Preparation and properties of novel gramicidin S analogs possessing a tri-, tetra- or pentamethylene bridge between ornithine side chains

Abstract: Gramicidin S (GS) analogs in which the N^δ atoms of the two Orn side chains are linked by an oligomethylene bridge [-(CH₂)_n-; n=3–5] were prepared via the bis(p-nitrobenzenesulfonyl) derivative [Orn(NBS)^2,2′]GS. For comparison the nonbridged secondary amino group-containing analog [Orn(Me)^2,2′]GS was also prepared. ¹H NMR and CD spectral analysis indicated that these analogs adopt the same β-sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene-bridged analog being the most potent.     

Properties of synthetic analogs of gramicidin S containing L-serine or L-glutamic acid residue in place of L-ornithine residue

In order to investigate the biological role of the δ-amino groups of the Orn residue in gramicidin S, the four analogs, [Ser²]-, [Ser^2,2′]-, [Glu²]- and [Glu^2,2′]-gramicidin S were synthesized by a solution method. Except for the last one, these analogs show antibiotic activity to a certain extent against gram-positive micro-organisms, but the activities are weaker than that of gramicidin S. These results indicate that the δ-amino group of the Orn residue is quite important for exhibiting of the activity of gramicidin S, but is not essential to the activity. NMR and CD studies of these analogs indicate that these analogs possess four intramolecular hydrogen bonds between the Val and Leu residues similar to those of GS, and the removal or diminution of the δ-amino group of ornithine residues give the considerable influence to the dihedral angle of N^αH-C^αH of D-Phe residues and consequently affect the type II′β-turn structure of the cyclic peptides. © Munksgaard 1996.     

Convenient preparation of [Orn(Tfa)2]- and [Orn(Boc)2, Orn(Tfa)2′]gramicidin S,versatile unsymmetrically protected derivatives of gramicidin S

Abstract: Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS. The mono(Tfa) derivative [Orn(Tfa)²]GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative [Orn(Boc)²,Orn(Tfa)^2′]GS from which another monoprotected derivative [Orn(Boc)²]GS was prepared in high yield. These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives. As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative [Orn(Boc)²,Orn(NBS)^2′]GS.     

Role of ring size on the secondary structure and antibiotic activity of gramicidin S

In order to investigate the contribution of ring size to the secondary structure and antibiotic activity of gramicidin S, many analogs consisting of 6, 7, 8, 9, 11, 12, 13, and 14 amino acid residues were synthesized. In the analogs with smaller ring sizes than that of gramicidin S, only des-Val¹-, des-Leu³- and des-Pro⁵-gramicidin S showed weak activity against the gram-positive micro-organisms tested. On the other hand, all analogs having larger rings, in which L- or D-Leu residues were inserted, were active. The activity of the analogs consisting of 10, 11 and 12 amino acid residues was stronger than those of the analogs with other ring sizes, and the activity of endo-Leu^2a-gramicidin S against S. epidermidis SP-al-1 and B. subtilis ATCC 6633 was twice of that of gramicidin S.     

Studies of peptide antibiotics. XLIV. Syntheses and biological activities of gramicidin S analogs containing δ-hydroxy-l-norvaline or glycine

The antibiotic gramicidin S (GS) has the structure of cyclo (-l -Val¹-L-Orn²-l -Leu³-d -Phe⁴-l -Pro⁵-L-Val¹′-l -Orn²′-l -Leu³′-d -Phe⁴′-l -Pro⁵′-) and is basic in character. Five GS analogs including [Gly^1,1′]-GS and the neutral [l -Hnv^2,2′]-GS (Hnv represents δ-hydroxynorvaline) were synthesized by the solid-phase method to evaluate the role of l -Val^1,1′ and l -Orn^2,2′ residues in GS. The hybrid analogs ([Gly¹]-GS and [l -Hnv²]-GS) and [d -Tyr^4,4′]-GS showed high antibacterial activities, whereas [Gly^1,1′]-GS and [l -Hnv^2,2′]-GS possessed no activity. Inhibitory effects by these analogs for the adsorption of ¹⁴C-labeled GS on cells of bacteria sensitive to GS were determined. The structure-activity relationship of GS is discussed on the basis of the results on these GS analogs.     

Conformationally stabilized gramicidin S analog containing dehydrophenylalanine in place of d-phenylalanine4,4′

Unsaturated gramicidin S analog, [ΔPhe^4,4′]gramicidin S, was synthesized by conventional solution method in order to evaluate the role of the dehydrophenylalanine residues replacing d -phenylalanine^4,4′ in stabilizing the bioactive β-shect conformation. The dehydrophenylalanine (ΔPhe) moiety was introduced by dehydroazlactonization of the β-phenylserine residue. The [ΔPhe^4,4′]gramicidin S prepared by this method showed very strong antimicrobial activities against Gram-positive bacteria, but not against Gram-negative ones. Several lines of spectroscopic evidence indicated that [ΔPhe^4,4′] gramicidin S has a reinforced β-sheet backbone conformation necessary for a full biological activity of gramicidin S. These results suggested that :α,β-dehydrogenation of the amino acid residue in a cyclic peptide can stabilize the turn structure.     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-α, β-diaminopropionic acid or α,β-dehydroalanine*

A gramicidin S (GS) analog ([d -Dpr^4,4] GS) containing d -α,β-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4′ positions was derived from [l -Orn(δ-formyl)^2,2, d -Dpr(β-Z)^4,4′]GS, which was synthesized by conventional method in solution. An analog [ΔAla^4,4′]GS was synthesized from [l -Orn(δ-Boc)^2,2′, d -Dpr^4,4′]GS through Hofmann degradation of the d -Dpr residues. Antimicrobial activities of these analogs were tested; [d -Dpr(β-Z)^4,4′]GS and [ΔAla^4,4′]GS showed high antimicrobial activities against Gram-positive bacteria. [d -Dpr^4,4′]-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [ΔAla⁴]semiGS were synthesized; these had no antimicrobial activity. Analogs containing ΔAla residues were hydrogenated, and the formation of l -Ala or d -Ala residues was determined. The ΔAla residues in [ΔAla^4,4′] GS were reduced to dl -Ala, and ΔAla in [ΔAla⁴]semiGS mostly to l -Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Effect of Drug substances on the microviscosity of lipid bilayer of liposomal membrane

The microviscosities of the lipid bilayers of liposomal membranes of phospholipids were measured by the intermolecular excimer, formation method employing pyrene as a fluorescence probe, and the effects ofn-alkanols and other local anesthetics on the microviscosity were investigated. The results showed that then-alkanols and the other local anesthetics effectively lowered the microviscosity of the lipid bilayer of the dipalmitoyl phosphatidycholine liposomal membrane in proportion to the concentration of the additives. Moreover, there was a fairly good correlation between the local anesthetic activities and the microviscosity-lowering activities of these drugs. This results suggests that the nerveblocking activity of local anesthetics might have some relation with their activity fluidizing the lipid bilayer of biomembrane.     

Interaction of glucagon with artificial lipid bilayer membranes

The enhancement of fluorescence emission from the tryptophan residue of glucagon, the quenching of that emission with acrylamide and with 5-doxyl and 16-doxyl stearic acid, circular dichroism spectra, the release of 6-carboxytluorescein, and polarized infrared attenuated total reflection (IR-ATR) spectra were used to study the interaction of glucagon with intact lipid vesicles and flat bilayers. Dimyristoylphosphatidylcholine bound the peptide only below the main transition temperature, thus confirming earlier results of Epand et al. (1977). However, the peptide is also bound by vesicles of unsaturated lipids above their transition temperature, suggesting an influence of lipid area on the binding process. Circular dichroism showed that binding to such vesicles also increases the helix content of glucagon. The IR-ATR study and a comparison with dynorphin-A-(I-13)-tridecapeptide revealed profound differences in orientation of the two peptides. The dichroic ratios and the derived order parameters indicated an isotropic orientation of the helical segments of glucagon, but did not exclude a principal orientation of the molecules lying flat on the nienibrane surface. In contrast, the axis of the dynorphin helix is clearly oriented normal to the interface. The two peptides also differ in their rates of 6-carboxyfluorescein release, suggesting a deeper penetration of the primary amphiphilic helix of dynorphin A-(I-13) than of the secondary amphiphilic helix of glucagon.     

Synthesis and NMR conformational analysis of a β-turn mimic incorporated into gramicidin S

The solution structure of a gramicidin S (GS) analog containing a β-turn mimic[BTD^4-5, Lys^2,2′]GS has been compared to that of native GS. The linear [BTD^4-5, Lys^2,2′]GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD^4-5, Lys^2,2′]GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular Hα-Hα NOE, and J^Nα coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' β-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test β-turn mimics.     

Proline residue‐modified polycationic analogs of gramicidin S with high antibacterial activity against both Gram‐positive and Gram‐negative bacteria and low hemolytic activity*

Abstract: Novel polycationic analogs of the cyclic decapeptide antibiotic, gramicidin S, possessing NH₂, d /l ‐Phe‐NH or l ‐Lys‐NH groups at the 4α‐ or 4β‐positions of the l ‐Pro residues, were synthesized. While l ‐Pro(4α/β‐NH₂)‐containing analogs exhibited much weaker antibacterial activity, the d /l ‐Phe and l ‐Lys‐substituted analogs exhibited higher antibacterial activity against Gram‐negative bacteria than the parent gramicidin S. All of these additional amino group‐containing analogs showed substantially reduced toxicity against human blood cells.     

Cationic liposome co-encapsulation of SMAC mimetic and zVAD using a novel lipid bilayer fusion loaded with MLKL-pDNA for tumour inhibition in vivo

The increase in multidrug resistance among colon cancer cells presents a challenge for the development of effective therapies. Small-molecule analogues of second mitochondria-derived activator of caspase (SMAC) mimetic in association with mixed lineage kinase domain-like protein (MLKL)-pDNA and z-VAD-fmk have shown ideal antitumor effects in colon cancer cells in vitro via induction of RIP3-dependent necroptosis. To achieve synergistic antitumor effects in vivo, liposomes loaded with SMAC mimetic, MLKL-pDNA and z-VAD-fmk have been developed using novel lipid fusion methods to co-localise the molecules of interest within the tumour cells. The co-encapsulation liposome (MLKL-zVAD-BV6-LP) had a high entrapment efficiency of approximately 95% for both zVAD and BV6 and was able to condense MLKL-pDNA very well. The vectors showed good biocompatibility, tumour targeting and small-molecule co-localisation. In a CT26 mouse model, the MLKL-zVAD-BV6-LP exhibited a tumour-suppression rate of over 60% in vivo, which was significantly higher than that of both the null-liposome and coadministration groups. Above all, the co-encapsulation system provided a novel approach to combination tumour therapy.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity

Abstract: A 25‐mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure–activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an α‐helical structure in the presence of DOPC/DOPG (3 : 1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye‐leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye‐leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that α‐helicity, rather than hydrophobicity, affects the properties and activity of the peptide.     

20S蛋白酶体抑制剂吡咯啉酮类似物的设计、合成及活性测试(英文)

本文设计并合成了一系列吡咯啉酮类似物,这些化合物可能作为Michael受体与蛋白酶体活性位点Thr1O~γ不可逆结合,从而抑制蛋白酶体的活性。虽然活性测试结果显示这些化合物对蛋白酶体的抑制活性很弱,但是可以通过吡咯啉酮类化合物进一步的结构优化来提高对蛋白酶体的抑制活性。     

Interaction of butylated hydroxytoluene (BHT) with phospholipid bilayer membranes: effect on 22Na permeability and membrane fluidity.

Butylated hydroxytoluene (BHT) is a small lipophilic molecule that is widely used as a food preservative. The interaction of this chemical with phospholipid bilayer membranes was monitored by measuring changes in ²²Na transport and hydrocarbon chain motion. Vesicles composed of saturated phospholipids display a marked increase in ²²Na permeability in the temperature region of the phase transition temperature of the component phospholipid. BHT greatly reduces this permeability increase. When a series of structural analogues was examined, there was found to be a poor correlation between lipid solubility and the capacity to decrease ²²Na transport. However, the presence of a hydroxyl group appeared to be an important structural requirement for the permeability change. In a parallel set of experiments, a spin labelled fatty acid ester was incorporated into similar vesicles and the mobility of the label used as a measure of lipid hydrocarbon chain motion. The phase transition temperature of a phospholipid is associated with a marked increase in fatty acyl chain motion. BHT lowers the temperature at which the lipid chains display significant motional freedom (i.e. melt). Since this change in membrane fluidity cannot account for the capacity of BHT to reduce ²²Na permeability some additional perturbation must be occurring. It is proposed that this additional perturbation involves an alteration at the interface.     

Preparation,characterization and tissue distribution of brucine stealth liposomes with different lipid composition

Objective: To improve the therapeutic index of brucine, the novel stealth liposomes (SLS-n), composed of naturally unsaturated and hydrogenated soy phosphatidylcholines, with significant difference of phase transition temperature, were developed to encapsulate brucine.

Methods: Brucine-loaded stealth liposomes with different lipid compositions were prepared and characterized for their entrapment efficiency (EE), particle size, zeta potential and in vitro drug release profile. Tissue distribution after intravenous administration of different brucine formulations was further compared in tumor-bearing mice.

Results: Compared with the conventional stealth liposomes composed of SPC (SLS-s) or HSPC (SLS-h), EE and zeta potential of SLS-n were increased slightly, and the size was decreased slightly. The results of drug release showed that SLS-n were more stable than SLS-s. After intravenous administration, tumor AUC in SLS-s, SLS-n and SLS-h treated animals were 1.33, 1.72 and 2.59-fold higher than in mice treated with the same dose of free brucine, respectively. Compared with brucine solution, administration of SLS-s and SLS-n could significantly decrease brucine concentration in brain, but administration of SLS-h resulting in significantly increased (2.75-fold) concentration in 10?min.

Conclusion: Since brucine has severe central nervous system toxicity, our study indicated that SLS-n could considerably improve the therapeutic index of brucine.     

Interaction of d-tubocurarine analogs with mutant 5-HT(3) receptors

Apomorphine has been introduced in the treatment of late-stage Parkinson's Disease (PD). The disadvantage of a short half-life of apomorphine is now overcome by the use of a continuous subcutaneous (s.c.) self-delivering system. We examined whether continuous s.c. infusion of apomorphine rescues nigro-striatal dopaminergic neurons from toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Apomorphine was continuously infused in mice by means of a s.c. minipump that delivered the drug at a rate of 0.5 or 3.15 mg/kg/day. MPTP induced a >80% reduction in striatal dopamine (DA) after one day. DA levels were still substantially reduced one month following MPTP injection, in spite of a partial recovery. Similarly, striatal immunoreactivity for tyrosine hydroxylase and dopamine transporter was markedly reduced at this time interval. Continuous s.c. infusion of apomorphine starting 40 h following MPTP injection rescued striatal dopaminergic terminals, as assessed by measurements of DA and its metabolites, as well as TH and DAT immunostaining after one month. The neurorescuing effect was more remarkable at a delivery rate of 3.15 mg/kg/day of apomorphine. In contrast, no rescue was observed when apomorphine was administered as a single daily s.c. bolus of 1 or 5mg/kg starting 40 h following MPTP. We conclude that apomorphine is able to rescue nigro-striatal dopaminergic neurons when continuously delivered at doses that are comparable to those delivered by minipumps in PD patients. These results suggest that continuous s.c. infusion of apomorphine not only relieves the symptoms, but also reduce the ongoing degeneration of nigro-striatal dopaminergic neurons in PD patients.     

Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers

Abstract: To investigate the role of peptide–membrane interactions in the biological activity of cyclic cationic peptides, the conformations and interactions of four membrane‐active antimicrobial peptides [based on Gramicidin S (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidylcholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic peptides include GS10 [Cyclo(VKLdY P)₂], GS12 [Cyclo(VKLKdY PKVKLdY P)], GS14 [Cyclo(VKLKVdY PLKVKLdY P)] and [d ‐Lys]⁴GS14 [Cyclo(VKLdK VdY PLKVKLdY P)] (underlined residues are d ‐amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. Interaction of the peptides with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the peptides induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [d ‐Lys]⁴GS14 > GS14 > GS12 > GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [d ‐Lys]⁴GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic peptides with lipid bilayers, the mechanism of their biological activity (and its relation to peptide–lipid interaction) proved to be versatile and complex, and dependent on the biophysical properties of both the peptides and membranes.