Host-defence skin peptides of the Australian Streambank Froglet Crinia riparia: isolation and sequence determination by positive and negative ion electrospray mass spectrometry

A combination of positive and negative ion electrospray mass spectrometry (ES-MS) together with automated Edman sequencing has been used to determine the amino acid sequences of the host-defence peptides from the skin glands of the froglet Crinia riparia. The peptides are called riparins. Of the eight peptides isolated, five are neuropeptides containing intramolecular disulfide linkages; e.g. the major peptide riparin 1.4 (FFLPPCAYKGTC-OH). Positive ion ES-MS identifies the five residues of riparin 1.4 outside the disulfide moiety, but provides no information on the sequence within the disulfide ring. In contrast, the negative ion dissociations of the [M-H]- ion of riparin 1.4 identify the --S-S-- link by loss of H2S2 from the [M-H]- ion, and also provide the sequence within the disulfide unit. Other peptides are riparin 2.1 [(IIEKLVNTALGLLSGL-NH2), a narrow-spectrum antibiotic], signiferin 3.1 [(GIAEFLNYIKSKA-NH2), an nNOS inhibitor] and riparin 5.1 [IVSYPDDAGEHAHKMG-NH2], which shows no neuropeptide, antibiotic or nNOS activity.     

Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method

We have utilized sequence information from an antiheme monoclonal antibody to develop novel porphyrin-binding peptides. Several peptides which have an intramolecular disulfide bond in different positions and different chain lengths were prepared. The affinities of peptides for meso-tetrakis(4-carboxyphenyl)porphyrin were increased by an appropriate conformational restraint using a disulfide bond. Detailed studies with a representative 12-peptide, 12C4, whose length was reduced from 20 residues of the complementarity-determining region (CDR), indicated that both the hydrophobic and electrostatic interactions were essential factors in the peptide-porphyrin binding. Moreover, two-dimensional 1H NMR spectroscopy revealed the conformation of the peptide and the critical residues for the porphyrin-binding. According to the obtained results, a further minimized 9-peptide, 9L, was successfully redesigned with a sequence capable of forming a beta-turn instead of a disulfide bond. Furthermore, affinity maturation studies of 9L were performed by using a combinatorial approach such as the spot-synthesis method. Peptides with an improved affinity for porphyrins were prepared by systematic amino acid replacement. Thus, the design of peptides targeted to porphyrins was demonstrated by the combination of antibody information and the rationally designed combinatorial method.     

Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation

Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.     

Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides

We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins). We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics. This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells. Here we examine some of the factors important for activity in this class of beta-peptides. An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive. The ratio of cationic to hydrophobic residues is also important. Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes. This class of beta-peptides is not degraded by proteases, which bodes well for biological applications.     

Artificial receptors that provides a preorganized hydrophobic environment: a biomimetic approach to dopamine recognition in water

[structure: see text] The recognition of dopamine in water has been achieved with tripodal oxazoline-based artificial receptors, capable of providing a preorganized hydrophobic environment by rational design, which mimics a hydrophobic pocket predicted for a human D2 receptor. The receptors show an amphiphilic nature owing to the presence of hydrophilic sulfonate groups at the periphery of the tripodal oxazoline ligands, which seems to contribute in forming the preorganized hydrophobic environment. The artificial receptors recognized dopamine hydrochloride in water with reasonable selectivity among various organoammonium guests examined. The observed binding behavior of the receptors was explained by evoking guest inclusion in the preorganized hydrophobic pocket-like environment and not by simple ion-pairing interactions. The rationally predicted 1:1 inclusion binding mode was supported by binding studies such as with a reference receptor that cannot provide a similar binding pocket, Job and VT-NMR experiments, electrospray ionization mass analysis, and guest selectivity data. This study implies that an effective hydrophobic environment can be generated even from an acyclic, small molecular artificial receptor. Such a preorganized hydrophobic environment, as being utilized in biological systems, can be effectively used as a complementary binding force for the recognition of organoammonium guests such as dopamine hydrochloride in water.     

Dicarba-closo-dodecaboranes as a pharmacophore. Novel potent retinoidal agonists.

The synthesis and biological evaluation of the dicarba-closo-dodecaborane (carborane) derivatives of retinoids are described. Retinoidal activity was examined in terms of the differentiation-inducing ability toward human promyelocytic leukemia HL-60 cells. High retinoidal activity (agonist or antagonist for the retinoid receptor RAR) requires a carboxylic acid moiety and an appropriate hydrophobic group located at a suitable position on the molecule. 4-[4-(1,2-Dicarba-closo-dodecaboran-1-yl)phenylamino]b enzoic acids and 4-[3-(1,2-dicarba-closo-dodecaboran-1-yl)phenylamino]b enzoic acids showed potent agonistic activity at concentrations of 10(-8)-10(-9) M. The results indicate that carboranes are applicable as the hydrophobic moiety of biologically active molecules.     

Ring Polymers of Tubulin Induced by Binding of Natural Antimitotic Peptides

A number of natural products with potent antimitotic activity are peptides and depsipeptides that bind to tubulin, provoke depolymerization of microtubules, and induce formation of single layer rings of tubulin dimers. These peptides are all hydrophobic and small relative to tubulin (3-5 amino acid residues), yet induce rings polymers with properties that can differ significantly in size and self-association. In addition, these compounds exhibit potent cytotoxicity that varies by several hundred fold from one compound to another. Cryptophycin induces unusually homogeneous rings, composed of eight tubulin dimers, that are stable to dilution at least to nanomolar tubulin concentrations.     

The role of the disulfide bond in amyloid-like fibrillogenesis in a model peptide system

Three terminally protected short peptides Bis[Boc-D-Leu1-Cys2-OMe] 1, Bis[Boc-Leu1-Cys2-OMe] and Bis[Boc-Val1-Cys2-OMe] 3 exhibit amyloid-like fibrillar morphology. Single crystal X-ray diffraction analysis of peptide 1 clearly demonstrates that it adopts an overall extended backbone molecular conformation that self-assembles to form an intermolecular hydrogen-bonded antiparallel supramolecular beta-sheet structure in crystals. Scanning electron microscopic (SEM) images, transmission electron microscopic (TEM) images and Congo red binding studies vividly demonstrate the amyloid-like fibril formation of peptides 1, 2 and 3. However, after reduction of the disulfide bridge of peptides 1, 2 and 3, three newly generated peptides Boc-D-Leu1-Cys2-OMe 4, Boc-Leu1-Cys2-OMe 5 and Boc-Val1-Cys2-OMe 6 are formed and all of them failed to form any kind of fibril under the same conditions, indicating the important role of the disulfide bond in amyloid-like fibrillogenesis in a peptide model system.     

Disulfide Click Reaction for Stapling of S-terminal Peptides

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC₅₀=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.     

The synthesis of 5,10-dihydro- and 2,3,5,10-tetrahydrothiazolo-[3,2-b] [2,4]benzodiazepines, 1,2,3,4,7,12-hexahydrobenzothiazolo-[3,2-b] [2,4] benzodiazepine,and 9,14-dihydro-6H-[1]benzothiopyrano-[4′,3′:4,5]thiazolo[3,2-b] [2,4] benzodiazepine via 1,2,4,5-Tetrahydro-3H-2,4-benzodiazepine-3-thione

Catalytic reductive scission of phthalazine (II) utilizing a two-stage palladium-Raney nickel procedure afforded o-xylene-α,α′-diamine (III) in 97% yield. Treatment of III with carbon disulfide gave [o-(aminomethyl)benzyl]dithiocarbamic acid (IV), which upon thermal cyclization furnished 1,2,4,5-tetrahydro-3H-2,4-benzodiazepine-3-thione (V). Reaction of V with 1,2-dibromoethane, chloro-2-propanone, ethyl 2-chloroacetoacetate, ethyl chloroacetate, and ethyl 2-bromohexanoate gave 2,3,5,10-tetrahydrothiazolo[3,2-b][2,4]benzodiazepine (VII) and substituted 5,10-dihydrothiazolo[3,2-b][2,4]benzodiazepines (Villa and b, IX, and X), respectively. Condensation of V with 2-chlorocyclohexanone and 3-bromothiochroman-4-one afforded 1,2,3,4,7,12-hexahydrobenzothiazolo[3,2-b][2,4]benzodiazepine (XII) and 9,14-dihydro-6H-[1]benzothiopyrano[4′,3′:4,5]thiazolo[3,2-b][2,4]benzodiazepine(XIll). None of the compounds possessed appreciable biological activity.     

The interaction of highly helical structural mutants with the NOP receptor discloses the role of the address domain of nociceptin/orphanin FQ

Nociceptin is a heptadecapeptide whose sequence is similar to that of Dynorphin A, sharing a message domain characterized by two glycines and two aromatic residues, and a highly basic C-terminal address domain but, in spite of these similarities, displays no opioid activity. Establishing the relative importance of the message and address domains of nociceptin has so far been hampered by its extreme conformational flexibility. Here we show that mutants of this peptide, designed to increase the helical content in the address domain, can be employed to explain the mode of interaction with the NOP receptor. Nociceptin analogues in which Ala residues are substituted with aminoisobutyric acid (Aib) show a substantial increment of activity in their interaction with the NOP receptor. The increment of biological activity was attributed to the well-documented ability of Aib to induce helicity. Here we have verified this working hypothesis by a conformational investigation extended to new analogues in which the role of Aib is taken up by Leu. The NMR conformational analysis confirms that all Ala/Aib peptides as well as [Leu(7,11)]-N/OFQ-amide and [Leu(11,15)]-N/OFQ-amide mutants (N/OFQ=nociceptin/orphanin FQ) have comparable helix content in helix-promoting media. We show that the helical address domain of nociceptin can place key basic residues at an optimal distance from complementary acidic groups of the EL(2) loop of the receptor. Our structural data are used to rationalize pharmacological data which show that although [Leu(11,15)]-N/OFQ-amide has an activity comparable to those of Ala/Aib peptides, [Leu(7,11)]-N/OFQ-amide is less active than N/OFQ-amide. We hypothesize that bulky residues cannot be hosted in or near the hinge region (Thr(5)-Gly(6)-Ala(7)) without severe steric clash with the receptor. This hypothesis is also consistent with previous data on this hinge region obtained by systematic substitution of Thr, Gly, and Ala with Pro.     

In Silico and In Vitro Structure–Activity Relationship of Mastoparan and Its Analogs

Antimicrobial peptides are an important class of therapeutic agent used against a wide range of pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses. Mastoparan (MpVT) is an α-helix and amphipathic tetradecapeptide obtained from Vespa tropica venom. This peptide exhibits antibacterial activity. In this work, we investigate the effect of amino acid substitutions and deletion of the first three C-terminal residues on the structure–activity relationship. In this in silico study, the predicted structure of MpVT and its analog have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bonds. The secondary structure and the biological activity of six designed analogs are studied. The biological activity assays show that the substitution of phenylalanine (MpVT1) results in a higher antibacterial activity than that of MpVT without increasing toxicity. The analogs with the first three deleted C-terminal residues showed decreased antibacterial and hemolytic activity. The CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 40% 2,2,2-trifluoroethanol (TFE). In conclusion, the first three C-terminal deletions reduced the length of the α-helix, explaining the decreased biological activity. MpVTs show that the hemolytic activity of mastoparan is correlated to mean hydrophobicity and mean hydrophobic moment. The position and spatial arrangement of specific hydrophobic residues on the non-polar face of α-helical AMPs may be crucial for the interaction of AMPs with cell membranes.     

Synthesis and cytotoxic activity of some new 2,6-substituted purines

A seriesof twenty four acyclic unsaturated 2,6-substututed purines 5a-20b were synthesized. These compounds were evaluated for cytotoxic activity against NCI-60 DTP human tumor cell line screen at 10μMconcentration. N?-[(Z)-4'-chloro-2'-butenyl-1'-yl]-2,6-dichloropurine(5a), N?-[4'-chloro-2'-butynyl-1'-yl]-2,6-dichloropurine(10a), N?-[(E)-2',3'-dibromo-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine(14)and N?-[4'-chloro-2'-butynyl-1'-yl]-6-(4-methoxyphenyl)-purine(19)exhibited highly potent cytotoxic activity with GI?? values in the 1-5 μM range for most human tumor cell lines. Other compounds exhibited moderate activity.     

Aladan scanning: The structure-activity relationship of dynorphin A

An unnatural amino acid, β-[6′-(N, N-dimethyl)amino-2′-naphthoyl]alanine (Ald) showing polarity-sen sitive fluorescence characteristics, was synthesized. A thorough Ald-scan of dynorphin A (Dyn A), the putative endogenous ligand for κ opioid receptors, was then performed. Replacement of the amino acid residues in positions 5, 8, 10, 12 or 14 of Dyn A(1-13)-NH2 with Ald resulted in compounds that had almost equal κ binding affinity compared with that of the parent compound; on the other hand, substi-tution o...     

Self-assembly of a ternary architecture driven by cooperative Hg2+ ion binding between cucurbit[7]uril and crown ether macrocyclic hosts

A novel supramolecular assembly comprising CB[7], styrylpyridinium dye (1) and Hg(2+) forms in aqueous solution based on the hydrophobic effect and metal-ligand and ion-dipole interactions. The binding of Hg(2+) to 1·CB[7] displays positive cooperativity relative to 1 itself.     

A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry

A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]⁺ or [M+n+n′ matrix+Na]⁺ (n = the number of cysteine residues, n′=1, 2,…, n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of 189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast, effective, and robust to identify disulfide bonds in proteins/peptides.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity

Conotoxins are a large family of disulfide‐rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33‐residue conotoxin Φ‐MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ‐MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β‐hairpins that resemble the N‐terminal domain of granulin. Similar to granulin, Φ‐MiXXVIIA promotes cell proliferation (EC₅₀ 17.85 μm ) while inhibiting apoptosis (EC₅₀ 2.2 μm ). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ‐MiXXVIIA expands the repertoire of disulfide‐rich conotoxins that recognize mammalian receptors.     

Enhanced detection of thiol peptides by matrix-assisted laser desorption/ionization mass spectrometry after selective derivatization with a tailor-made quaternary ammonium tag containing maleimidyl group

The goal of this study was to develop a new quaternary ammonium tag for the selective labeling of thiol peptides to improve their detection by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The tag, 1-[3-(4-maleimidylphenoxy)propyl]trimethylammonium bromide (MPPTAB), was designed by using the maleimidyl moiety as a selective labeling group for thiols, and a positively charged quaternary ammonium moiety for improving ionization efficiency; the two moieties were combined by a bifunctional linker p-aminophenol. The properties of the tag were investigated with glutathione as a model peptide, and the derivatization conditions were also optimized with reversed-phase high-performance liquid chromatography. The results show that the effective derivatization of thiol peptides can be achieved at the reactant ratio of 3:1 (MPPTAB to the peptide) with a reaction time of 2 h at 37 degrees C, and the excess MPPTAB can be removed with 2-mercaptoethanol. The comparative study by mass spectrometry indicates that, after derivatization, the signal intensity of thiol peptides with more polar residues such as GPVEDAITAAIGRVAC is increased by 3-5 times, while that with less polar residues like CLLLGGTMV and LCMFVPGYT can be enhanced by more than 100 times. This may be explained in terms of the introduction of the positive charge and the improvement of hydrophobicity: the former raises the ionization efficiency of the tagged peptides, and the latter facilitates their solubility in a hydrophobic matrix, which in turn increases the signal intensity. The present strategy may have a potential use in detecting low-abundant thiol peptides.     

Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds

Cyclic disulfide‐rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X‐ray structures of these peptides to assist in drug design applications, but disulfide‐rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L ‐ and D ‐forms of three prototypic cyclic disulfide‐rich peptides: SFTI‐1 (14‐mer with one disulfide bond), cVc1.1 (22‐mer with two disulfide bonds), and kB1 (29‐mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi‐racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.