A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established as cyclo(PPFFILLX), where X is a non-proteinaceous amino acid, N-methyl-4-aminoproline. This amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanar cis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysed cis/trans amide-Pro bond isomerisation.     

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

Summary We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established ascyclo(PPFFILLX), where X is a non-proteinaceous amino acid,N-methyl-4-aminoproline. this amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanarcis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysedcis/trans amide-Pro bond isomerisation.     

Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres

Fmoc-protected Phe-Gly-type (Z)-alkene dipeptide isostere (ADI) and (E)-fluoroalkene dipeptide isostere (FADI) were synthesized and applied to Fmoc-based solid-phase peptide synthesis (SPPS). These cis-peptide bond mimetics were introduced into a bioactive pentapeptide [H-Amb-Phe-Gly-Leu-Arg-Trp-NH(2); Amb = 4-(aminomethyl) benzoic acid], which has potent GPR54 agonistic activity. The resulting pentapeptide derivatives showed low GPR54 agonistic activity, as compared with the parent peptide and (E)-ADI-containing derivative. This suggests that the trans-amide conformer of Phe-Gly peptide bond of the parent peptide would be significantly important for bioactivity. Contrary to our expectations, a (Z)-FADI-containing derivative exhibited essentially no activity, revealing the necessity of critical validation of FADI-bioisosterism.     

Somatostatin analogues containing o-aminomethylphenylacetic acid as a bridge unit

Summary A new cis-peptide bond mimetic, -benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues.     

An active insect kinin analog with 4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif

The insect kinins are potent diuretic peptides that preferentially form a cis-Pro, type VI beta-turn. An insect kinin analog containing (2S,4S)-4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif, demonstrates significant activity in the physiological range in a cricket diuretic assay. This is the first instance of a 4-aminopyroglutamate analog of a peptide with a preference for a type VI turn that demonstrates significant bioactivity. The results provide further confirmatory evidence for the active conformation of the insect kinins, and a new scaffold with which to design biostable, peptidomimetic analogs capable of disrupting critical insect kinin-regulated processes in insects.     

Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus

The multifunctional 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala; and are associated with the regulation of diuresis in a variety of species of insects. We previously reported the functional expression of a southern cattle tick (Boophilus microplus) G protein-coupled receptor that is activated by insect kinins. Four different stereochemical variants of each of the 4-aminopyroglutamic acid (APy) and tetrazole moieties, mimics of a cis-peptide bond, type VI beta-turn in insect kinins were now evaluated on the expressed tick receptor using a calcium bioluminescence plate assay. This study represents the first investigation of the interaction of restricted-conformation analogs incorporating components that mimic specific conformations and/or peptide bond orientations in an expressed arthropod neuropeptide receptor. Analog Ac-RF[APy]WGa (2R,4S) was at least 10-fold more active than the other analogs, thus identifying the optimal stereochemistry for tick receptor interaction. The optimal stereochemistry for the tetrazole insect kinin analogs in the tick receptor assay was identified as (D,L). The APy is superior to the tetrazole as a scaffold for the design of mimetic insect kinin analogs. These biostable analogs provide new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin regulated processes.     

The cyclic contryphan motif CPxXPXC, a robust scaffold potentially useful as an omega-conotoxin mimic

Contryphan-R, from venom of the cone-shell Conus radiatus, represents a novel cyclic peptide scaffold onto which residues may be grafted to mimic unrelated protein surfaces. Three substitutions were made at the x and X positions of the disulfide-bridged motif CPxXPXC, where X and x represent any L- and D-handed residues, respectively, P represents proline or hydroxyproline, and C a half-cystine. These substitutions were designed to mimic part of the pharmacophore of the unrelated globular polypeptide omega-conotoxin GVIA, which blocks N-type calcium channels. The structure of this engineered contryphan, YNK-contryphan-R ([D-Tyr4, Asn5, Lys7]contryphan-R), is shown to be similar to that of native contryphan-R (Pallaghy et al., Biochemistry, 1999, Vol. 38, pp. 13553-13559), confirming that the scaffold is robust with respect to the multiple substitutions. In particular, the alpha-beta bond vectors characterising the orientation of the side chains relative to the backbone are similar in contryphan-R, YNK-contryphan-R, and omega-conotoxin GVIA, which is the required result for a scaffold-based approach to molecular design. The solution structure of YNK-contryphan-R has an N-terminal, nonhydrogen-bonded, chain reversal centered on Hyp3-D-Trp4, and a C-terminal type I beta-turn. A minor form due to cis-trans isomerism of the Hyp2-Cys3 peptide bond is present in YNK-contryphan-R in a larger proportion than in contryphan-R. It is evident, particularly from the (3)J(HalphaHN) coupling constants, that YNK-contryphan-R is more flexible than contryphan-R, probably due to the absence in YNK-contryphan-R of the Pro-Trp packing present in the native molecule. Nevertheless, the structure confirms that cyclic peptide molecular designs can achieve the intended conformations.     

The role of the protein core in the inhibitory power of the classic serine protease inhibitor,chymotrypsin inhibitor 2

A synthetic cyclic peptide, reported to be a tight-binding inhibitor of serine proteases, is instead found to be a good substrate, as is the linear peptide of the same sequence. Both of the peptides, designed to mimic the binding loop of chymotrypsin inhibitor 2 (CI2), were cleaved by subtilisin primarily at the CI2 reactive-site Met-59-Glu-60 bond, revealing that the sequence, in the absence of the structural context of the inhibitor, provides sufficient specificity for hydrolysis of this bond. Insights from the crystal structure of the CI2/subtilisin complex, together with biochemical analysis of a CI2 Gly-83 deletion mutant, have allowed us to identify key features that make CI2 an effective inhibitor, while the cyclic and linear peptides are substrates.     

Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis-peptide containing bacterial luciferase family

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO(2) reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene-H(4)MPT) to methyl-H(4)MPT with coenzyme F(420)H(2), which is a reduced 5'-deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis-peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F(420) at 2.6 A resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re-face of F(420) by a bulge that contains the non-prolyl cis-peptide bond. The bindingmode of F(420) is very similar to that in F(420)-dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F(420) to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis-peptide bond in an equivalent position.     

Molecular dynamics study of disulfide bond influence on properties of an RGD peptide.

Three 1 ns length molecular dynamics simulations of an RGD peptide (Ac-Pen-Arg-Gly-Asp-Cys-NH2, with Pen denoting penicillamine) have been performed in aqueous solution, one for the disulfide bridged, and two for the unbridged form. The trajectories were analyzed to identify conformations explored by the two forms and to calculate several properties: NMR vicinal coupling constants, order parameters, dipole moments and diffusion coefficients, in an effort to describe the physical role of the disulfide bond. The cyclic peptide was able to explore several distinct backbone conformations centered around a turn-extended-turn structure. However, its flexibility was limited and it appeared to be 'locked in' into a a family of structures characterized by a high dipole moment and a well-defined conformation of the pharmacophore, which has been previously identified as biologically active. Excellent agreement between the simulated and observed NMR vicinal coupling constants indicates that realistic structures were sampled in the cyclic peptide simulation. The linear form of the peptide was much more flexible than the cyclic one. In the two independent 1 ns simulations of the linear form the explored conformations could be roughly grouped into two classes, of cyclic-like and extended type. Within each simulation the peptide switched between the two classes of structures several times. Exact matches between conformations in the two linear peptide simulations were not found; several conformational regions with backbone rms deviations below 1A were identified, suggesting that representative structures of the linear form have also been identified. In the linear peptide simulations the RGD pharmacophore is able to adopt a wide range of conformations, including the one preferred by the cyclic form. The lower biological activity of the linear peptide compared to the cyclic one may be correlated with the lower population of this structure in the absence of the disulfide bond.     

In situ maleimide bridging of disulfides and a new approach to protein PEGylation

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.     

Synthesis of cyclic peptides and peptide libraries on a new disulfide linker.

A new cysteine-based disulfide linker for Fmoc solid phase peptide synthesis was developed (Fmoc-Cys(3-mercapto-3-methylbutanoic acid)OPp) that allows the on-resin assembly and side chain deprotection of cyclic peptides. Model peptides and a cyclic peptide library of the structure [a-a-x-x-a-a-c] composed of D-amino acids were assembled and the synthesis and cleavage conditions studied. The best cyclization results were obtained with PyBOP/HOAt/diisopropylethyl amine. Racemization rates of the cysteine in the analyzed model sequences were between 5.2 and 12.3%. Cleavage of the disulfide bond was best carried out with DTT in 50% 2-propanol/100 mM ammonium bicarbonate. The cleaved peptides can be used directly in biological assays.     

Membranolytic selectivity of cystine-stabilized cyclic protegrins.

To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin-1 (PG-1), an 18-residue cationic peptide with a broad-spectrum antimicrobial activity. These cyclic protegrins bear end-to-end peptide bonds together with varying numbers (zero to three) of cross-strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low- and high-salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram-positive and Gram-negative bacteria, fungi and one tested virus, HIV-1. Compared to PG-1, the cyclic tricystine ccPG 3 displayed approximately a 10-fold decrease in hemolytic activity against human cells and 6- to 30-fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [DeltaSS]cPG 8, a cyclic protegrin with no disulfide bond, and [DeltaCys6,15]cPG 5, a cyclic mimic of PG-1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG-1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of beta-strand structures in water/trifluoroethanol or phosphate-buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.     

Inhibition of a common human anti-hepatitis B surface antigen idiotype by a cyclic synthetic peptide.

A common human anti-hepatitis B surface antigen idiotype-anti-idiotype reaction was partially inhibited by a cyclic synthetic hepatitis B surface antigen peptide. Reduction of the intrachain disulfide bond and subsequent alkylation destroyed its inhibitory activity, suggesting that a conformation-dependent group alpha epitope was associated with this cyclic peptide.     

In vitro activity profiles of cyclic and linear enkephalin pseudopeptide analogs

The peptide bond in the 4-5 position of the cyclic and linear enkephalin analogs H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] and H-Tyr-D-Ala-Gly-Phe-L(or D)-Leu-OH was replaced by a thiomethylene ether linkage. Each of the configurational isomers of the cyclic pseudopeptide H-Tyr-cyclo[-D-Lys-Gly-Phe psi [CH2S]L(or D)-Leu-] showed high potency in both the guinea pig ileum and the mouse vas deferens assay and, therefore, had no preference for either mu- or delta-opioid receptors, in contrast to the cyclic parent peptides H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] which are mu-receptor selective. The loss of selectivity observed with the cyclic pseudopeptides may be due to the greater flexibility of their 18-membered ring structures as a consequence of the peptide bond substitution. The linear pseudopeptide analogs were both less potent and less delta-receptor selective than their parent compounds. These results indicate that thiomethylene ether peptide bond replacements can have a pronounced effect on the activity profile of peptide hormones and neurotransmitters.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Parallel solid-phase synthesis of a small library of linear and hydrocarbon-bridged analogues of VEGF(81-91): potential biological tools for studying the VEGF/VEGFR-1 interaction

The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system.     

Thyrotropin-releasing hormone and cyclic AMP activate distinctive pathways of protein phosphorylation in GH pituitary cells

The studies reported here were undertaken to clarify the cellular mechanism of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), in clonal, hormone-responsive GH pituitary cells and to assess the possibility of a role for cyclic AMP as a mediator of TRH action. We investigated patterns of protein phosphorylation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of high speed supernatant and pellet fractions from untreated and treated GH cells. Brief treatment of cells with agents which elevate or mimic cellular cyclic AMP (8-bromo cyclic AMP, dibutyryl cyclic AMP, vasoactive intestinal polypeptide or cholera toxin) stimulated the phosphorylation of five supernatant peptides (41, 45, 47, 72, and 82 kilodaltons) and one pellet peptide (135 kilodaltons) and decreased the phosphorylation of one supernatant peptide (55 kilodaltons). In contrast, TRH promoted the phosphorylation of four different supernatant peptides (two 59, 65, and 80 kilodaltons). In addition, TRH also stimulated the phosphorylation of cyclic AMP-responsive 41-, 45-, and 82-kilodalton supernatant peptides and 135-kilodalton pellet protein and decreased the phosphorylation of 55-kilodalton supernatant peptide. Altered labeling of 47- and 72-kilodalton supernatant peptides, however, was not observed with TRH. Time course studies, as well as the overlapping biological action of TRH and vasoactive intestinal polypeptide, lead us to conclude that these peptide hormones utilize distinct, parallel pathways which converge at some late step. Furthermore, the results indicate that effects of TRH are mediated by a cyclic AMP-independent pathway.     

Synthesis and biological activity of the first cyclic biphalin analogues

Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH<--Phe<--Gly<--D-Ala<--Tyr). In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. d-Alanine residues in positions 2, 2' of the parent peptide were replaced by d- and l-cysteine and an intramolecular disulfide bond between the cysteine thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles.     

Cyclopeptides of Linum usitatissimum.

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.