Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes

The brush border membrane of the insect midgut is an initial site for interaction of insecticidal proteins. We have investigated the possibility that it may contain a target site for two insecticidal fungal toxins, destruxin and efrapeptin, both of which are ATPase inhibitors. We have studied the effects of the toxins on the hydrolytic activity of a vacuolar type ATPase (V-ATPase) that we have identified from Galleria mellonella midgut columnar cell brush border membrane vesicles (BBMV) by its cation and pH dependence, sensitivity to proton pump inhibitors and K(m) (0.49 mM ATP). Efrapeptin strongly inhibited the BBMV V-ATPase but destruxin had little effect. We compared the effects of the inhibitors on known plant membrane hydrolytic enzymes, and although the vacuolar pyrophosphatase and plasma membrane ATPase were not inhibited by the toxins, the V-ATPase from mung bean, but not barley, was inhibited (50%) by 10 microM concentrations of both compounds. Different forms of the toxins were tested on the ATPases and destruxin B and efrapeptin F were the most effective. Kinetic analysis showed that the purified forms of both compounds inhibited the V-ATPases uncompetitively and modelling of data for inhibition of the BBMV V-ATPase by efrapeptin at concentrations of 0.06--12 microM yielded a K(i) of 0.125 microM.     

Molecular modeling, synthesis, and screening of new bacterial quorumsensing antagonists

Abstract A new series comprising 7 analogs of N-(sulfanyl ethanoyl)-L-HSL derivatives, 2 analogs of N-(fluoroalkanoyl)- L-HSL derivatives, N-(fluorosulfonyl)-L-HSL, and 2,2-dimethyl butanoyl HSL were synthesized using a solid-phase organic synthesis method. Each of the 11 synthesized compounds was analyzed using NMR and mass spectroscopies, and molecular modeling studies of the 11 ligands were performed using SYBYL packages. Thereafter, a bacterial test was designed to identify their quorum-sensing inhibition activity and antifouling efficacy. Most of the synthesized compounds were found to be effective as quorum-sensing antagonists, where antagonist screening revealed that 10 among the 11 synthesized ligands were able to antagonize the quorum sensing of A. tumefaciens.     

Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.     

An apparatus for simultaneous manual solid-phase synthesis of multiple peptide analogs

A new design of reaction vessel for simultaneous manual solid-phase synthesis of multiple peptide analogs is described. Simultaneous use of four of these vessels attached to a single rotary mixer has been successfully applied to synthesis of two sets of four decapeptide amide analogs. Efficient coupling was indicated by chemical determination at the end of each synthesis cycle and overall final yields of between 78 and 84% were obtained. The products obtained were of a high quality, as assessed by high-performance liquid chromatography and amino acid analysis. This system allows expeditious synthesis of multiple peptide analogs for structure-function studies with economical use of efficiently ventilated laboratory space.     

The destruxins: synthesis, biosynthesis, biotransformation, and biological activity

Destruxins, secondary metabolites first reported in 1961, are cyclic hexadepsipeptides composed of an alpha-hydroxy acid and five amino acid residues. The name "destruxin" is derived from "destructor" from the species Oospora destructor, the entomopathogenic fungus from which these metabolites were first isolated. Individual destruxins differ on the hydroxy acid, N-methylation, and R group of the amino acid residues; where established, the configurations of the amino acid residues are S, and those of the hydroxy acids are R. Destruxins exhibit a wide variety of biological activities, but are best known for their insecticidal and phytotoxic activities. The great interest in destruxins derives from their potential role as virulence factors in fungi, whether such microorganisms are useful insect biocontrol agents or detrimental, causing great plant disease epidemics. Reports on isolation, chemical structure determination, total synthesis, transformation by diverse organisms, and biological activity of destruxins and related metabolites are reviewed for the first time.     

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.     

Synthesis of allose-templated hydroxyornithine and hydroxyarginine analogs

Conformationally constrained amino acid analogs are widely used to probe the bioactive conformation of peptides. In this paper we report on the synthesis of hexafunctional allose-templated l- and d-hydroxyornithine and l- and d-hydroxyarginine analogs in which the allose-based polyol scaffold constrains the side chain of hydroxyornithine and hydroxyarginine in an extended conformation. The partially protected building blocks were selected for future use in solid-phase peptide synthesis using the Fmoc-strategy. The synthesis starts from a previously prepared C-glucosyl glycine analog. Multiple chemical protection-deprotection steps and an oxidation are used to prepare 3-keto-C-glucosyl analogs that serve as a precursor to install an amino function via reductive amination. Guanidinylation of the amino group provides access to allose-templated hydroxyarginine analogs. Both hexafunctional building blocks are further chemically modified to provide suitable protection for solid-phase peptide synthesis using the Fmoc-strategy.     

Antifeedant Properties of Destruxins and their Potential Use with the Entomogenous Fungus Metarhizium anisopliae for Improved Control of Crucifer Pests

Destruxins A, B and E, produced by the entomogenous fungus Metarhizium anisopliae, are insecticidal but comparatively low doses have antifeedant properties. Treatment of cabbage leaf discs with destruxins significantly reduced feeding by larvae of Plutella xylostella and Phaedon cochleariae in both choice and no-choice assays. The Antifeedant Index (AI) was dose related and there were significant differences between treated and untreated leaves. The AI and acute toxicity assays suggest that insect death was due to a combination of the starvation and toxicity effects of destruxins. In whole plant experiments, adults and larvae of P. cochleariae were found to be more susceptible to infection by M. anisopliae V245 if it was used in conjunction with a crude destruxin mixture. Destruxins drove larvae off the plant, irrespective of which leaf surface was treated. Adults could be forced to the adaxial or abaxial surface of leaves using the crude destruxin. Mortality was usually more consistent and generally greater if adults were forced to abaxial than adaxial surfaces inoculated with the fungus. High humidity on the abaxial surface favoured conidia germination and infection. Mortality was also greater for adults dusted with the pathogen and forced to the abaxial rather than to the adaxial leaf surface. The increased movement and starvation associated with destruxin treatment may also have stressed the insects making them more susceptible to infection.     

Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.

Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes for enzyme activation, we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17-mer peptide corresponding to residues 187-203 of SNAP-25. A more stable substrate, Nle202SNAP-25 [187-203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time-dependent inhibition. Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described. These substrate-derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.     

The pro-oligonucleotide approach: solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates.

A modified phosphoramidite method has been designed for the solid-phase synthesis of two dodecathymidine phosphotriesters and two dodecathymidine thionophosphotriesters. In these analogs, each internucleoside link bears an S -acyl-2-thioethyl (Me-SATE or tBu-SATE) group removable upon esterase activation. Efficient synthesis of these lipophilic analogs was achieved thanks to the use of a photolabile linker anchored to the solid support in combination with thymidine-3'- O -phosphoramidites having a SATE group in place of the regular 2-cyanoethyl one. Both dodecathymidine phosphotriester and thionophosphotriester having S -acetyl-2-thioethyl groups were found to be stable in the presence of snake venom and calf spleen phosphodiesterases whereas, upon incubation in CEM cell extracts, they were selectively hydrolyzed to the anionic parent dodecathymidylate and dodecathymidine phosphorothioate, respectively. In addition, Me-SATE-protected dodecathymidine thionophosphotriester was stable in mouse and human sera as well as in human gastric juice. These results depict the potential of SATE-protected oligonucleotides as prodrugs of antisense oligonucleotides.     

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.     

Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E.

Two microbial metabolites, bafilomycin B1 and destruxin E, have been found to inhibit significantly the oxidized low density lipoprotein (LDL)-induced accumulation of lipid droplets at 3 nM and 0.5 microM, respectively, in macrophage J774. The incorporation of [14C]oleate into cholesteryl esters in the cells incubated with oxidized LDL was inhibited to the same extent by the two compounds. Both compounds had no effect on the cell surface binding at 4 degrees C and the internalization of oxidized 125I-LDL as well as on the activity of acyl-CoA:cholesterol acyltransferase. However, when incubated with these compounds at 37 degrees C, receptors for oxidized LDL were partially trapped within the cell. In accordance with receptor accumulation, ATP-dependent acidification of endosomes and lysosomes was significantly inhibited by 50 nM bafilomycin B1 and 1 microM destruxin E, respectively. From these results it was concluded that the inhibition of ATP-dependent acidification of endosomes and lysosomes by bafilomycin B1 and destruxin E resulted in the reduction of oxidized LDL-induced synthesis of cholesteryl ester and thereby caused a reduced accumulation of lipid droplets in macrophage J774.     

Hydrolysis of individual isomers of fluorogenic pyrethroid analogs by mutant carboxylesterases from Lucilia cuprina

We previously showed that wild-type E3 carboxylesterase of Lucilia cuprina has high activity against Type 1 pyrethroids but much less for the bulkier, alpha-cyano containing Type 2 pyrethroids. Both Types have at least two optical centres and, at least for the Type 1 compounds, we found that wild-type E3 strongly prefers the less insecticidal configurations of the acyl group. However, substitutions to smaller residues at two sites in the acyl pocket of the enzyme substantially increased overall activity, particularly for the more insecticidal isomers. Here we extend these analyses to Type 2 pyrethroids by using fluorogenic analogs of all the diastereomers of cypermethrin and fenvalerate. Wild-type E3 hydrolysed some of these appreciably, but, again, not those corresponding to the most insecticidal isomers. Mutations in the leaving group pocket or oxyanion hole were again generally neutral or deleterious. However, the two sets of mutants in the acyl pocket again improved activity for the more insecticidal acyl group arrangements as well as for the more insecticidal configuration of the cyano moiety on the leaving group. The activities of the best mutant enzyme against the analogs of the most insecticidal isomers of cypermethrin and fenvalerate were more than ten and a hundred fold higher, respectively, than those of wild-type. The implications for resistance development are discussed.     

A solid-phase approach to novel purine and nucleoside analogs

This paper describes a method for the preparation of purine analogs using the solid-phase approach. Nucleoside bases were constructed on Merrifield resin by sequential displacement of purine dichloride with amines, and after detachment, the purine analogs were condensed with d,l-ribofuranoside compounds by the Vorbrüggen method. Thereof, l-ribofuranoside was prepared from l-arabinose via the selective oxidation-reduction procedure of the 2-OH group. Some compounds exhibited moderate activity against HIV-1 in PBM cells.     

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure-activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.     

Inhibition of Sporulation of Genus Bacillus by Various Microbial Protease Inhibitors

Several analogs modifying the 6-methoxy-2-methoxymethyl-3-(3,4-methyienedioxyphenyl)-1,4-benzodioxan-7-yl group of haedoxans were synthesized and their insecticidal activity was examined. 2-(2,6-Dimethoxyphenoxy)-1-hydroxy-6-(2-methoxy-5-methoxyethoxyphenyl)-3,7-dioxabicyclo-[3.3.0]octane, which lacked the 3-(3,4-methylenedioxybenzyloxy) moiety of the benzodioxanyl group, was not insecticidal, but caused prolonged paralysis of the housefly. A compound replacing the 6-(2-methoxy-5-methoxyethoxyphenyl) by 6-(5-butoxy-2-methoxyphenyl) exhibited insecticidal activity comparable to one thirty-thousandth of that of haedosan A. It became evident that the 1,4-benzodioxane framework charging the 3-(3,4-methylenedioxy)phenyl group is important for the insecticidal activity of haedoxans.     

Structural requirements for the biological activity of thymopentin analogs

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.     

Improved synthesis and purification of Alzheimer's Aβ 1–42 and analogs

Fmoc-amino acid fluorides are highly efficient coupling agents for solution and solid-phase peptide synthesis and this property was used advantageously for the manual solid-phase assembly of the Alzheimer's Aβ 1–42 peptide [Milton et al., In Marshak, D. (Ed.) Techniques in Protein Chemistry, Vol. VIII, Academic Press, Orlando, FL, 1997, pp. 865–873]. Further studies comparing this methodology in the preparation of Aβ 1–42 peptide analogs employing a fully automated continuous-flow peptide synthesizer are reported.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.