The synthesis of immunomodulating peptide alloferon, the active principle of antiviral drug allokine-alpha

Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The solid phase variant of the synthesis was shown to be more technological; it allows the preparation of the product at a higher total yield (40% vs. 17% for conventional synthesis in solution from the starting derivatives of the C-terminal dipeptide). The by-products formed during the synthesis of alloferon were identified.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

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.     

Inhibitory Effect of Alloferons in Combination with Human Lymphocytes on Human Herpesvirus 1 (HHV-1) Replication In Vitro

Over the past two decades there has been intense study of compounds from vertebrates, microorganisms, plants, mushrooms, marine sponges, worms, etc. as well as insects in terms of their antiviral activity. Insects produce a variety of biologically active peptides. One of them is alloferon. The in vitro and in vivo experiments demonstrate that synthetic alloferon has an immunomodulatory properties. It was reported that alloferon and its analogues (alloferon I and II) have antimicrobial properties, as well. The aim of this study was to evaluate in vitro the effect of alloferon I and II, either alone or in combination with human lymphocytes, on human herpesvirus type 1 (HHV-1) McIntyre strain replication. On the base of results we can conclude that alloferon I and II inhibit the replication of HHV-1 McIntyre strain in HEp-2 cells. Enhanced antiviral activity was observed when infected cells were treated with alloferons and unstimulated or phytohemagglutinin PHA-stimulated lymphocytes simultaneously. After application of alloferons and PHA-stimulated lymphocytes to the HHV-1 infected HEp-2 culture, the mean HHV-1 titer reduction for alloferon and II, when used at the highest dose—400 µg/mL, were 3.69 and 3.27 log₁₀/TCID_50/mL, respectively.     

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH₂)¹]‐ and [Phe(p‐OMe)¹]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.     

Studies of insect peptides alloferon,Any‐GS and their analogues. Synthesis and antiherpes activity

The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as alloferon, Any‐GS and their analogues. The synthesis of 14 peptides was performed by the solid‐phase method. Biological effect of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV‐1) in vitro using a Vero cell line. It was found that the investigated peptides inhibit the replication of HHV‐1 in Vero cells. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Mononuclear copper(II) complexes of alloferons 1 and 2: a combined potentiometric and spectroscopic studies

Mononuclear copper(II) complexes of the alloferon 1 His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, alloferon 2 Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, Ac-alloferon 1 Ac-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly and Ac-alloferon 2 Ac-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly have been studied by potentiometric, UV-vis, CD and EPR spectroscopic methods. The potentiometric and spectroscopic data shows that acetylation of the amino terminal group induces significant changes in the coordination properties of the Ac-alloferons 1 and 2 compared to the alloferons 1 and 2, respectively. The presence of four (Ac-alloferon 1) or three (Ac-alloferon 2) histidyl residues provides a high possibility for the formation of macrochelates via the exclusive binding of imidazole-N donor atoms. The macrochelation suppresses, but cannot preclude the deprotonation and metal ion coordination of amide functions and the CuH₋₃L species with {N_Im, 3N⁻} bonding mode at pH above 8 are formed. The N-terminal amino group of the alloferons 1 and 2 takes part in the coordination of the metal ion and the 4N complex with {NH₂, 3N_Im} coordination mode dominates at physiological pH 7.4 for alloferon 1 and the 3N {NH₂, CO, 2N_Im} binding mode for alloferon 2. However, at higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions.     

Role of the Cytoplasmic Membrane in the Synthesis of Ribonucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Disrupted spheroplast preparations of Pseudomonas schuylkilliensis strain P contained fragments of cytoplasmic membrane and approximately 82% of the total cellular phospholipid. The protoplast-bursting factor (PB-factor), partially purified from pig pancreas, and a heat-treated pancreatic lipase fraction both inhibited ribonucleic acid (RNA) synthesis by disrupted spheroplasts but did not inhibit or only slightly inhibited RNA synthesis by intact cells or intact spheroplasts. The PB-factor preparation and the heat-treated pancreatic lipase fraction catalyzed partial (15 to 50%) deacylation of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine in disrupted spheroplasts but not in intact spheroplasts. Phospholipase A activity was demonstrated in the PB-factor preparation by use of isolated phospholipids as substrates. Treatment of disrupted spheroplasts with the PB-factor preparation caused a 70% inhibition in oxidative phosphorylation and RNA synthesis, but had little effect on electron transport. Addition of adenosine-5'-triphosphate or adenosine-5'-diphosphate and a mixture of ribonucleosides after treatment with the PB-factor preparation partially restored oxidative phosphorylation but did not relieve the inhibition in RNA synthesis. The most reasonable explanation for the latter observation appears to be that the concentrations of newly synthesized nucleotides retained by the preparations with partially deacylated membrane phospholipids were insufficient to permit the synthesis of RNA.     

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.     

Synthesis of the dodecapeptide corresponding to domain III of bovine brain calmodulin: alpha-beta shift side reactions during the synthesis by the classical method in solution

We report details of the chemical synthesis of the dodecapeptide corresponding to the calcium binding loop III of bovine brain calmodulin (sequence 93-104) and its fragments 96-04, 93-98, and 99-104. The preparation of the peptides employed classical solution methods and a fragment-condensation strategy. The major difficulties were encountered during the synthesis of the peptides containing the N-terminal sequences -Gly-Asn-Gly- and -Asp-Lys-Asp-Gly-Ans-Gly-, in which alpha-beta shift side reactions were observed.     

Preparation of ADP-Glucose with an Enzyme from Arthrobacter simplex

For the purpose of enzymatic preparation of ADP-glucose (ADPG), bacterial screening was performed to find a strain having a high activity of ADPG pyrophosphorylase which catalyzes the synthesis of ADPG from ATP and glucose-1-phosphate. A cell-free extract of Arthrobacter simplex IFO 12069 showed a strong enzyme activity for the synthesis of ADPG, which was isolated from the reaction solution by ion-exchange column chromatography and identified by paper and thin-layer chromatography. The enzyme activity of the bacterium reached a maximum in the late logarithmic phase under aerobic growth conditions. Some factors affecting the ADPG synthesis, e.g. reaction pH, substrate concentrations, divalent cations, inhibitors and activators, were studied with an ammonium sulfate fraction, 30~50% saturation as the enzyme preparation.     

Effect of different doses of a chalone-containing alcohol precipitate of Ehrlich ascitic tumor on the mitotic activity and DNA synthesis in this tumor

Chalone-containing preparation has been obtained from ascitic Ehrlich's tumour by alcohol precipitation and the effect of various preparation doses on mitotic activity and DNA synthesis in the tumour has been studied. The preparation was shown to suppress tumour cell proliferation, acting on mitosis initiation and mitotic S phase as well as on DNA synthesis in the cells at S phase of mitotic cycle. The effect of the preparation on DNA synthesis in phase S cells was more pronounced than on cells entering DNA synthesis phase. The changes in all the parameters examined were dose-dependent. The preparation effect was tissue-specific.     

Experiments towards the synthesis of the ergot alkaloids and related structures. Part 8. The total synthesis of 4-methyl-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carbonitrile hydrochloride hemihydrate,an immediate precursor of the despyrrole analogues of lysergic acid and its amides

Exposure of the N-methoxycarbonyl-bicyclic-keto-acid 5 (improved preparation) to the Barnick beta-keto-acid synthesis yielded an aqueous solution of the sodium salts of the beta-keto-acids 26 and 27 which on heating at 60-65 degrees C furnished the N-methoxycarbonyl-tricyclic-ketone 9 (55%) plus the hydroxy-ketone 28 which on acid treatment raised the yield of 9 to 68%. Reduction (NaBH4) of 9 yielded the alcohol 32 (94%) which was treated with thionyl chloride followed by copper (I) cyanide and sodium iodide in acetonitrile to give the tricyclic-N-methoxycarbonyl nitrile 35 whose relative configuration was obtained by X-ray analysis. Attempts to remove the N-methoxycarbonyl group from 35 were unsuccessful. Conversion of the alcohol 32 to its methoxypropyl ether 41 followed by reaction with ethereal MeLi-LiBr yielded the amino-alcohol 39 (75%) converted to the N-formyl-tricyclic alcohol 42 with formic-acetic anhydride (70%). The alcohol 42 was then converted into the N-formyl nitrile 44 via the chloride 43 as employed in the earlier synthesis of the nitrile 35. Removal of the N-formyl group from the nitrile 44 was achieved by refluxing methanolic hydrochloric acid to give the required amino-nitrile hydrochloride 46 (91%) whose structure was confirmed by X-ray analysis. Reaction of the free base with methyl iodide in ethyl acetate in the presence of calcium carbonate furnished the N-methyl base 48 isolated as its hydrochloride, hemihydrate 49 (59%). The overall yield of 49 via this eleven-step synthesis was 3.4%.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

Experiments Towards the Synthesis of the Ergot Alkaloids and Related Structures. Part 8.17 The Total Synthesis of 4-Methyl-2,3,4,4a,5,6-hexahydro-benzo[f]quinoline-2-carbonitrile Hydrochloride Hemihydrate,an Immediate Precursor of the Despyrrole Analogues of Lysergic Acid and Its Amides

Exposure of the N -methoxycarbonyl-bicyclic-keto-acid 5 (improved preparation) to the Barnick β -keto-acid synthesis 1 yielded an aqueous solution of the sodium salts of the β -keto-acids 26 and 27 which on heating at 60-65°C furnished the N -methoxycarbonyl-tricyclic-ketone 9 (55%) plus the hydroxy-ketone 28 which on acid treatment raised the yield of 9 to 68%. Reduction (NaBH 4) of 9 yielded the alcohol 32 (94%) which was treated with thionyl chloride followed by copper (I) cyanide and sodium iodide in acetonitrile to give the tricyclic- N -methoxycarbonyl nitrile 35 whose relative configuration was obtained by X-ray analysis. Attempts to remove the N -methoxycarbonyl group from 35 were unsuccessful. Conversion of the alcohol 32 to its methoxypropyl ether 41 followed by reaction with ethereal MeLi-LiBr yielded the amino-alcohol 39 (75%) converted to the N -formyl-tricyclic alcohol 42 with formic-acetic anhydride (70%). The alcohol 42 was then converted into the N -formyl nitrile 44 via the chloride 43 as employed in the earlier synthesis of the nitrile 35. Removal of the N -formyl group from the nitrile 44 was achieved by refluxing methanolic hydrochloric acid to give the required amino-nitrile hydrochloride 46 (91%) whose structure was confirmed by X-ray analysis. Reaction of the free base with methyl iodide in ethyl acetate in the presence of calcium carbonate furnished the N -methyl base 48 isolated as its hydrochloride, hemihydrate 49 (59%). The overall yield of 49 via this eleven-step synthesis was 3.4%.     

Effects of increasing extracellular pH on protein synthesis and protein degradation in the perfused working rat heart.

Increasing the extracellular pH over the range pH 7.4-8.9 stimulated protein synthesis by about 60% in the rat heart preparation anterogradely perfused in vitro. Protein degradation was inhibited by this pH increase. The magnitudes of the effects at pH 8.9 on protein synthesis and degradation were similar to those of high concentrations of insulin. Cardiac outputs were increased, as were cardiac phosphocreatine contents, indicating that the alterations in extracellular pH did not adversely affect the physiological viability of the preparation. ATP contents were unaltered. The creatine kinase equilibrium was used to assess the magnitude of the change in intracellular pH induced by these treatments. The increase in intracellular pH was about 0.2 for a 1-unit increase in extracellular pH. Thus small changes in intracellular pH have dramatic effects on cardiac protein turnover.     

Studies on the cell-free biosynthesis of beta-lactam antibiotics.

Cell walls of Cephalosporium acremonium mycelia were lysed by enzyme preparations from either Helix pomatia (snail) digestive juice or Cytophaga. The yield of protoplasts depended on the lytic-enzyme preparation and the age of the culture, and it increased after the mycelia were pretreated with dithiothreitol. A cell-free preparation, obtained by osmotic lysis of protoplasts, synthesized labelled penicillin N from L-[14C]valine. Approx. 0.03-0.06% of the amino acid was incorporated into penicillin N. Under conditions of penicillin N synthesis, the broken-protoplast preparation failed to produce significant amounts of cephalosporin C or its precursors, deacetylcephalosporin C and deacetoxycephalosporin C.     

Synthesis and use of peptide substrates for the isolation and characterization of proenkephalin processing enzymes

Classical methods of peptide synthesis in solution were used for the preparation of the two tetrapeptides alanyl-lysyl-arginyl-tyrosine and acetyl-alanyl-lysyl-arginyl-tyrosine-(N-methylamide). The two compounds were able to be recognized as substrates by proenkephalin processing enzymes and were used for the development of a quantitative assay for these enzymes. The first substrate proved to be convenient, although it was also partially degraded by amino- and carboxypeptidases under the conditions of the assay. The second was found to be hydrolyzed by the endopeptidases at too slow a rate to allow its routine use in the assay.     

Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.     

Effect of sulfochloranthine on protein and nucleic acid biosynthesis in Escherichia coli

Sulfochloranthine was shown to be bacteriostatic for Escherichia coli B cells grown in a chemically defined medium at a concentration of 0.002%, sublethal at a concentration of 0.005%, and lethal at 0.01% (0.000312, 0.00078 and 0.00156% of active chlorine, respectively). Protein synthesis by E. coli B cells was noticeably inhibited when the concentration of the preparation was 0.002%, and stopped completely at a 0.01% concentration of the preparation. Biosynthesis of nucleic acids, in particular DNA, was inhibited to a lesser extent. The bacteriostatic concentration of the preparation had virtually no effect on DNA biosynthesis, but inhibited RNA biosynthesis by 50%. Sulfochloranthine used at sublethal doses inhibited synthesis of both DNA and RNA by 75%; DNA and RNA biosynthesis ceased at the lethal concentration of the preparation.