Design and synthesis of biologically active peptides: a 'tail' of amino acids can modulate activity of synthetic cyclic peptides

In earlier work, we synthesized a cyclic 9-amino acid peptide (AFPep, cyclo[EKTOVNOGN]) and showed it to be useful for prevention and therapy of breast cancer. In an effort to explore the structure–function relationships of AFPep, we have designed analogs that bear a short ‘tail’ (one or two amino acids) attached to the cyclic peptide distal to its pharmacophore. Analogs that bore a tail of either one or two amino acids, either of which had a hydrophilic moiety in the side chain (e.g., cyclo[EKTOVNOGN]FS) exhibited greatly diminished biological activity (inhibition of estrogen-stimulated uterine growth) relative to AFPep. Analogs that bore a tail of either one or two amino acids which had hydrophobic (aliphatic or aromatic) side chains (e.g., cyclo[EKTOVNOGN]FI) retained (or had enhanced) growth inhibition activity. Combining in the same biological assay a hydrophilic-tailed analog with either AFPep or a hydrophobic-tailed analog resulted in decreased activity relative to that for AFPep or for the hydrophobic-tailed analog alone, suggesting that hydrophilic-tailed analogs are binding to a biologically active receptor. An analog with a disrupted pharmacophore (cyclo[EKTOVGOGN]) exhibited little or no growth inhibition activity. An analog with a hydrophilic tail and a disrupted pharmacophore (cyclo[EKTOVGOGN]FS) exhibited no growth inhibition activity of its own and did not affect the activity of a hydrophobic-tailed analog, but enhanced the growth inhibition activity of AFPep. These results are discussed in the context of a two-receptor model for binding of AFPep and ring-and-tail analogs. We suggest that tails on cyclic peptides may comprise a useful method to enhance diversity of peptide design and specificity of ligand–receptor interactions.     

Biological and physical properties of a beta-endorphin analog containing only D-amino acids in the amphiphilic helical segment 13-31

Our approach to the modeling of beta-endorphin has been based on the proposal that three basic structural units can be distinguished in the natural peptide hormone: a highly specific opiate recognition sequence at the N terminus (residues 1-5) connected via a hydrophilic link (residues 6-12) to a potential amphiphilic helix in the C-terminal residues 13-31. Our previous studies showed the validity of this approach and have demonstrated the importance of the amphiphilic helical structure in the C terminus of beta-endorphin. The present model, peptide 5, has been designed in order to evaluate further the requirements of the amphiphilic secondary structure as well as to determine the importance of this basic structural element as compared to more specific structural features which might occur in the C-terminal segment. For these reasons, peptide 5 retains the three structural units previously postulated for beta-endorphin; the major difference with regard to previous models is that the whole C-terminal segment, residues 13-31, has been built using only D-amino acids. In aqueous buffered solutions as well as in 2,2,2-trifluoroethanol-containing solutions, the CD spectra of peptide 5 show the presence of a considerable amount of left-handed helical structure. Enzymatic degradation studies employing rat brain homogenate indicate that peptide 5 is stable in this milieu. In delta- and mu-opiate receptor-binding assays, peptide 5 shows a slightly higher affinity than beta-endorphin for both receptors while retaining the same delta/mu selectivity. In opiate assays on the guinea pig ileum, the potency of peptide 5 is twice that of beta-endorphin. In the rat vas deferens assay, which is very specific for beta-endorphin, peptide 5 displays mixed agonist-antagonist activity. Most remarkably, peptide 5 displays a potent opiate analgesic effect when injected intracerebroventricularly into mice. At equal doses, the analgesic effect of peptide 5 is less than that of beta-endorphin (10-15%) but longer lasting. In conjunction with our previous model studies, these results clearly demonstrate that the amphiphilic helical structure in the C terminus of beta-endorphin is of predominant importance with regard to activity in rat vas deferens and analgesic assays. The similarity between the in vitro and in vivo opiate activities of beta-endorphin and peptide 5, when compared to the drastic change in chirality in the latter model, demonstrates that even a left-handed amphiphilic helix formed by D-amino acids can function satisfactorily as a structural unit in a beta-endorphin-like peptide.     

Synthesis and properties of the first all-aza analogue of a biologically active peptide

The synthesis of the first all-aza-amino acid analogue ( 2 ) of a peptidic renin inhibitor is described. The X-ray structural analysis and molecular modelling investigations of this novel compound reveal interesting conformational features which have a significant impact on its biological activity. In addition, insight into conformational features of azapeptides in general in comparison with the corresponding purely peptidic compounds is given.     

Changes with aging in the levels of amino acids in rat CNS structural elements I. Glutamate and related amino acids

Glutamate and related amino acids were determined in 53 discrete brain areas of 3-and 29-month-old male Fischer 344 rats microdissected with the punch technique. The levels of amino acids showed high regional variation-the ratio of the highest to lowest level was 9 for aspartate, 5 for glutamate, 6 for glutamine, and 21 for GABA. Several areas were found to have all four amino acids at very high or at very low level, but also some areas had some amino acids at high, others at low level. With age, in more than half of the areas, significant changes could be observed, decrease occurred 5 times more frequently than increase. Changes occurred more often in levels of aspartate and GABA than in those of glutamate or glutamine. The regional levels of glutamate and its related amino acids show severalfold variations, with the levels tending to decrease in the aged brain.     

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.     

Localization of the receptor-binding region of Clostridium perfringens enterotoxin utilizing cloned toxin fragments and synthetic peptides. The 30 C-terminal amino acids define a functional binding region.

In this study a short sequence encoding the receptor-binding activity of the much larger 35-kDa enterotoxin elaborated by Clostridium perfringens was localized by recombinant DNA techniques. Defined fragments corresponding to portions of the enterotoxin gene were cloned into an Escherichia coli expression vector system, and these lysates were analyzed for their ability to compete for binding with native C. perfringens enterotoxin (CPE). The lysate containing CPE290-319 (CPE sequence encompassing residues 290-319) was shown to compete with 125I-CPE for specific binding sites on rabbit intestinal brush border membranes. To confirm this finding, a peptide corresponding to the CPE amino acid sequence 290-319 was synthesized and found to completely block CPE specific binding. To demonstrate directly that CPE290-319 can act as a competitive antagonist of CPE cytotoxicity for physiologic receptors, Vero cells were preincubated with either E. coli lysates containing CPE290-319 or the synthetic peptide corresponding to this sequence. Preincubation of Vero cells with either the lysate or the peptide completely protected these cells from CPE challenge. This information localizes the C-terminal 30 residues of CPE (CPE290-319) as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor.     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

Application of rare-earth elements as labels in studying biologically active compounds. I. Luminescence spectra of solutions of complexes of pyridoxaldiene amino acids with europium]

The luminescence spectra of qater solutions of europium complexes with azometines formed by condensation of amino acids with pyrydoxal are investigated. The differences in luminescence spectra connected with the variations in the complexes structures are found. The possibilities of application of the europium ions luminescence for biochemical reactions investigation and for structure studies and identification of biologicaly active compounds are discussed.     

Design and biological activity of a new generation of synthetic C3a analogues by combination of peptidic and non-peptidic elements.

Based on published X-ray crystallographic data of the anaphylatoxic complement peptide C3a, we have synthesized a series of peptides with appropriate amino acid exchanges and a maximal length of 13 amino acids. N-terminal acylation of these optimized structures with epsilon-aminohexanoic acid and complex aromatic structures like fluorenylmethoxycarbonyl, 2-nitro-4-azidophenyl, fluoresceinyl and rhodaminyl leads to a dramatic increase in biological activity. The culmination of our synthetic efforts is a C3a analogue with 13 amino acid residues and a biological activity six times that of native C3a.     

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH₂) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.     

Interaction of phospholipids with proteins, peptides and amino acids. New advances 1987-1989

1. The review deals with the recent achievements in the study of the various interactions of phospholipids with proteins, peptides and amino acids. The interactions are classified according to the hydrophobic, hydrophilic or mixed character of the interactive forces. The effect of the interaction on the structure and biological activity of the interacting biomolecules is discussed.     

Methyl 5'-(6-aminopurin-9-yl)-5'-deoxy-beta-D-ribofuranoside 2',3'-cyclic monophosphate: a novel, biologically active, structural analogue of cyclic AMP

A novel structural analogue of cyclic AMP has been synthesized. This compound has been found to activate protein kinase from skeletal muscle (Ka 5.0 microM). It is virtually resistant to degradation by beef heart cAMP phosphodiesterase. It is an inhibitor of this enzyme with an [I]50 of 47.0 microM. The proliferation of cancer cells (HT-29) is inhibited by this compound. It represents the first example of a 2',3'-cyclic nucleotide with marked biological activity.     

Azure B and a synthetic structural analogue of methylene blue,ethylthioninium chloride,present with antidepressant-like properties

Aims

The phenothiazinium compound, methylene blue (MB), possesses diverse pharmacological actions and is attracting attention for the treatment of bipolar disorder and Alzheimer's disease. MB acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and possesses antidepressant activity in rodents. The goal of this study was to synthesise a structural analogue of MB, ethylthioninium chloride (ETC), and to evaluate the effects of the structural changes on the MAO inhibitory and antidepressant properties of MB. This study also investigated the antidepressant properties of azure B, the major metabolite of MB, versus MB and imipramine as active comparators.

Main methods

ETC and azure B were firstly evaluated as inhibitors of human MAO, and secondly for antidepressant-like activity in the acute forced swim test (FST) in rats, and compared to saline, imipramine and MB.

Key findings

The results document that ETC is a reversible inhibitor of MAO-A and MAO-B with IC₅₀ values of 0.510 μM and 0.592 μM, respectively, and that it is a weaker MAO-A inhibitor than MB and azure B. ETC and azure B were more effective than imipramine and MB in reversing immobility in the FST without inducing locomotor effects, with evidence supporting a serotonergic action. Of interest is the finding that ETC is more toxic for cultured cells than MB.

Conclusion

Azure B may therefore be a contributor to the antidepressant effect of MB. Small structural changes made to MB retain its antidepressant effect, even though the resulting phenothiazinium compound possesses reduced MAO-A inhibitory potency.     

Identification of a follicle-stimulating hormone receptor-binding region in hFSH-beta-(81-95) using synthetic peptides.

Pituitary and placental glycoprotein hormones are heterodimers with alpha-subunits of identical primary structure, but dissimilar beta-subunits. Regions of structural similarity between the beta-subunits might be involved in interaction with the homologous alpha-subunits, and regions of structural dissimilarity could, therefore, be candidates for receptor interactions. A restrained matrix dot-plot analysis identified hFSH-beta-(8-32) and hFSH-beta-(55-65) as candidates for interaction with alpha-subunit. Therefore, by subtraction, hFSH-beta-(33-54) and hFSH-beta-(66-111) seemed candidates for regions of interaction with receptor. In a previous report we demonstrated that hFSH-beta-(33-53) represented a receptor-binding region of hFSH-beta. Analysis of structural parameters (flexibility, surface probability, secondary structure prediction, etc.) indicates similarities between hFSH-beta-(33-53) and hFSH-beta-(85-95), suggesting the latter might be the component of hFSH-beta-(61-111) interacting with the receptor. Testing of 11 synthetic peptides, corresponding to the primary structure of hFSH-beta, demonstrated that hFSH-beta-(31-45)-peptide amide, were unique in ability to inhibit 125I-follicle-stimulating hormone binding to receptor. hFSH-beta-(81-95)-peptide amide also stimulated estradiol biosynthesis in Sertoli cell cultures. The correlation between binding inhibition and surface probability, flexibility, and predicted secondary structure (alpha, extended, and turn) was highly significant (R2 = 0.87, p less than 0.0001). Regression significance for these parameters, taken individually, were very poor. Receptor-binding regions, therefore, appear to be characterized by a particular and complex arrangement of secondary structure motifs, surface probability, and flexibility.     

Substitution of basic amino acids in the basic region stabilizes DNA binding by E12 homodimers.

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 has previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrophoretic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein complexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 can increase the intrinsic DNA-binding activity of E12 and stabilize the binding complexes, thus alleviating the repression from the inhibitory domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bHLH proteins, which all possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the myogenic bHLH proteins may need stronger DNA binding domains.     

Application of rare-earth elements as labels in studying biologically active compounds. III. Natural and magnetic circular dichroism of complexes of rare-earth elements with pyridoxalidene amino acid complexes]

Circular dichroism of pyrodoxalidenaminoacid complexes with rare earths and other metals and magnetic circular dichroism of several neodimium complexes are investigated. CD spectra of the complexes of the rare earths with azometines made of different amino acids could be classified into four groups. Some of the CD spectra are shown to be individual. The dependence of the CD spectra on the rare earch ion dimension is considered. Coordination of the rare earth ions is shown to be the same as in the case of zinc, cobalt, iron or copper.     

Eliminating the six N-terminal amino acids of the caspase 3 large subunit improved production of a biologically active IL2-Caspase3 chimeric protein

Designing a chimeric protein and developing a procedure for its stable production as a biologically active protein, are key steps in its potential application to clinical trails. IL2-Caspase3 chimeric protein designed to target activated T lymphocytes was found to be a promising molecule for targeted treatment, however was found to be difficult to produce as a biological active molecule. Thus, we designed a new version of the molecule, IL2-Caspase3s, in which six amino acids (aa 29-34) from the N-terminus of the large subunit of caspase 3 were excluded. Repeated expressions, productions, and partial purifications of the IL2-Caspase3s yielded reproducible batches with consistent results. We found that IL2-Caspase3s causes cell death in a specific, dose-, and time-dependent manner. Cell death due to IL2-Caspase3s is caused by apoptosis. This improved and biologically stable IL2-Caspase3s chimeric protein may be developed in the future for clinical trails as a promising therapy for several pathologies involving activated T-cells. Moreover, this truncated caspase 3 sequence, lacking the N-terminal six amino acids of its large subunit, may be used in other caspase 3-based chimeric proteins targeted against various human diseases, using the appropriate targeting moiety.     

Development of a bioactive fiber with immobilized synthetic peptides designed from the active site of a beetle defensin

The 9-mer peptides RLYLRIGRR and RLLLRIGRR were immobilized to amino-functionalized cotton fibers by a modification of the SPOT synthesis technique. The antibacterial activities of the peptide-immobilized cotton fibers against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) were investigated. Antibacterial assays revealed that these fibers inhibit the growth of MRSA and the antibacterial activities were maintained after washing and sterilization by autoclaving. The anticancer effect of the peptide-immobilized fiber was also investigated with mouse myeloma cells and human leukemia cells. These results indicate that these fibers have strong growth inhibition activity against bacteria and cancer cells.