Synthesis, conformational analysis and biological activities of lanthionine analogs of a cell adhesion modulator.

Cell adhesion is critical for many biological processes, such as hemostasis, wound healing, tumor metastasis and inflammation. Integrins are important mediators of cell adhesion. The integrin alpha4beta1, also known as VLA-4, is a cell surface receptor involved in inflammation. A cyclic peptide, 1-FCA-Arg-c[Cys-Asp-Thz-Cys]-OH, is a potent antagonist to VLA-4 with an IC50 of 2.4 nM. In the current study, we synthesized the lanthionine analogs of 1-FCA-Arg-c[Cys-Asp-Thz-Cys]-OH and determined the conformations of both the parent compound and its lanthionine analog in solution by NMR and computer simulations. The lanthionine analog retains its selectivity to VLA-4 with high nanomolar potency. Both molecules adopt similar topological arrangements in their conformations, while some important differences remain in the sulfur bridge region, which may cause the difference in potency.     

Synthesis and conformational study of a cyclic hexapeptide analog of somatostatin containing dehydrophenylalanine

A cyclic hexapeptide analog of somatostatin, cyclo-(Pro-delta z-Phe-D-Trp-Lys-Thr-Phe) (II) has been synthesized by a combination of solid phase and solution methodology. It shows a potency for inhibition of growth hormone release in vitro about one-tenth that of the corresponding saturated analog, cyclo-(Pro-Phe-D-Trp-Lys-Thr-Phe) (I). N.m.r. studies indicate comparable backbone conformations for analogs I and II. However, the sum of our findings from biological evaluation and solution physical data suggest that on the receptor the position-7 phenyl ring of I is adopting a conformation which differs from that of one of the major solution conformers defined previously by n.m.r. studies.     

Binding crevice for TT-232 in a homology model of type 1 somatostatin receptor

Somatostatin receptor type 1 was modelled based on the atomic structure of bovine rhodopsin. Possible ways of binding interaction between somatostatin receptor type 1 and TT-232, a cycloheptapeptide analogue of somatostatin with broad therapeutic potential, were analysed by molecular docking. The twelve TT-232 conformations, obtained by NMR measurements in H(2)O-D(2)O mixture, were similar, disclosing a consensus backbone conformation. Several residues interacting with TT-232, such as Val133, Asp137 (helix 3), Arg197 (helix 4), Phe287, Gln291, Asn294 (helix 6), Ser305, and Tyr313 (helix 7), were found. In accordance, in vitro binding experiments indicated high-affinity binding of TT-232 to (125)I labelled somatostatin sites in brain membranes. The single binding crevice obtained by docking may allow the design and discovery of new peptidomimetics of TT-232 in the future.     

Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.     

Synthesis and conformational analysis of arabinofuranosides, galactofuranosides and fructofuranosides

Many microorganisms produce biologically important polysaccharides containing galactofuranosyl, arabinofuranosyl and/or fructofuranosyl residues. Recent interest in identifying antibiotics that act by inhibiting the biosynthesis of these glycans has resulted in the development of new and efficient methods for the assembly of oligosaccharides containing furanose residues. In general, it is now possible to synthesize any furanose-containing oligosaccharide with reasonable efficiency. In conjunction with these synthetic investigations, an increasing number of studies have probed the conformation of furanose rings, and a solid appreciation of the conformational preferences of key furanoside ring systems is now available.     

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.     

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues.

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17, Lys21]hCt (1), which had a 5-10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We hypothesized that a stabilized, possibly type I beta turn/beta sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD spectra indicated increased type I beta turn and antiparallel beta sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in 'freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a beta turn/beta sheet conformation in region 17-21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt-based drug for the treatment of osteoporosis and other bone-disorder-related diseases.     

Synthesis and conformational analysis of AzAsx-containing oligopeptides

Summary For the sake of improving synthetic methods and evaluating the conformational perturbation induced by the substitution of AzAsx for the Asx residue in the cognate dipeptide R-CO-Asx-Pro-NHR, we prepared the AzAsx-dipeptide sequence by making use of the crystalline triphosgene. This reagent allows, in situ and under very mild experimental conditions, both the carbonylation and the activation of the properly substituted and N-protected hydrazine before coupling with the proline partner. With regard to conformational behaviour, the azadipeptide sequence displays a -fold, unlike the cognate dipeptide which adopts an Asx-turn.     

Theoretical conformational analysis of a μ-selective cyclic opioid peptide analog

The allowed conformations of the μ-receptor-selective cyclic opioid peptide analog were determined using a grid search through the entire conformational space. Energy minimization of the 13-membered ring structure lacking the exocyclic Tyr¹ residue and the Phe³ side chain using the molecular mechanics program Maximin resulted in only four low-energy conformations. These four ring structures served as templates for a further energy minimization study with the Tyr¹ residue and Phe³ side chain added to the molecule. The results indicated that the Tyr¹ and Phe³ side chains enjoy considerable orientational freedom, but nevertheless, only a limited number of low-energy side-chain configurations were found. The obtained low-energy conformers are discussed in relation to various proposed models of the bioactive conformation of enkephalins and morphiceptin.     

Synthesis and conformational analysis of the insulin-like 4 gene product.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Synthesis, conformational analysis and CB1 binding affinity of hairpin-like anandamide pseudopeptide mimetics.

We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.     

beta-Nicotinamide-alpha-adenine dinucleotide. Synthesis and properties of a coenzyme analog.

It was not possible to synthesize beta-nicotinamide-alpha-adenine dinucleotide (NalphaAD) via the NAD pyrophosphorylase-catalyzed reaction of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-triphosphate. It was therefore prepared by reacting a mixture of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-monophosphate in aqueous pyridine, using N,N'-dicyclohexylcarbodiimide as condensing agent. NalphaAD can replace NAD in the lactate-dehydrogenase-catalyzed oxidation of lactate to pyruvate with LDH 4M from hog muscle as well as LDH 4H from pig heart. Km values are found to be about one order of magnitude higher for NalphaAD than for NAD. The turnover number for NalphaAD with LDH 4H is similar to that of NAD (95%), whereas it is reduced to one third (35%) in the reaction with LDH 4M. Experiments findings are discussed on the lines of a NAD-equivalent conformation of NalphaAD in the holoenzyme complex.     

Synthesis, lipophilicity and anti-HIV activity of a new brominated analog of zidovudine.

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5'- epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.     

Synthesis and conformational analysis of a series of galactosyl enkephalin analogues showing high analgesic activity.

Two galactosyl derivatives of [DMet2,Pro5] enkephalin-amide (compound 1), namely [DMet2,Pro5] enkephalin [N1.5-beta-D-galactopyranosyl] amide (compound 2) and O1.5-(beta-D-galactopyranosyl) [DMet2,Hyp5] enkephalin-amide (compound 3) have been synthesized. Such glycosylpeptides have been shown to be extremely potent analgesic agonists. The conformational analysis of these three compounds in DMSO-d6 solution has been carried out using two-dimensional NMR methods. Both the parent compound (1) and the beta N-galactosyl derivative (2) show similar NMR parameters which are consistent with fairly rigid beta-strands at both the N-terminus and C-terminus, connected by a glycine residue that displays a mixture between multiple conformational states. Thus, although the beta N-galactosyl derivative (2) has been shown to be significantly more potent than the parent compound (1) in the tail immersion and paw pressure tests of analgesia, no correlation can be established between the conformation of (1) and (2) in DMSO and the difference in analgesic activity. In contrast, important conformational differences with respect to (1) and (2) have been detected in the beta O-galactosyl derivative (3). In this case, only one of the likely conformations for (1) and (2) are consistent with the experimental data. These data show that the position of the galactose residue in compound (3) causes Gly3 to loose flexibility leading to a more rigid folded conformation. Such a change in conformation could be related to the difference in analgesic activity between (2) and (3).     

Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN. Design of a potent endozepine antagonist.

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo1-8 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1-8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1-8 [d-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics. Cyclo1-8 OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo1-8 [d-Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.     

Synthesis and bioactivity of fatty acid-conjugated GnRH derivatives.

Transdermal delivery of peptidic drugs is usually inefficient, notably due to their hydrophilic character which makes it difficult to cross the hydrophobic layers of the skin. In order to obtain transdermally deliverable analogs of gonadotropin releasing hormone (GnRH), we have synthesized related hydrophobic derivatives by attaching various aliphatic acids to the N(epsilon)-amino side chain of [D-Lys]6GnRH, a superactive GnRH agonist. It was found that the affinity toward the GnRH receptor gradually decrease with increased hydrophobicity, i.e. increase in chain length of the attached aliphatic acid. Nevertheless, analogs with 12-carbon or shorter aliphatic acids were shown to be GnRH superagonists, with in vitro and in vivo potencies similar to that of [D-Lys]6GnRH. [D-Lys-lauryl]6GnRH was shown to have a longer duration of action in vivo, as compared to [D-Lys]6GnRH. The transdermal penetration of the peptides was evaluated by in vivo functional experiments in rats. According to these studies the efficiency of penetration is gradually lowered in increasingly hydrophobic analogs. These results are discussed with respect to the circular dichroism spectra of the peptides in trifluoroethanol. The spectra of the aliphatic acid-conjugated superagonists examined do not express a significant tendency towards a beta-turn conformation, typical of GnRH and its agonists. This finding contradict previous publications which suggested a correlation between the conformations of GnRH analogs in trifluoroethanol and their biological activities.