Synthesis and biological evaluation of cyclic and branched peptide analogues as ligands for cholecystokinin type 1 receptor

A library of cyclic CCK8 analogues, containing unnatural amino acids in the peptide sequence, is prepared using solid-phase synthesis. The structure of these cyclic peptides is based on a previously synthesised compound, cyclo-CCK8, selective for CCK(1) receptor. Structure-activity investigations are performed by evaluating the binding properties of the new analogues. In particular, the binding ability of the cyclic CCK8 analogues is tested by nuclear medicine studies on cell line transfected with CCK(1) receptor. Compounds named cyclo-A4-cyclo-A7 show binding constant in the range 6.0-8.0 microM, with an improved affinity over the previous described cyclo-CCK8, but almost comparable IC(50) values among new analogues towards CCK(1) were obtained.     

Interaction of tryptophan-modified analogues of cholecystokinin-octapeptide with cholecystokinin receptors on pancreatic acini

None of six different tryptophan-modified analogues of the C-terminal octapeptide of cholecystokinin differed from the unaltered peptide in terms of their efficacies for stimulating amylase secretion from dispersed acini prepared from guinea-pig pancreas. Replacementof hydrogen with fluorine in position 5 or 6 on the indole ring of the tryptophan residue did not alter the potency with which the peptide stimulated amylase secretion; however, replacement of hydrogen by fluorine in positions 4, 5, 6, and 7 of the indole ring, of modifying or replacing the indole nitrogen caused a 30- to 300-fold decrease in potency. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of ¹²⁵I-labeled cholecystokinin. Our findings indicate that reducing the ability of the tryptophan residue to donate electrons produced a greater decrease in the affinity of the peptide for the cholecystokinin receptors than did abolishing the ability of tryptophan to form hydrogen bonds, and modifications that altered both abilities caused a greater decrease in affinity than did modification of only one ability. Finally, in the tryptophan residues of cholecystokinin octapeptide, tetrafluorination of the indole ring or replacing the indole nitrogen by oxygen reduced the ability of the peptide to cause residual stimulation of enzyme secretion, probably by accelerating the rate at which bound peptide dissociated from its receptors when the acini were washed and resuspended in fresh incubation solution.     

Characterization of central cholecystokinin receptors using a radioiodinated octapeptide probe

We have developed a binding assay for 125I-Bolton-Hunter-labeled cholecystokinin octapeptide (125I-(BH)CCK8) using mouse cerebral cortex membrane preparations. This ligand interacts with cortical membrane preparations in a saturable, high-affinity manner, satisfying the requirements for specific cholecystokinin receptor labeling. Salt is required for maximal binding and BSA is specifically inhibitory with cerebral cortical but not with pancreatic sites. Cholecystokinin peptides as small as CCK30-33 displace binding at low nanomolar concentrations. Dissociation of 125I-(BH)CCK8 is biphasic in both mouse and guinea pig cortex. Pretreatment of membranes at 37 degrees C results in a marked loss of recognition sites, suggesting that the sites may be rapidly metabolized in vivo. After 37 degrees C pretreatment, the loss of CCK recognition sites corresponds to a selective loss of the slow component of dissociation curves. This selective elimination of one dissociation population, as well as the biphasic dissociation kinetics, suggests that at least two distinct CCK receptor subtypes exist in the brain.     

Design of cyclic and other templates for potent and selective peptide alpha-MSH analogues

For over three decades, the design of linear peptide ligands often has incorporated cyclic constraints to improve potency, receptor selectivity, proteolytic stability and biodistribution. Its importance has been so well established that modern day schemes for ligand-based drug design often start with cyclization of linear peptides to rigidify peptide structure, to limit its conformational possibilities, and to find key pharmacophore elements in three-dimensional space. In the past several years, cyclic constraints have been used to develop ligands with improved efficacy, binding affinity, biostability and receptor selectivity for alpha-melanocyte-stimulating hormone (alpha-MSH). Furthermore, potent cyclic alpha-MSH analogues, such as MT-II and SHU-9119, have made structure-activity relationship studies and molecular modeling more useful for creating new three-dimensional, topographical pharmacophore templates.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and antifungal activities of cyclic octa-lipopeptide burkholdine analogues

Synthesis and antifungal activity of cyclic octapeptide derivatives of burkholdines are described. To construct cyclic octapeptides, the combination of Fmoc-SPPS and cyclization with DIC/HOBt in the solution phase was employed. Synthesized peptides were evaluated for antifungal activity with MIC values against Saccharomyces cerevisiae, Aspergillus oryzae, and Candida viswanathii. As a result, the lipid side chain and the stereochemistry of each amino acid of Bk-1097 analogues significantly affected antifungal activity.     

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.     

Synthesis of cyclic analogues of loop 4 of nerve growth factor

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of theβ-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52–83% yields by the solid-phase method with the use of the Fmoc/Bu ^t strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10^?3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five-and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.     

Synthesis and SAR of highly selective MMP-13 inhibitors

The structure-based design and synthesis of a series of novel biphenyl sulfonamide carboxylic acids as potent MMP-13 inhibitors with selectivity over MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Aggrecanase 1, and TACE are described.     

Development of highly potent and selective dynorphin A analogues as new medicines.

Dynorphin A (Dyn A), a 17 amino acid peptide H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH, is a potent opioid peptide which interacts preferentially with kappa-opioid receptors. Research in the development of selective and potent opioid peptide ligands for the kappa-receptor is important in mediating analgesia. Several cyclic disulphide bridge-containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A(1-11)-NH2 cyclic analogues incorporating the sulphydryl-containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the mu- and delta-receptors) as the parent Dyn A(1-11)-NH2 peptide in the guinea-pig brain (GPB), but exhibited a much lower activity in the guinea-pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure-activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at kappa-opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand-based drug design.     

Synthesis of analogues of the Des-Phe-NH2 C-terminal hexapeptide of cholecystokinin showing gastrin antagonist activity

Four analogues of Z-CCK-27-32-NH2, Z-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2, a cholecystokinin receptor antagonist have been synthesized by solution methodology. In these analogues, Z-Tyr(SO3-)-Nle-Gly-Trp-Met-Asp-NH2 16, Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-NH2 17, BOC-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2 24 and Boc-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp-NH2 25 methionyl residues were replaced by norleucyl residues. Preliminary biological activity on gastrin-induced acid secretion, in rat, are reported. These derivatives proved to antagonize the action of gastrin, with ED 50 of between 0.5 and 3 mg/kg.     

Synthesis of potent and highly selective inhibitors of human tryptase

The serine protease tryptase has been implicated in allergic and inflammatory diseases and associated with asthma. The synthesis and SAR of a series of N1-activated-4-carboxy azetidinones are described, resulting in identification of BMS-363131 (2) as a potent inhibitor of human tryptase (IC(50)<1.7 nM) with high selectivity (>3000-fold) for tryptase versus related serine proteases including trypsin.     

Synthesis and in vitro evaluation of human FP-receptor selective prostaglandin analogues

The in vitro evaluation of a series of saturated prostaglandins revealed that compounds with omega chain aromatic rings retain nanomolar potency for the human prostaglandin F receptor (hFP receptor), exemplified by compound 8. In contrast, the double bonds are required for activity in the series with an acyclic omega chain as in PGF2alpha.     

Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH(2)) showed potent binding and agonist activities at delta and mu opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands.     

Synthesis of proline analogues as potent and selective cathepsin S inhibitors

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure–activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.     

Synthesis and biological evaluation of analogues of the marine cyclic depsipeptide obyanamide

On the basis of the total synthesis of obyanamide, 20 analogues of this marine cyclic depsipeptide have been synthesized by (i) preparation of the tripeptide fragments in the western hemisphere using Z/OtBu protocol; (ii) preparation of the dipeptide fragments in the eastern hemisphere using Boc/OMe protocol; and (iii) fragments coupling, removal of protecting groups (Boc and OtBu, in one pot), and macrocyclizaion in the last step. The cytotoxic test showed that three synthetic compounds exhibited moderate activities against HL‐60, KB, LOVO, and A549 cell lines. According to the results, the β‐amino acid residue was found to play a critical role in the biological activities. Additionally, the ester bond along with the Ala(Thz) moiety was also essential for biological activities. However, it seems too early to draw a conclusion that the N‐methylation of Val/Phe can lead to higher or lower cytotoxic activities. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Heterogeneity of cholecystokinin receptors in pancreas

Specific labeling of a major Mr 85-95 K protein was obtained using the SH, NH2 heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) to affinity label cholecystokinin (CCK) receptors on rat pancreatic plasma membranes, pancreatic acinar cells and acinar cell tumor membranes with 125I-CCK-33. Endoglycosidase F (endo F) digestion of this species in gel slices indicated that at least two components were present which contain N-linked glycans. The smaller protein of Mr approximately 85 K was digested by endo F to a final product of approximately Mr 62 K while the larger Mr approximately 95 K protein generated two endo F products of Mr 55 K and Mr 43 K. These findings suggest that the receptor for CCK on pancreatic acinar cells exhibits an oligomeric structure, possessing two distinct CCK-binding proteins.     

Discrimination between CCK receptors of guinea-pig and rat brain by cyclic CCK8 analogues

The properties of high affinity CCK8 binding sites of guinea-pig and rat brain cortex were compared using [3H]pCCK8. Large differences were observed, with the KD value being significantly higher in the rat (KD = 1.25 nM) than in guinea-pig brain (KD = 0.18 nM). Both sites exhibited different specificities for various CCK8 analogues, the selectivity factors KI rat/KI guinea-pig varied from 0.9 for CCK4 to 64 for cyclic CCK8-related compounds. Significant differences in the inhibition of [3H]pCCK8 binding by monovalent and nucleotides cations were also observed. These results could be explained by a difference in receptor environment or by a species difference in the proportion of CCK8 receptor-subtypes.