

[THE THYROID STATUS OF RATS IMMUNIZED WITH PEPTIDES DERIVED FROM THE EXTRACELLULAR REGIONS OF THE TYPES 3 AND 4 MELANOCORTIN RECEPTORS AND THE 1B-SUBTYPE 5-HYDROXYTRYPTAMINE RECEPTOR].

The activity of the hypothalamic-pituitary-thyroid (HPT) axis is controlled by the brain neurotransmitter systems, including the melanocortin signaling system. Pharmacological inhibition of type 4 melanocortin receptor (M4R) leads to disruption of the functioning of HPT axis and to reduction of the level of thyroid hormones. At the same time, the data on how prolonged inhibition of M4R affects this axis and on its role in regulation of M3R are absent. The relationship between the thyroid status and the activity of 1B-subtype 5-hydroxytryptamine receptor (5-HT1BR) is scarcely explored. The aim of this work to study the effects of chronic inhibition of M3R, M4R and 5-HT1BR induced by immunization of rats with BSA-conjugated peptide derived from the extracellular regions of these receptors on the thyroid status and the activity of thyroid stimulating hormone (TSH)-sensitive adenylyl cyclase signaling system (ACSS) in the thyroid glarid (TG) of the immunized animals. In rats immunized with the peptides K-[TSLHL WNRSSHGLHG11-25]-A of M4R, A[PTNPYCICTTAH269-280]-A of M3R and. [QAKAEE-EVSEC(Acm)-VVNTDH189-205]-A of 5-HT1BR levels of thyroid hormones such as fT4, tT4 and tT3 were significantly reduced. In rats immunized with M4R and M3R peptides, an increase of TSH was detected whereas in the animals immunized with 5-HT1BR peptide the level of TSH, on the contrary, was reduced. In the TG of rats immunized with M4R and M3R peptides, the stimulatory effects of hormones (TSH, PA-CAP-3 8) and GppNHp on adenylyl cyclase activity were attenuated, and the changes were most pronounced in the case M4R peptide immunization. After immunization with 5-HT1BR peptide the stimulatory effects of TSH, PACAP-38 and GppNHp were retained. Thus, the main cause of thyroid hormones deficit in rats immunized with M4R and M3R peptides was the decreased sensitivity of ACSS thyrocytes to TSH, whereas in rats iimunized with 5-HT1BR peptide the deficit of thyroid hormones was associated with decreased level of TSH. Our data on the negative impact of long-term immunization of rats with BSA-conjugated peptides derived from the extracellular regions of M4R, M3R.and 5-HT1BR on their thyroid status is a strong argument in favor of participation of these receptors and intracellular signaling pathways associated with them in the regulation of HPT axis.

The immunization with peptide 189-205, a derivative of serotonin receptor subtypes 1B, changes the sensetivity of adenylyl cyclase to hormones in the rat brain.

The aim of this work was to study the effect of multiple (during 12 months) immunization of rats with BSA-conjugated peptide 189-205 corresponding to the second extracellular loop of rat HT1BR on ACSS activity in the brain of immunized animals (group HT1BR) and its regulation by hormones.

[Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine].

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

Palmitoylated peptide 562-572 of luteinizing hormone receptor increases testosterone level in male rats.

We studied the effect of intraperitoneal and intratesticular administration of NKDTKIAKKNle-A(562-572) peptide and its palmotoylated analog NKDTKIAKK-Nle-A(562-572)-K(Palm)A to male rats on adenylate cyclase activity in the testicular membranes in vitro and on plasma testosterone levels. Peptide NKDTKIAKK-Nle-A(562-572)-K(Palm)A stimulated basal adenylate cyclase activity and reduced activity of the enzyme in testicular membranes stimulated by chorionic gonadotropin. After intratesticular administration in a dose of 200 µg/kg, it significantly increased testosterone so that 1, 3 and 5 h after administration its level was increased by 74, 44 and 35%, respectively. Administered intraperitoneally in a lower dose (50 µg/kg), the peptide had little effect on testosterone level. Unmodified peptide was inactive.

Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor.

[Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable to the 612-627-K(Pal)A in the ability to inhibit the AC effect of TSH, but, although to a lesser extent that it decreased the AC effects of other hormones, demonstrating the low receptor specificity. Thus, these data point to the high efficiency of peptide 612-627-K(Pal)A, as a regulator of TSHR, and the prospects of creating the drugs based on it to control the thyroid functions in pathology.

[N-palmitoylated peptide 232-245 of rat type 4 melanocortin receptor possessing agonistic activity].

Melanocortin receptors of the type 4 (M4R) play a key role in the regulation of feeding behavior, neuroendocrine functions, and energy metabolism. The alterations in their functional activity induce obesity, metabolic syndrome, depression, and mental disorders, which makes the search of selective regulators of M4R to be one of the actual problems of molecular endocrinology. Promising for the development of such regulators is to design peptides corresponding to functionally important regions of M4R. The purpose of this study was to study the influence of synthesized N-palmitoylated peptide Palm-Thr-Gly-Thr-Ile-Arg-Gln-Gly-Ala-Asn-(Nle)-Lys-Gly-Ala-Ile232-245-amide (Palm-232-245) structurally corresponding to the C-terminal half of the third intracellular loop (ICL-3) of rat M4R on functional activity of adenylyl cyclase signaling system (ACSS) in the fractions of synaptosomal membranes isolated from the brains of male rats. It has been shown that, at a concentration of 10(-7) M and higher, Palm-232-245 stimulates the basal activity of adenylyl cyclase (AC) in the synaptosomal membranes and increases the basal level of GTP binding with the EC50 values of 71 and 267 nM, respectively. Under the combined action of low concentrations of the peptide (10(-7)-10(-6) M) and M4R agonists, α-melanocyte-stimulating hormone (α-MSH) and THIQ (10(-7) M), we observed an additivi stimulatory effect on AC, which disappeared when the peptide concentration was increased to 10(-4)-10(-3) M. In the synaptosomal membranes preincubated with 10(-5) M peptide, the maximum stimulatory effect of M4R agonists on AC activity was lower than that in controls, and EC50 values for this effect, on the contrary, increased. In the case of combined action of the peptide and hormones (γ-MSH, serotonin, PACAP-38) that activate AC via the other receptors, the additivity of their stimulating effects on the ACSS persisted throughout the range of peptide concentrations. The effect of the peptide was not observed in myocardial and testicular membranes no in which there is M4R homologous to the peptide. Thus, N-palmitoylated peptide Palm-232-245 specifically activates the ACSS in the rat brain by acting as intracellular M4R agonist. This may be used to create drugs regulating brain melanocortin system and physiological processes that depend on it.

[The influence of immunization of rats with BSA-conjugated peptide 269-280 of type 3 melanocortin receptor on the metabolic parameters and thyroid functions].

One of the approaches to study the role of the brain hormonal signaling systems in the regulation of biochemical and physiological processes is their shutdown using the antibodies generated to peptides corresponding to extracellular regions of receptors. The brain type 3 melanocortin receptors (M3R) play an important role in the central regulation of the metabolism and the endocrine system. However, the influence of prolonged inhibition of M3R on energy metabolism, insulin resistance, and thyroid gland (TG) function is practically not studied. The aim of the study was to investigate the influence of prolonged repeated immunization of male rats with the BSA-conjugated peptide Ala-[Pro-Thr-Asn-Pro-Tyr-Cys-Ile-Cys-Thr-Thr-Ala-His269-280]-Ala (A[269- 280]A) corresponding to the third extracellular loop of M3R on their metabolic parameters and functional activity of TG. 9 months after the first immunization, the weight of rats was reduced and after 12-13 months was significantly lower than in controls. The weight of abdominal and brown adipose tissues, on the contrary, increased. At the same timeline there was an increase in the fasting glucose and insulin levels, and increase of the HOMA-IR index (by 75%) indicating that immunized animals develop insulin resistance. The rats have increased glucose utilization due to an increase of insulin production by pancreatic ß-cells. 12 months after the first immunization, the increase of the triglycerides level (by 74%) and the ratio of LDL- and HDL-cholesterol (by 36%) were revealed. 13 months after the start of immunization, the levels of free and total thyroxine and total triiodothyronine significantly decreased. In the TG plasma membranes of immunized rats the weakening adenylyl cyclase stimulating effect of thyroid-stimulating hormone was detected. Thus, long-term decrease in the bra- in M3R activity due to repeated immunization of rats with BSA-conjugated peptide A[269-280]A induces the disturbances of the peripheral metabolism and TG function.

[Regulation of adenylyl cyclase activity in the rat testes by acylated derivatives of peptide 562-572 of luteinizing hormone receptor].

One of directions of the search of hormonal signaling systems regulators is the development of peptides that correspond to the cytoplasmic regions of G-protein-coupled receptors (GPCR). Modification of the pepti- des with hydrophobic radicals increases their efficiency and selectivity. But currently is not studied as the acti- vity of the peptide depends on the localization of the hydrophobic radicals, their number and the chemical natu- re. The aim of this work was the synthesis of modified by fatty acid radicals derivatives of peptide 562-572 corresponding to the C-terminal region of luteinizing hormone receptor (LHR), and the study of regulatory ef- fects of the acylated LHR-peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in the rat tissues. To elucidate the effect of localization of hydrophobic radicals and their number the modification of peptide 562-572 only at the N- or C-terminus or at both ends was carried out. To study the effect of hydrop- hobicity the residues ofpalmitic (Pal) and decanoic (Dec) acids were selected. Using a solid phase strategy we have synthesized unmodified peptide NDTKIAKK-Nle-A562-572-KA (1) and five of its acylated analogues, such as N[K(Dec)]DTKIAKK-Nle-A562-572-KA (2), NKDTKIAKK-Nle-A562-572-[K(Dec)]A (3), N[K(Dec)] DTKIAKK-Nle-A562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A562-572-KA (5), and NKDTKIAKK-Nle- A562--572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the ba- sal AC activity and reduced AC stimulating effect of human chorionic gonadotropin (hCG) in the testes of rats, peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the un- modified peptide 1, while peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by the tissue and the receptor specificity. Thus, the modification of LHR-peptide 562-572 with fatty acid radicals at the C-terminus increases its regulatory effect on the functional activity of the adenyla- te cyclase system in the rat testes, indicating promising the modification of GPCR-peptides with hydrophobic radicals. These data support the hypothesis that the hydrophobic radical to be localized in the locus of GPCR-peptide, where a transmembrane domain is located in the receptor.

[The secondary structure of peptides derived from the third intracellular loop of the serpentine type receptors and its interrelation with their biological activity].

We and other authors have shown that synthetic peptides corresponding to regions of the third intracellular loop (ICL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and trigger them in the absence of hormone. To create on the basis of these peptides the selective regulators of hormonal signaling systems the relationship between their biological activity and secondary structure are studied. It is assumed that most suitable is a helical conformation, which allows the peptide effectively interact with signaling proteins. The aim of this study was to test the biological activity and secondary structure of synthesized by us linear peptides and their dimeric and palmitoylated analogs, corresponding to C-terminal region of the ICL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine receptor of the type 6 (5-HT6R). It is shown that LHR-peptides at the micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in the plasma membranes of rat testes, while 5-HT6R-peptides activate AC and G-proteins in the synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in the tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR-peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while 5-HT6R-peptides the effect of 5-HT6R-agonist, EMD-386088, and the action of the peptides was not found in the case of non-homologous receptors. Using circular dichroism spectroscopy it is shown that in neutral (pH 7) and acidic (pH 2) medium all the peptides are exist predominantly in the antiparallel beta-sheet (37-42%) and disordered conformations (33-35%). In alkaline medium (pH 10) in the case palmitoylated peptides the increase of the contribution of the helical conformation to 12-27% was observed. In the presence of trifluoroethanol (10-80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs to 14%), however, in this case the antiparallel beta-sheet and disordered conformation prevailed. The conclusion was made that the lack of clearly expressed ability to form helices in peptides derived the ICLs of receptors did not significantly affect their activity. This is consistent with proposed mechanism of peptides action, whereby peptide interacts with the complementary regions of homologous receptor that does not require the helix formation.

[Development of non-hormonal regulators of adenylyl cyclase signaling system on the basis of peptides, derivatives of the third intracellular loop of somatostatin receptors].

In the majority of the serpentine type receptors the third intracellular loop (ICL-3) is responsible for interaction with heterotrimeric G-proteins and for transduction of hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, in consequence, can be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to C-terminal regions 255-269 and 240 254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som1R and Som2R). Micromolar concentrations of these peptides activated Gi-proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255-269 of Som1R is a selective antagonist of Som1R, and the peptide 240-254 of Som2R is an agonist of Som1R. So, the peptide 255-269 of Som1R decreased the regulatory effects of somatostatin and selective Som1R-agonist CH-275 realized via the receptor homologous to them, while the peptide 240-254 of Som2R, on the contrary, increased AC inhibitory action of CH-275. Both peptides insignificantly influenced regulatory effects of the Som2R-agonist octreotide. Summing up, the peptides studied by us are selective regulators of somatostatin-sensitive AC system. Using the peptides it was shown that ICL-3 of Som1R and Som2R includes the main molecular determinants that are responsible for activation of Gi-proteins and regulation of AC system by somatostatin and its analogues.

[Specific features of laryngeal paresis following surgical treatment of diffuse toxic goiter (a prospective longitudinal passive study)].

Specific manifestations of postoperative laryngeal paresis observed with the use of indirect laryngoscopy are described in 53 patients subjected to the surgical treatment of diffuse toxic goiter. Laryngeal paresis was shown to develop both in the early (up to 7 days) and in the late (over 14 days) postoperative periods. The delayed form of pathology accounted for 13% of the total number of the cases of postoperative laryngeal paresis. The standard treatment of transient postoperative laryngeal paresis resulted in the complete recovery of vocal cord mobility within 1-6 months after the onset of therapy, regardless of the state of the cords at the time of diagnosis of the disease. Persistent postoperative laryngeal paresis developed by the end of the 15 month observation period. Phonation was found to be preserved in 66% of the patients in whom laryngeal paresis (unilateral abduction paresis) had been diagnosed by indirect laryngoscopy. In all the remaining patients, phonation recovered 15 months or more after surgery. The authors argue that neither the recovery nor the preservation of phonation can be a criterion for the absence of complications. Also, the outcome of surgical intervention unsupported by the results of laryngoscopy performed within 1, 6, and 15 months after the treatment does not reflect the true structure of postoperative complications.

[Low-molecular regulators of polypeptide hormones receptors containing LGR repeats].

During the last years the low-molecular non-peptidic regulators of the polypeptide hormones receptors containing LGR-repeats were identified. In the review the data on the structure and the molecular mechanisms of action of these regulators as agonists and antagonists of the luteinizing, follicle-stimulating and thyrotropin hormones are analyzed and systematized. The regulators interact with the serpentine domain of LGR-receptor and trigger the signaling cascades coupled with the receptor. Low-molecular agonists and antagonists of the LGR-receptors are considered as a new generation of the drugs that regulates the functional activity of sensitive to pituitary glycoprotein hormones signaling systems with high efficiency and selectivity. These regulators are more accessible compared to the hormones and can be use orally.