[Antiaggregation activity of arachidonic acid conjugates with neurotropic peptides proglyprol and semax].

The influence two original derivatives of a therapeutically important peptide, bearing arachidonic acid residue with semax and proglyprol, upon platelet aggregation have been studied in vitro. It is established that both derivatives, in contrast to the parent peptide, possess moderate anti-aggregant properties and produce a dose-dependent decrease in the interplatelet interaction induced by ADP, epinephrine, and arachidonic acid within the concentration range of 0.018 - 1.8 mM. This activity was more pronounced for arachidonoylsemax in comparison with arachidonoylproglyprol.

[Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor].

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.

[Kinetics of Semax penetration into the brain and blood of rats after its intranasal administration].

The radioactive peptide analogue Semax corresponding to the ACTH(4-10) sequence (Met-Glu-His-Phe-Pro-Gly-Pro) with a molar radioactivity of 56 Ci/mmol labeled with tritium at the C-terminal Pro was prepared. The labeled peptide was used for studying the kinetics of Semax penetration into rat brain and blood after its intranasal administration (50 microg/kg, 20 microl of solution) to nonbred white rats of body mass 200-250 g. It was demonstrated that 0.093% of the total introduced radioactivity per gram can be found in the rat brain 2 min after the administration, 80% of this radioactivity belonged to Semax, and the rest, to its metabolites. The peptide undergoes rapid enzymatic degradation, with the tripeptide Pro-Gly-Pro prevailing in biological samples relative to the total content of Semax and its metabolites.

Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties.

Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chemical and biological properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiological pH, the main complex species formed by Ac-Semax, [CuLH-2]2-, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulphur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH-2]2- formal redox potential, which is more positive than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are associated either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.

[Effect of modification of the N-terminal region of molecule on the expression of neotropic effect of semax analogues].

A comparative study of neotropic activity of semax (MEHFPGP), an analogue of the ACTH(4-10), and some of its derivatives in which the N-terminal methionine was modified or substituted with other amino acid residues was performed. The effect of these peptides on learning of albino rats in tests with positive (alimentary) and negative (pain) reinforcement was studied. In the case of modification of methionine by attachment of the gluconic-acid residue or substitution of methionine with lysine, the neotropic effect of the peptide was retained. The substitution of methionine with tryptophan or serine resulted in a decrease in the neotropic activity. The substitution of methionine with glycine, threonine, or alanine caused a complete loss of the neotropic activity of the peptide. Therefore, the amino acid residue located at position 1 of the heptapeptide analogue semax, plays a key role in retaining the neotropic effects of the peptide and determines the degree of their expression.

Synthesis of a corticotropin analog that retains full biological activity after iodination.

In order to circumvent the drastic decrease in biological activity that accompanies iodination of corticotropin, we have synthesized an analog of the human hormone containing phenylalanine in place of tyrosine in position 2 and norleucine in place of methionine in position 4 to give [Phe2,Nle4]ACTH-(1-38). This analog, referred to as Phe2,Nle4-ACTH-(1-38), was purified by ion exchange chromatography and partition chromatography. Phe2,Nle4-ACTH-(1-38) was found to be indistinguishable from synthetic human ACTH in its ability to stimulate corticosterone production in isolated rat adrenocortical cells. Iodination of Phe2,Nle4-ACTH-(1-38) resulted in the formation of monoiodo-Tyr23,Phe2,Nle4-ACTH-(1-38) and diiodo-Tyr23,Phe2,Nle4-ACTH-(1-38), which were completely separated from each other and unmodified Phe2,Nle4-ACTH-(1-38) by reverse phase high performance liquid chromatography. Monoiodo-Tyr23,Phe4,Nle4-ACTH-(1-38) was also found to be as potent as ACTH in stimulating steroidogenesis. These results show that Phe2,Nle4-ACTH-(1-38) can be used for the preparation of the radioligand with full biological activity for studying physiologically relevant corticotropin receptors and for use in RIA.

Adrenocorticotropin. 49.1 Synthesis and biological activity of [2-delta-aminovaleric acid, 5-arginine-a1adrenocorticotropin-(2-19).

An adrenocorticotropin analogue, [2-delta-aminovaleric acid, 5-arginine]adrenocorticotropin-(2-19), has been synthesized by the solid-phase method and its biological activity has been determined. It was found that substitution of arginine for glutamic acid at position 5 of [2-delta-aminovaleric acid]adrenocorticotropin-(2-19) increased the steroidogenic potency in idolated rat adrenal cells and the lipolytic potency in isolated rat fat cells but decreased the lipolytic potency in isolated rabbit fat cells. The synthetic analogue had only 2% of the melanotropic potency of the parent molecule.

Adrenocorticotropin. 47. Synthesis and biological activity of adrenocorticotropic peptides modified at the tryptophan position.

Three biologically active peptides, [9-beta-(1-naphthyl)alanine]-ACTH-(1-19), [9-Ni-formyltryptophanl-ACTH-1(1-19), and (8-lysine,9-phenylalaninel-ACTH-1(1-9), have been synthesized by the solid-phase method. All of the synthetic peptides showed diminished biological activity compared to ACTH-(1-19). It was also shown that the steroidogenic and lipolytic activities of ACTH-(1-19) were not inhibited by [8-lysine,.-phenylalanine]-ACTH-(1-19).

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells.

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.

Adrenocortical function testing in dairy cows and its effect on milk yield.

Administration of 6IU synthetic ACTH1-24 intravenously to six Holstein-Friesian cows resulted in a cortisol peak concentration after 1 hour of 148 +/- 34.2 ng/ml. Basal plasma cortisol concentration (4.84 +/- 0.83 ng/ml) was reached 5 hours after ACTH injection. Until 7 days after ACTH administration no effect on milk yield was recorded. So it is concluded that a dose of 6 IU ACTH1-24 is sufficient for a conspicuous release of cortisol without any alteration in milk production. This dose can be used as a standard test for the evaluation of adrenocortical function in lactating cows when administered intravenously at 9 a.m. and when plasma samples for cortisol assay are collected just prior to administration and at 10 a.m.

[Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment]. / Khimiko-fermentativnyi sintez oligonucleotidnoi matritsy analoga fragmenta 4-10 adrenokortikotropnogo gormona.

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).

[Structural and functional organization of ACTH: synthesis and properties of analogs of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexa-amino acids instead of a natural sequence of ACTH 19-24 amino acids]. / Strukturno-funktsional'naia organizatsiia ACTH: sintez i svoistva analogov ACTH-(11-24)-tetradeka- i ACTH-(1-24)-tetrakozopeptidov, soderzhashchikh geksaaminokisloty vmesto prirodnoi posledovatel'nosti aminokislot ACTH 19-24.

To assess the role of amino acid sequence ACTH 19-24 in the corticotropin structure and steroidogenic activity, the analogues of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexaglycine, hexaphenylalanine, hexaglutamic acid or hexalysine instead of the natural 19-24 sequence have been synthesized by conventional methods. All these compounds in water have the CD curves characteristic of random coil, CD spectra of analogue ACTH-(1-24)-tetracosapeptide and hexalysine-containing analogue ACTH-(11-24)-tetradecapeptide in trifluoroethanol indicate the presence of alpha-helices. The latter compound manifested higher steroidogenic activity than ACTH-(11-24)-tetradecapeptide. All the other analogues were either less active than ACTH-(1-24)-tetracosapeptide or inactive over the concentration range 10(-5)-10(2) mg/ml, thereby testifying to functional importance of the 19-24 sequence for manifesting full steroidogenic activity.

[Synthesis and biological properties of a cyclic analog of ACTH-(5-14)-decapeptide]. / Sintez i biologicheskie svoistva tsiklicheskogo analoga ACTH-(5-14)-dekapeptida.

A cyclic analogue and the corresponding linear segment of the corticotropin molecule, namely ACTH-(5-14)- and [cyclo (Glu gamma-epsilon Lys)]ACTH-(5-14)-decapeptide, both including the specific and unspecific active centers of the ACTH molecule, have been synthesized and studied. The cyclic structure is fixed by amide bond between the glutamic acid and lysine side chains. Condensation of fragments has been realized by azide or DCC/HOBT methods. Cyclization has been achieved using diphenylphosphorylazide. The cyclic analogue has full steroidogenic activity, while its melanotropic activity is 3 orders of magnitude higher than that of the linear decapeptide.

[Synthesis and analysis of potent cyclic analogs of the ACTH active center]. / Sintez i issledovanie vysokoaktivnykh tsiklicheskikh analogov aktivnogo tsentra kortikotropina.

Linear and cyclic analogues of the specific active center of the ACTH molecule have been synthesized, viz. [Lis5]ACTH-(5-10)-, [Lys5, cyclo (Gly10----epsilon Lys5)]-ACTH-(5-10)-hexapeptides, [Lys5 (Gly)]ACTH-(5-10)- and [Lys5, Gly11, cyclo (Gly11----epsilon Lys5)]-ACTH-(5-11)-heptapeptides. The cyclic structures are fixed by covalent bond between the COOH-group of the C-terminal glycine and epsilon-amino group of a lysine residue. Azide method, DCC/HOBT or pentafluorophenyl esters are used for fragment coupling, while cyclization is achieved by means of diphenylphosphoryl azide or pentafluorophenyl esters. Cyclic compounds are 2-3 orders of magnitude more active than their linear counterparts as revealed by assaying their melanocyte-stimulating activity in vitro on frog skin. Only the title heptapeptide possesses a steroidogenic activity similar to that of ACTH-(5-10)-hexapeptide. The results obtained are in accord with the idea implying the formation in the hormone-receptor complexes of quasi-cyclic structures in the region of the specific active center of ACTH molecule.

[Synthesis of [cyclo(Glu gamma-----epsilon Lys(Gly)]ACTH-(5-14) undecapeptide. Biological and physico-chemical properties of analogs of ACTH-(5-10)- and ACTH-(5-14)-peptides]. / Sintez [tsiklo(Glu gamma----epsilon Lys(Gly)]-ACTH-(5-14) undekapeptida. Biologicheskie i fiziko-khimicheskie svoistva analogov ACTH-(5-10)- i ACTH-(5-14)-peptidov.

Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures.