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Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity.

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels., Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors., Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses. They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational gamma-carboxyglutamic acid (Gla) residues. Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form alpha-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold. We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and 1H NMR spectroscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from 1H NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of alpha- and 310 helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+) to form a stable alpha-helix, while conantokin-T adopts a stable alpha-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+).

The three-dimensional solution structure of mu-conotoxin GIIIB, a 22-residue polypeptide from the venom of the piscivorous cone snail Conus geographus, has been determined using 2D 1H NMR spectroscopy. GIIIB binds with high affinity and selectivity to skeletal muscle sodium channels and is a valuable tool for characterizing both the structure and function of these channels. Structural restraints consisting of 289 interproton distances inferred from NOEs and 9 backbone and 5 side chain dihedral angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. In addition to the 1H NMR derived information, the 13C resonances of GIIIB were assigned at natural abundance, and hydroxyproline C beta and C gamma chemical shifts were used to distinguish between the cis and trans peptide bond conformations. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 1.22 A for the backbone atoms and 2.48 A for all heavy atoms. For the well-defined region encompassing residues 3-21, the corresponding values were 0.74 and 2.54 A, respectively. GIIIB adopts a compact structure consisting of a distorted 310-helix, a small beta-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the helix and the beta-sheet, forming a structural core with similarities to the CS alpha beta motif [Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M., & Vovelle, F. (1995) Structure 3, 435-448]. This motif is common to several families of small proteins including scorpion toxins and insect defensins. Other structural features of GIIIB include the presence of eight arginine and lysine side chains that project into the solvent in a radial orientation relative to the core of the molecule. These cationic side chains form potential sites of interaction with anionic sites on sodium channels. The global fold is similar to that reported for mu-conotoxin GIIIA, and the structure of GIIIB determined in this study provides the basis for further understanding of the structure-activity relationships of the mu-conotoxins and for their binding to skeletal muscle sodium channels.

A proline-rich peptide was isolated and purified to homogeneity from an extract of bovine neutrophil granules using semi-preparative RP-HPLC. The relative molecular mass of the peptide (called Bac-X) was determined by ionspray MS to be 5149 +/- 0.5. The amino acid composition of the peptide was characterized by its limited number of amino acid types, which included a high proline (43.3%) and arginine content (20.3%), and hydrophobic residues. Bac-X had similar characteristics to Bac-5, a previously characterised bactenecin of bovine neutrophil granules, with respect to its proline, arginine and hydrophobic amino acid content, molecular mass and antibacterial specificity. Tryptic and N-bromosuccinimide digestion of Bac-X produced fragments with masses (M(r) 785 and 4224 and 3100 respectively) consistent with those expected from a peptide with the reported sequence of Bac-5. Bac-X differed from Bac-5 in the number of amino acid residues (43 for Bac-X versus 42 for Bac-5) and contained glycine which Bac-5 did not. However, the calculated molecular mass of the peptide, based on the amino acid compositional data, did not match the experimental value. The purified peptide could not be sequenced by Edman degradation due to apparent blockage of the N-terminus. Partial sequence information, obtained by LC-MS and collision induced dissociation MS-MS analysis of a M(r) 785 tryptic fragment of Bac-X, showed that this peptide contained a six residue sequence (-RFPPIR-) not found in Bac-5 which, based on its reported sequence, contained a M(r) 785 tryptic fragment with the sequence -FRPPIR-. This difference in sequence of Bac-X compared with Bac-5 illustrates the application of electrospray (ionspray) MS techniques to the detection and identification of minor differences in related protein/peptide forms.