Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d ‐amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so‐called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all‐D‐AII and all‐D‐VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro‐AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a β‐turn conformation in different solutions. In the presence of membrane‐mimetic micelles, AII had also a β‐turn conformation, while retro‐AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro‐AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro‐AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of Amino Acid Deletion on the Antiplasmodial Activity of Angiotensin II

Malaria is an infectious disease for which effective treatment and prevention strategies remain limited. Our group recently reported that angiotensin II (AII) presents antiplasmodial activity and inhibits the development of Plasmodium gallinaceum in Aedes aegypti. However, details concerning role of each amino acid residue in the antiplasmodial activity of the peptide and information about the minimal structure responsible for this activity remain unknown. In this work, we investigated the effects of specific deletions (i.e., mono-, di-, tri- and tetra-deletions) of AII amino acids on the antiplasmodial activity of this molecule. The peptides were synthesized on solid phase method using the t-Boc strategy, purified using high performance liquid chromatography and characterized using mass spectrometry. The lytic activity of the peptides was assessed in vitro using mature sporozoites extracted from the salivary glands of infected Aedes aegypti mosquitoes. The results demonstrate that all of the deletions reduced antiplasmodial activity compared to native AII and that active analogs tend to adopt β-turn conformations; however, the deletion of bulky hydrophobic residues causes greater reductions of bioactivity than the deletion of hydrophilic residues. Corroborating previous studies, we observed that analog extremities are susceptible to changes and can be carefully modified without compromising the activity of this compound. This research contributes to our understanding of the role of each AII amino acid residue in activity against Plasmodium gallinaceum and identifies two short analogs with similar antiplasmodial activity to AII. These analogs may be candidates for additional antimalarial assays because they are inexpensive and easy to synthesize.     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

Relative potency of the forms of GnRH and their analogs on LH release in sea bass

The in vivo and in vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release luteotropic hormone (LH) was studied in sea bass Dicentrarchus labrax in particular the hypothalamic fish‐specific sea bream GnRH form (sbGnRH) and the general mesoencephalic form chicken GnRH‐II (cGnRH‐II). The potencies of the natives and their analogs (GnRHas) were referred to that of [D‐Ala⁶, Pro⁹Net]‐mGnRHa (LHRHa) at equivalent doses. Analogs of the native peptides [D‐Arg⁶, Pro⁹Net]‐cGnRH‐II, [D‐Ala⁶, Pro⁹Net]‐cGnRH‐II, [D‐Trp⁶, Pro⁹Net]‐sbGnRH and [D‐Ala⁶, Pro⁹Net]‐sbGnRH were effective in inducing in vivo LH release (at 15 µg kg^?1 body mass), exhibiting longer lasting activity than their corresponding native forms. Injection of sbGnRH and cGnRH‐II provoked a small but significant peak of circulating LH at 1·5 h after treatment (a.t.) decreasing down to basal levels at 4 h a.t. [D‐Arg⁶, Pro⁹Net]‐cGnRH‐II, [D‐Ala⁶, Pro⁹Net]‐cGnRH‐II and [D‐Ala⁶, Pro⁹Net]‐mGnRHa evoked a higher and a more sustained elevation of LH, peaking at 12 h a.t. and returning to basal levels between 48 and 72 h a.t. [D‐Trp⁶, Pro⁹Net]‐sbGnRH and [D‐Ala⁶, Pro⁹Net]‐sbGnRH also induced a significant surge of LH in plasma at 4 h a.t. turning to the basal levels at 24 h a.t. These rises, however, were of less amplitude and duration than the observed after treatment with cGnRH‐II analogs and [D‐Ala⁶, Pro⁹Net]‐mGnRHa. The in vitro stimulation of dispersed pituitary cells with the different native and modified forms of GnRH resulted in a dose‐dependent increase in the quantity of LH released at 24 h a.t. [D‐Arg⁶, Pro⁹Net]‐cGnRH‐II and [D‐Ala⁶, Pro⁹Net]‐cGnRH‐II induced the highest response of LH in vitro release followed by salmon GnRH (sGnRH), [D‐Ala⁶, Pro⁹Net]‐mGnRHa and [D‐Trp⁶, Pro⁹Net]‐sbGnRH. The lowest activity was exhibited by sbGnRH. Collectively, the in vitro biological activity (compared by their EC₅₀) can be ordered as follows: [D‐Arg⁶, Pro⁹Net]‐cGnRH‐II > [D‐Ala⁶, Pro⁹Net]‐cGnRH‐II > sGnRH > [D‐Ala⁶, Pro⁹Net]‐mGnRHa > [D‐Trp⁶, Pro⁹Net]‐sbGnRH > [D‐Ala⁶, Pro⁹Net]‐sbGnRH > cGnRH‐II > sbGnRH.     

Angiotensin II restricted analogs with biological activity in the erythrocytic cycle of Plasmodium falciparum

The anti‐plasmodial activity of conformationally restricted analogs of angiotensin II against Plasmodium gallinaceum has been described. To observe activity against another Plasmodium species, invasion of red blood cells by Plasmodium falciparum was analyzed. Analogs restricted with lactam or disulfide bridges were synthesized to determine their effects and constraints in the peptide–parasite interaction. The analogs were synthesized using tert‐butoxycarbonyl and fluoromethoxycarbonyl solid phase methods, purified by liquid chromatography, and characterized by mass spectrometry. Results indicated that the lactam bridge restricted analogs 1 (Glu‐Asp‐Arg‐Orn ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) and 3 (Asp‐Glu‐Arg‐Val‐Orn ‐Tyr‐Ile‐His‐Pro‐Phe) showed activity toward inhibition of ring formation stage of P. falciparum erythrocytic cycle, preventing invasion in about 40% of the erythrocytes. The disulfide‐bridged analog 10 (Cys‐Asp‐Arg‐Cys ‐Val‐Tyr‐Ile‐His‐Pro‐Phe) was less effective yet significant, showing a 25% decrease in infection of new erythrocytes. In all cases, the peptides presented no pressor activity, and hydrophobic interactions between the aromatic and alkyl amino acid side chains were preserved, a factor proven important in efficacy against P. gallinaceum. In contrast, hydrophilic interactions between the Asp¹ carboxyl and Arg² guanidyl groups proved not to be as important as they were in the case of P. gallinaceum, while interactions between the Arg² guanidyl and Tyr⁴ hydroxyl groups were not important in either case. The β‐turn conformation was predominant in all of the active peptides, proving importance in anti‐plasmodial activity. This approach provides insight for understanding the importance of each amino acid residue on the native angiotensin II structure and a new direction for the design of potential chemotherapeutic agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells

Neurotensin (NT) analogs, modified at Arg⁸ and Arg⁹, were used to assess the role of Arg in NT binding to mast cells. [D-Arg⁸]- and [D-Arg⁹]-NT bound 4–5 times better than NT, whereas [D-Arg^8,9]-NT had the same binding affinity as NT. Binding of [Ala⁸]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala⁹]-NT had 32% binding. C-terminal peptides, NT_8–13 and NT_9–13, had about 65% binding. These data suggest that Arg⁸ plays a greater role than Arg⁹ in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys^2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

Syntheses and biological evaluation of analogs of luteinizing hormone-releasing hormone (LH-RH) modified in position 2, 3, 4 or 5

Syntheses by the conventional methods as well as the chemical, physical and biological properties are described of the following analogs of the LH-releasing hormone (LH-RH): [Leu³]-LH-RH, [Phe³]-LH-RH, [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH, Des-His²-[Phe⁵]-LH-RH, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH. $\text{In vivo}$ assays showed that [Leu³]-LH-RH did not release LH in doses as high as 5 – 25 μg, having less than 0.0008% of LH-RH activity, while [Phe³]-LH-RH had 0.43% of the LH-RH activity of natural LH-RH. The LH-RH activities of [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH and Des-His²-[Phe⁵]-LH-RH were extremely low. On the other hand, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH had significant LH-RH activity. The structure-activity relationship of LH-RH is discussed on the basis of these findings.     

The pro-apoptotic action of new analogs of the insect gonadoinhibiting peptide Neb-colloostatin: Synthesis and structure–activity studies

Neb-colloostatin (SIVPLGLPVPIGPIVVGPR), an insect oostatic factor found in the ovaries of the flesh fly Neobellieria bullata, strongly induces apoptosis in insect haemocytes. To explain the role of Ser¹ and Pro⁴ residues of Neb-colloostatin in the pro-apoptotic activity of this peptide, the synthesis of a series of analogs was performed, such as: [Ac-Ser¹]- (1), [d-Ser¹]- (2), [Thr¹]- (3), [Asp¹]- (4), [Glu¹]- (5), [Gln¹]- (6), [Ala¹]- (7), [Val¹]- (8), [d-Pro⁴]-(9), [Hyp⁴]- (10), [Acp⁴]- (11), [Ach⁴]- (12), [Ala⁴]- (13), [Ile⁴]- (14), and [Val⁴]-colloostatin (15). All peptides were bioassayed in vivo for the pro-apoptotic action on haemocytes of Tenebrio molitor. Additionally, the structural properties of Neb-colloostatin and its analogs were examined by the circular dichroism in water and methanol. Peptides 1, 4, 5, 7, 8, 10, 12, 14, and 15 strongly induce T. molitor haemocytes to undergo apoptosis and they show about 120–230% of the Neb-colloostatin activity at a dose of 1 nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.     

Some analogs of luteinizing hormone releasing hormone (LH-RH) having intense ovulation-inducing activity

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly¹⁰-[Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide, des-Gly¹⁰-[α-aminoisobutyric acid⁶]-LH-RH-ethylamide, des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Ile⁵, D-Ala⁶]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly¹⁰-[D-Ala⁶]-LH-RH-ethylamide and des-Gly¹⁰-[Phe⁵, D-Ala⁶]-LH-RH-ethylamide, were 50 times or more active than the original molecule.     

Human somatotropin: Noncovalent interactions of the natural NH2-terminal 1–134 segment with synthetic analogs of the COOH-terminal fragment

Three analogs of the COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰,Ala^165,182]-HGH-(150–187), [Nle¹⁷⁰,Ala^165,182]-HGH-(152–187), and [Nle¹⁷⁰,Ala^165,182]-HGH-(154–187), have been synthesized by the solid-phase method. The synthetic analogs were complemented with the natural NH₂-terminal fragment [Cys(Cam)⁵³]-HGH-(1–134) to form recombinants with HGH activities, as revealed by the rabbit liver membrane receptor binding and the Nb2 lymphoma cell assays.     

Further studies on the antiviral activity of alloferon and its analogues

The subject of our studies was the synthesis, biological evaluation, and conformational studies of insect tridecapeptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) and its analogues such as: [des‐His¹]‐, [Lys¹]‐, [Arg¹]‐, and [Ala¹]‐alloferon. These peptides were synthesized to check the influence of the His residue at position 1 of the alloferon chain on its antiviral activity. Two aspects of the biological effects of these peptides were determined: (i) the cytotoxicity in vitro in the Vero, LLC‐MK2, and HEp‐2 cell lines, and (ii) the antiviral activity in vitro in respect to DNA and RNA viruses. We found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackie virus replication, whereas [Lys¹]‐alloferon exhibited a high inhibitory action towards both viruses. Moreover, the peptides did not show any cytotoxic activity against the Vero, LLC‐MK2, and HEp‐2 cells. The preliminary circular dichroism conformational studies showed that the peptides investigated seem to prefer an unordered conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris-phosphate (IP₃), intracellular calcium, [Ca²⁺]_i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP₃ and a rapid increase in [Ca²⁺]_i in cells loaded with the Ca²⁺-responsive fluorescent dye fura-2. The prompt AII-induced [Ca²⁺]_i spike was not affected by incubating HCEC in Ca²⁺-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca²⁺ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca²⁺ from intracellular stores. The AII-triggered increases in IP₃, [Ca²⁺]_i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar¹, Val⁵, Ala⁸-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca²⁺]_i and independent of PKC. © 1996 Wiley-Liss, Inc.     

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct ³H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.     

Novel analogs of alloferon: Synthesis,conformational studies,pro-apoptotic and antiviral activity

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1_MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala⁹]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys⁹]-, [Phg⁹]-, [Lys¹²]-, and [Phe¹²]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.     

Preferred solution and calculated conformations of dermorphin and analysis of structure–conformation–activity relationships in the series [A1an]-dermorphin

We describe the solution (¹H-nmr) and calculated conformations of the opiatelike peptide dermorphin and the analysis of structure–conformation–activity relationships in the series [Alaⁿ]-dermorphin. We used ¹H-nmr spectroscopy to study dermorphin and its analogs [Alaⁿ]-dermorphin (with n = 1, 2…7) dissolved in dimethylsufoxide. Conformational energy calculations using semiempirical partitioned energy function methods were then carried out on dermorphin and its [L -Ala²]-analog. Agreement between calculation and experiment is satisfying, both suggesting predominance of a type I β-turn around Pro⁶-Ser⁷ at the C-terminus and of an extended structure in the central sequence Phe³-Gly⁴-Tyr⁵. Detailed analysis by step-by-step substitutions with Ala indicates that intraresidue interactions dominate over medium-range interactions (between adjacent residues), although the latter may also have a noticeable influence in shaping conformations. As a general feature, the effects of substitutions on the arrangement of side chains are always larger on the succeeding residue than on the preceding residue. Almost all the variations of activity observed in the analogs can be explained from conformational changes occurring in the aromatic side chains of the biologically important Tyr¹, Phe³, and Tyr⁵ on substitutions effected on adjacent residues (fluctuations via medium-range interactions).     

Synthesis and biological properties of (Leu-6)-LH-RH and (D-Leu-6,desGly-NH210)-LH-RH ethylamide

[Leu⁶,desGly-NH₂¹⁰]-LH-RH ethylamide and [Leu⁶]-LH-RH, two analogs of LH-RH, were prepared by the solid phase method. LH- and FSH-releasing activity of these peptides was assayed against LH-RH by subcutaneous administration in immature male rats. When the integrated levels of LH and FSH over a 6 hr period after the injection were used as parameter of the LH- and FSH-releasing activities, the [Leu⁶]-LH-RH showed LH-releasing activity of 9 times and FSH-releasing activity of 5 times greater than that of LH-RH. [Leu⁶,desGly-NH₂¹⁰]-LH-RH ethylamide showed LH- and FSH-releasing activities of 53.6 and 14.5 times greater, respectively, than that of LH-RH.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the natural NH2-terminal fragment with a synthetic analog of the COOH-terminal fragment

Complementation of the natural [Cys (Cam)⁵³]-HGH-(1–134) fragment with synthetic analogs of COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰, Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) has been investigated. It was found that the synthetic fragment, [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187), gave a recombinant with about 40% HGH radioreceptor-binding activity. When compared with the natural recombinant, the semisynthetic hormone exhibited similar receptor-binding activities. The natural and semisynthetic recombinants were indistinguishable in radioimmunoassay. The α-helical content of the semisynthetic recombinant was completely restored in comparison with that of the native hormone as revealed by circular dichroism spectra. On the other hand, attempts to obtain a recombinant with the synthetic [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) were unsuccessful. The synthesis of [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) is herein described.     

Studies of the aminopeptidase proteolysis of semax analogues with different <Emphasis Type="Italic">N</Emphasis>-terminal amino acid residues

Proteolysis of semax (Met-Glu-His-Phe-Pro-Gly-Pro, Sem) and its analogues with the substitution of Ala, Gly, Thr, or Trp for the N-terminal Met was studied. This substitution was shown to change the degradation rate of these peptides by leucine aminopeptidase (EC 3.4.11.2, Sigma, Type VI, 9.2 activity units/mg). [Ala¹]Sem, [Gly¹]Sem, and [Thr¹]Sem (the semax analogues) proved to be more stable to the proteolysis than semax itself. It was demonstrated that the primary product of the proteolysis was His-Phe-Pro-Gly-Pro (Sem-5). In the case of [Trp¹]Sem, the comparable amount of Glu-His-Phe-Pro-Gly-Pro (Sem-6) was found to be formed along with Sem-5. In was established that all the studied semax analogues could be used as inhibitors of its proteolysis.     

Bioactive Neolignans and Other Compounds from Magnolia grandiflora L.: Isolation and Antiplasmodial Activity

Bioassay‐guided fractionation of a methanol extract of Magnolia grandiflora against Plasmodium falciparum yielded two new ( 1 and 2 ) and six known ( 3 – 8 ) bioactive compounds. The structures of the new compounds were assigned by mass spectrometric and 1D‐ and 2D‐NMR data. Known compounds were identified by comparison of ¹H‐NMR and MS data with literature data. The two known neolignans 3 and 4 showed moderate antiplasmodial activity with the IC₅₀ values of 2.8 ± 0.1 and 3.4 ± 0.1 μm , respectively. Weak antiplasmodial activity was recorded for compounds 1 , 2 , 5 , 6 , 7 , and 8 , with the IC₅₀ values of 38 ± 2, 23 ± 2, 16.5 ± 0.2, 86 ± 1, 44 ± 4, and 114 ± 9 μm , respectively.