Search

A series of secondary and tertiary pyridyl amides as potential central nicotinic acetylcholine receptors (nAChRs) ligands were prepared. Amides displayed negligible or very low affinity, whereas two amines achieved by reduction of corresponding secondary amides, showed affinity in the nanomolar range for nAChRs.

A series of HIV-1 protease inhibitors based on the lead compound Pc (IC50 = 165 nmol/l) with structural modifications at P1/P1' substituents or with a lengthening at its core unit were synthesized from amino acid starting materials. All analogues were less active than Pc against the protease.

Because of the encouraging results obtained using vinyl ester derivatives, we synthesized and tested a novel series of peptide-based proteasome inhibitors bearing a new pharmacophore unit at the C-terminal. N-Acylpyrrole moiety is a potential substrate for Michael addition by catalytic threonine. Several analogues have demonstrated a selective inhibition of the multicatalytic complex beta1 subunits, the capacity to permeate cellular membrane, and good pharmacokinetics properties.

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.

Dermorphinoyl(DMR)-glycine, DMR-sarcosine and DMR-glycyl-arginine have been prepared in order to examine the effect of C-terminal extension of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) on opioid activity. On GPI preparation the addition of Gly, Sar, or Gly-Arg to the carboxyl terminus of dermorphinoic acid was detrimental to mu activity: dermorphinoyl-derivatives, in fact, retain only 5-20% of dermorphin potency. Following intracerebroventricular administration (tail-flick test), whereas the analgesic activities of compounds showed the trend dermorphin greater than DMR-Sar greater than DMR-Gly-Arg greater than DMR-Gly greater than morphine, the nonapeptide displayed highest activity after subcutaneous injection in mice: DMR-Gly-Arg was 2.5 and 10 times more potent than dermorphin and morphine, respectively.

Using the potent cyclic peptide T analog-Thr-Thr-Asn-Tyr-Thr-Asp- as parent compound, a series of analogues were synthesized and their potencies in a monocyte chemotaxis assay were compared with those of correspondingly modified linear peptides. Structure-activity relationships observed with cyclic compounds did not always parallel those determined with linear analogues. -Thr-Hse-Asn-Tyr-Thr-Asp- showed the highest affinity to CD4 receptor of monocytes of any peptide thus far studied. It also proved to be highly resistant to degradation by plasma or brain enzymes.

Dehydropeptide analogs of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and N-terminal fragments containing one or two dehydrophenylalanine residues in the 3rd and/or 5th position, have been investigated by means of CD spectroscopy. The results indicate that the above dehydropeptides can adopt different conformations in alcohol and water solutions. In methanol and trifluoroethanol, the CD spectra are mainly consistent with the presence of folded structures, probably stabilized by intramolecular hydrogen bonds. In water, conversely, CD data indicate disruption of ordered structures and formation of preferentially extended flexible conformations. Models of the involved folded structures are tentatively proposed, taking into account the geometric features of dehydro residues and their tendency to favor hydrogen-bonded 10-membered rings.

The synthesis, by fragment condensation, of protected peptides related to the N-terminal (positions 1-14) and C-terminal (positions 36-52) portions of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The alpha-carboxyl function of the lysyl residue in position 14 was used as the free acid, or protected by the tert-butyloxycarbonylhydrazide. Two different synthetic pathways were used in the preparation of the 36-52 sequence of the inhibitor. The identity and purity of the synthesized peptide derivatives were established by amino acid analysis of acid hydrolysates, optical rotation and this layer chromatography in two solvent sytems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values and enzymic digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (demorphin), and opiate-like peptide, and tri-, tetra-, penta- and hexapeptide-amide analogs, were synthesized by conventional methods in solution, to determine the minimum peptide chain-length, required for analgesic activity.

The 20S proteasome is a multicatalytic protease complex responsible for the degradation of many proteins in mammalian cells. Specific inhibition of proteasome enzymatic subunits represents a topic of great interest for the development of new drug therapies. Following our previous development of a new class of peptide-based inhibitors bearing a C-terminal vinyl ester residue as a pharmacophoric unit that are able to interact with the catalytic threonine, we report here the synthesis and biological properties of a new series of vinyl ester cyclopeptide analogues. Some of these derivatives were shown to inhibit the chymotrypsin-like activity of the proteasome at nanomolar concentration and their potency was found to depend on the size of the tetrapeptidic cyclic portion.

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8-30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 +/- molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10-17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.

We describe the synthesis and biological activities of a series of methyl 3,4-epoxypiperidine-3-carboxylate tripeptide derivatives that inhibit the chymotryptic and tryptic active sites of the 20S proteasome. Of the series, compound 2 which contains 3-hydroxy-2-methylbenzoyl group at its N-terminal position, displayed the greatest inhibitory potency (IC(50)

The 26S proteasome is a multicatalytic protease complex that plays an essential role in intracellular protein degradation. We have synthesized and tested a series of arecoline peptide derivatives where the peptide portion derives from a screening of tripeptide sequences, and the arecoline moiety has been considered as a potential substrate for catalytic threonine. Derivatives 17-19 are the best compounds of the series, showing chymotryptic-like (beta5) inhibition (IC(50) congruent with 1 microM) and favorable pharmacokinetic properties.

In order to improve the immunotherapeutical potential of H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide, an Epstein–Barr virus (EBV) subdominant epitope derived from the membrane protein LMP2, we have synthesized and tested CLG analogues containing cis- and/or trans- 4-aminocyclohexanecarboxylic acid (ACCA) replacing Gly-Gly and/or Thr-Met dipeptide units. All pseudopeptides were tested for metabolic stability and for their capacity to bind HLA-A2 molecules and to sensitize target cells to lysis. All new compounds exhibited higher enzymatic resistance compared to the original CLG and some trans -ACCA-derivatives were able to associate HLA-A2 and to efficiently stimulate CTL responses directed against the CLG natural epitope.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN-α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope.

Inhibition of the proteasome, the multicatalytic protease complex responsible for the turnover of many cellular proteins, represents an attractive target in the development of new drug therapies, proteasome inhibitors being potentially useful tools for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Based on our previous development of a new class of inhibitors bearing a C-terminal VE cluster able to interact with catalytic threonine, we report herein the synthesis and activity of new VE-based peptide analogs bearing an additional allyl pharmacophore unit at the C- or N-terminal position of the pseudotripeptide sequence. In the new series, the structural modification carried out to the prototype determine a decrease of proteasome inhibition. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

A series of deltorphin C (H-Tyr- d -Ala-Phe-Asp-Val-Val-Gly-NH 2 ) analogues were synthesized to assess the consequences of changing anionic and hydrophobic residues on δ receptor selectivity. Analogues with altered C-terminal groups, inverted sequences, or esterified with tert -butyl, benzyl, or ethyl groups revealed that high δ selectivity required an unmodified amino acid sequence. Shifts of Asp and hydrophobic residues decreased δ selectivity due to loss in δ affinity (5- to ≈ 700-fold); μ affinity was unchanged or increased 14-fold. Suppression of charge or deamidation diminished δ selectivity through reduced δ and modified μ affinities. Data provide evidence that a negative charge does not a priori guarantee high selectivity and specific alignment of anionic and hydrophobic residues might facilitate optimum spatial configuration which complements the δ receptor binding site.

The synthesis and biological properties of vinyl ester peptide-based molecules bearing linear N-terminal amino acids are reported. Compounds were tested in vitro for their capacity to inhibit the chymotryptic-, tryptic-like, and post-acidic activities of the proteasome. Some analogues showed selective inhibition of post-acidic (PGPH) activity, which is attributed to the β1 subunit. Interestingly, active compounds demonstrated higher inhibitory activity toward ‘standard’ proteasomes than toward immunoproteasomes. The inhibitory potency was found to be related to the amino acidic sequence and to the length of the N-terminal residues. The new inhibitors demonstrated resistance to plasmatic proteases and a good capacity to permeate the cell membrane.

Here we report the study of a new series of vinyl ester cyclopeptide analogues synthesized on the basis of our previous development of a class of cyclopeptides derived from our linear prototype inhibitors. In these compounds, the exocyclic pharmacophoric unit Leu-VE was linked to the gamma-carboxyl group of the glutamic acid residue at the C-terminal. The best analogues of the series have been shown to inhibit the caspase-like activity of the proteasome at nanomolar concentrations and have also demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

Here we report the synthesis and biological properties of peptide-based molecules bearing constrained analogues of phenylalanine at the C-terminal. Compounds were tested as proteasome subunits' inhibitors. Dehydro-peptides showed good inhibition, in particular against trypsin-like (T-L) proteasome activity while some C-terminal Tic-derivatives inhibit only caspase-like activity in enzymatic beta1 subunits with a certain degree of efficacy. The best analogues of the series demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands.

The Gly 4 and/or Tyr 5 residues in dermorphin hexapeptide (H-Tyr- d -Ala-Phe-Gly-Tyr-Pro-OH) were replaced by Nα-methyl- or d -amino acids in order to examine the effect on opioid activity. Two pseudopeptides (H-Tyr- d -Ala-Phe-Gly-ψ (NHCO)-Xaa-Pro-OH, Xaa — Tyr or Phe) in which the Gly 4 Xaa bond is reversed, were also prepared. Metabolic stability, analgesia and selectivity of these compounds for different receptor populations have been investigated. Results suggest that the 12 new analogues showed a negligible affinity for the K binding site and some selectivity for μ- or δ receptors. In some cases the analgesic potencies seems to be related to enzymatic stability of the peptides.

The octapeptide sequence of peptide T is contained within the envelope of HIV and seems to mediate the viral binding to CD4 expressing cells, including monocytes. The biological activity of the alpha-aminobutyric acid pentapeptide derived from the C-terminal sequence of peptide T, in which the polar side chain of threonine in position 4 is substituted by a hydrophilic group, is measured by the monocyte chemotaxis assay. The chemotactic activity of human monocytes is assessed by determining the concentration at which the pentapeptide analog is maximally active and the effectiveness at that concentration, in comparison with peptide T and two shorter homologs, the pentapeptide and tetrapeptide. These experiments suggest that the synthetic analog is a potent chemotactic factor active at picomolar concentrations and that it competes with peptide T for the monocyte binding site.

Here we report the study of a new series of peptide-based proteasome inhibitors with a vinyl ester moiety at C-terminal. The presence of Tic, a rigid analogue of phenylalanine, in the central portion of some derivatives is not favourable for the activity. The best analogue of the series shows a potent and selective inhibition for the beta2 subunit and good enzymatic stability.

Two small libraries of tripeptidic-based vinyl ester derivative proteasome inhibitors were synthesized and tested, starting with the Hmb-Val-Gln-Leu-VE prototype. The P3 and P4 positions were investigated with a complete set of amino acid residues, some of which showed remarkable selective inhibition of the trypsin-like (beta2) subunit. In both positions, aromatic and hydrophobic residues were preferred.

Here we report the synthesis and biological activities of new tripeptidic-based vinyl ester derivative proteasome inhibitors. Starting from Hmb-Val-Ser-Leu-VE prototype, we investigated P2 position and N-terminal substitution. The more effective inhibitors of the series showed remarkable inhibition and selectivity for the trypsin-like (beta2) subunit and were revealed to be specific for the proteasome. In vitro metabolic stability studies of the new vinyl ester analogues are also reported here.

The proteasome is a multicatalytic proteinase complex which plays a central role in intracellular protein degradation. We report here the synthesis and biological activities of a new class of specific proteasome inhibitors selective for trypsin-like activity. These tripeptide-based compounds bearing a C-terminal vinyl ester are nontoxic, and do not affect cell proliferation, but are able to modulate the generation and presentation of immunogenic peptides presented by MHC class I molecules.

Syntheses are described of new dermorphin and [D-Ala2]deltorphin I analogues in which the phenylalanine, the tyrosine or the valine residues have been substituted by the corresponding N-alkylglycine residues. Structural investigations by CD measurements in different solvents and preliminary pharmacological experiments were carried out on the resulting peptide-peptoid hybrids. The contribution from aromatic side chain residues is prominent in the CD spectra of dermorphin analogues and the assignment of a prevailing secondary structure could be questionable. In the CD spectra of deltorphin analogues the aromatic contribution is lower and the dichroic curves indicate the predominance of random conformer populations. The disappearance of the aromatic contribution in the [Ntyr1,D-Ala2]-deltorphin spectrum could be explained in terms of high conformational freedom of the N-terminal residue. The kinetics of degradation of the synthetic peptoids digestion by rat and human plasma enzymes were compared with that of [Leu5]-enkephalin. The binding to opioid receptors was tested on crude membrane preparations from CHO cells stably transfected with the mu- and delta-opioid receptors. The biological potency of peptoids was compared with that of dermorphin in GPI preparations and with that of deltorphin I in MVD preparations. All the substitutions produced a dramatic decrease in the affinity of the peptide-peptoid hybrids for both the mu- and delta-opioid receptors. Nval5 and/or Nval6 containing hybrids behaved as mu-opioid receptor agonists and elicit a dose-dependent analgesia (tail-flick test) when injected i.c.v. in rats.

We describe the synthesis and biological activities of a series of methyl 3,4-epoxypiperidine-3-carboxylate tripeptide derivatives that inhibit the chymotryptic and tryptic active sites of the 20S proteasome. Of the series, compound 2 which contains 3-hydroxy-2-methylbenzoyl group at its N-terminal position, displayed the greatest inhibitory potency (IC(50)1 microM). All derivatives showed favourable pharmacokinetic properties.

A series of N -benzyl pseudopeptides was designed, synthesized and tested as HIV-1 protease inhibitors. The ability of the new compounds containing N -benzyl hydroxyalkylamino acid core structure to inhibit HIV replication in cell culture is comparable to their capacity to inhibit the isolated enzyme, a result compatible with good pharmacokinetic properties of these derivatives. The pseudotripeptide Fmoc-Leu-N(Bzl)Hse-Met-NH- t Bu was the best inhibitor of the series (IC 50 =170 nM) showing promising inhibition of viral replication (ED 50 =52 nM). All new compounds exhibit high enzymatic resistance and stability against cell cultures and plasma enzymes.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

Two series of peptidomimetics containing a novel C(2) pseudosymmetrical hydroxyalkyldiamino core structure were prepared from amino acid starting materials and tested for inhibitory activity against the HIV-1 protease (HIV-1 Pr) and the virus in cell culture. In the 2,3-diamino-1-hydroxypropane series, compound 6a, containing P1/P1(I) benzyl and P2/P2(I) Fmoc substituents, displayed modest HIV-1 Pr inhibition (IC(50) = 430 nM). The corresponding 2,4-diamino-1-hydroxybutane derivative (6b) was the best inhibitor of the series (IC(50) = 160 nM). Interestingly, 6a and 6b showed satisfactory inhibition of HIV replication in cell culture (ED(50) = 340 and 110 nM, respectively), a result which suggests good cell membrane penetration by this class of compounds.

A series of thirty 2-(3-pyridylaminomethyl)azetidine, pyrrolidine and piperidine analogues as nicotinic acetylcholine receptor (nAChR) ligands was explored. In general, pyrrolidinyl and many azetidinyl compounds were found to bind with enhanced affinity relative to the piperidines. In the three series, the parallel structural changes (stereochemistry, N-methylation and/or chloro substitution) do not consistently lead to parallel shifts in affinity. The more active compounds (K(i) affinity values ranging from 8.9 to 90 nM) were about as analgesic as nicotine in a tail-flick assay in mice after subcutaneous injections.

Two series of peptidomimetics containing an N -hydroxyamino acid core structure were prepared by mixed solution solid-phase synthesis and tested for inhibitory activity against the human immunodeficiency virus (HIV-1) protease (Pr) and the virus in cell culture. In general, N-hydroxy Gly containing pseudopeptides displayed modest HIV Pr inhibition (IC 50 ≥930 nM). In the N -hydroxy Phe derivatives, Fmoc-Phe-ψ[CO–N(OH)]-Phe-Pro-NHtBu was the best inhibitor of the series (IC 50 =144 nM) showing satisfactory inhibition of HIV replication in cell culture (ED 50 =98 nM) and remarkable stability against cell culture and plasma enzymes.

Major histocompatibility complex (MHC) class I molecules bind and display oligopeptides deriving from viral proteins in infected cells. MHC-peptide complexes form the antigens that can be recognized by specific T cell receptors (TCR) expressed on cytotoxic T lymphocytes (CTL). In this way CTL can identify and kill infected cells selectively, sparing healthy cells. We focused our analysis on the H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide, an Epstein-Barr virus (EBV) epitope which represents the target of HLA-A2 restricted EBV-specific cytotoxic T lymphocytes responses [1]. EBV is a lymphotropic virus associated with a number of human malignancies. The CLG nonamer represents a good target for the immunotherapy of EBV-associated malignancies, since it is expressed and conserved in nasopharyngeal carcinoma and Hodgkin’s disease biopsies. However, the CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses to this epitope. Moreover, this epitope shows low enzymatic stability being rapidly hydrolyzed by plasma enzymes. In order to improve the immuno-therapeutical potential of this epitope we have synthesized and tested CLG analogues containing cis- and trans-4-ACCA (4-aminocyclohexanecarboxylic acid) in N- and/or C-terminal portions of the sequence (Table 1). All analogues were tested for metabolic stability and for their capacity to bind HLA-A2 molecules and to induce specific CTL activation.

Nociceptin (NC)/orphanin FQ (H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH) is a neuropeptide, which modulates several biological functions by activating the ORL1 receptor [1]. In the past years, we have performed systematic structure activity studies on NC that have established: a) NC residues 1–13 are sufficient both for high ORL1 receptor binding and biological activity; b) The basic residues in the C-terminal sequence, especially Arg8, appear to be instrumental for ORL1 receptor recognition; c) Contrary to opioid peptides, which strictly require Tyrfor opioid receptor activation, the active core of NC that stimulates the ORL1 receptor, is the Phe4; d) Specific changes of the N-terminal sequence, expecially at Phe, led to the discovery of a partial agonist, [PheΨ(CH2-NH)Gly2]NC(1–13)-NH2, and [Nphe]NC(1–13)-NH2, a pure antagonist of the ORL1 receptor [2]. In the present investigation, we describe the effect of substitutions with a halogen series of the Phe4aromatic side chain of the NC(1–13)-NH2, and identification of new ligands, which behave as superagonists of the ORL1 receptor.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHO NCR ) were investigated. The compounds of the first series (a series) have an ordinary Xaa 1 -Gly 2 bond, those of the second series (b series) have a Xaa 1 Ψ(CH 2 -NH)Gly 2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa 1 -Gly 2 ) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe 1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO NCR cells with different potencies depending on the first amino acid residue, [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-17)NH 2 and [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-13)NH 2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe 1 ]NC(1-17)NH 2 and [Nphe 1 ]NC(1-13)NH 2 , which behaved as NC receptor antagonists both in the isolated tissue and in CHO NCR cells (pK B 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe 1 in the NC sequence as obtained with the pseudopeptide bond between Phe 1 and Gly 2 or with the displacement of the benzyl side chain by one atom, as in Nphe 1 , lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe 1 Ψ-(CH2-NH)Gly 2 ]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe 1 ]NC(1-13)NH 2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.

The octapeptide D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH 2 ([D-Ala 1 ]TNH 2 ), an analog of peptide T (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH) associated with CD 4 /T 4 receptors involved in human immunodeficiency virus infection, was combined with the chelating polyazamacrocycle 1,4,8,11-tetraazacyclotetradecane (cyclam) to afford the bifunctional ligand cyc-[D-Ala 1 ]TNH 2 . This was then reacted with [ 99m TcO 4 ] − and Sn 2+ to yield the monocationic complex [ 99m Tc(O) 2 (cyc-[D-Ala 1 ]TNH 2 )] + . Biological activity of both the cyclam-peptide conjugate and the resulting Tc-99m complex were evaluated by measuring their chemotactic indexes. Results showed that N-cyclam acylation and subsequent labeling with Tc-99m of [D-Ala 1 ]TNH 2 were tolerated, and both cyc-[D-Ala 1 ]TNH 2 and [ 99m Tc(O) 2 (cyc-[D-Ala 1 ]TNH 2 )] + retained the high chemotactic capacity of the original octapeptide. Biodistribution of the Tc-99m complex was carried out in rats. Fast blood clearance and no accumulation in organs of interest were observed.

Biotechnology Center, University of Ferrara, Ferrara, ItalyThe latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus(EBV)-associated tumors and consequently it may represent a target for specific cytotoxic Tlymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is lim-ited by the poor immunogenicity of the protein that induces weak CTL responses directed tothe CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We havenow demonstrated that selective peptide stimulation of peripheral blood lymphocytesinduced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable targetfor specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine thestrength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analo-gues carrying single or combined amino acid substitutions to increase HLA/peptide stability.Among the analogues tested we identified two peptides which, compared to the natural epi-tope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes.These peptides demonstrated a potent, specific stimulatory capacity and could be used forselective CTL-based therapies.

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.

Two series of peptidomimetics containing a novel C2 symmetrical hydroxyalkylgem-diamino core structure were prepared, from amino acid starting materials, and evaluated as inhibitors of HIV-1 protease (HIV-1 Pr). 1, 1-Diamino-3-hydroxypropane (gHse) derivatives showed weak inhibitory potency (IC50> 10 μM). In the 1, 1-diamino-2-hydroxyethane (gSer) series, a compound containing P1/P1’benzyl and P2/P2’Fmoc substitueras, displayed a significant HIV-1 Pr inhibition (IC50= 440 nM). © Munksgaard 1997.

The achiral symmetric alpha-aminoisobutyric acid (Aib) replaced the critical N-terminal residues of the amphibian skin opioid deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) without detriment to the physicochemical requirements for delta opioid receptor recognition. Substitutions by the alpha, alpha-dialkyl amino acid in place of D-Ala2 or Phe3, or both, exhibited high delta receptor affinity (Ki delta = 0.12-3.6 nM) and 5-9-fold greater selectivity (Ki mu/Ki delta = 5000-8500) than the parent compound. This is the first definitive demonstration that the D-chirality of alanine and the aromaticity of phenylalanine are replaceable by an achiral alpha, alpha-dialkylated residue without detrimental effects on ligand binding. Incorporation of the mono-alpha-alkyl amino acid L- or D-Ala at the third position also produced highly selective delta ligands (Ki mu/Ki delta = 2000-3500), albeit with reduced delta affinities (Ki delta = 6-15 nM). Replacement of the anionic residue Asp4 by Aib yielded an opioid peptide that fit two-site binding models for the delta receptor (eta = 0.763; P0.0001) and displayed dual high affinity for both delta and mu receptors, emphasizing the repulsive effect by a negative charge at mu receptor sites and the insignificance of Asp for delta affinity. Molecular dynamics conformation analyses suggested that Aib residues caused distinct changes in deltorphin C secondary structure when substituted for D-Ala2, Asp4, and simultaneously D-Ala2 and Phe3 but not when substituted for Phe3. These conformational changes might be critical factors for the proper orientation of reactive constituents of residues in the N-terminal region of deltorphin C. Disparities between binding data and functional bioassays of [Aib3] indicated that Phe3 was required for bioactivity in mouse vas deferens but not for interaction with delta opioid receptors in rat brain membranes.

The delta opioid antagonist H-Dmt-Tic-OH (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) exhibits extraordinary delta receptor binding characteristics [Ki delta = 0.022 nM; Ki mu/Ki delta = 150,000] and delta antagonism (pA2 = 8.2; Ke = 5.7 nM). A change in chirality of Dmt at C alpha (1, 2, 6, 8, 10, 13) curtailed delta receptor parameters, while replacement of its alpha-amino function by a methyl group (3) led to inactivity; Tyr-Tic analogues 4 and 11 weakly interacted with delta receptors. N-Alkylation of H-Dmt-Tic-OH and H-Dmt-Tic-Ala-OH with methyl groups produced potent delta-opioid ligands with high delta receptor binding capabilities and enhanced delta antagonism: (i) N-Me-Dmt-Tic-OH 5 had high delta opioid binding (Ki delta = 0.2 nM), elevated delta antagonism on mouse vas deferens (MVD) (pA2 = 8.5; Ke = 2.8 nM), and nondetectable mu activity with guinea pig ileum (GPI). (ii) N,N-Me2-Dmt-Tic-OH (12) was equally efficacious in delta receptor binding (Ki delta = 0.12 nM; Ki mu/Ki delta = 20000), but delta antagonism rose considerably (pA2 = 9.4; Ke = 0.28 nM) with weak mu antagonism (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:5640). N-Me-(9) and N,N-Me2-Dmt-Tic-Ala-OH (15) also augmented delta opioid receptor binding, such that 15 demonstrated high affinity (Ki delta = 0.0755 nM) and selectivity (Ki mu/Ki delta = 20132) with exceptional antagonist activity on MVD (pA2 = 9.6; Ke = 0.22 nM) and weak antagonism on GPI (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:7180). Although the amidated dimethylated dipeptide analogue 14 had high Ki delta (0.31 nM) and excellent antagonist activity (pA2 = 9.9; Ke = 0.12 nM), the increased activity toward mu receptors in the absence of a free acid function at the C-terminus revealed modest delta selectivity (Ki mu/Ki delta = 1655) and somewhat comparable bioactivity (GPI/MVD = 4500). Thus, the data demonstrate that N,N-(Me)2-Dmt-Tic-OH (12) and N,N-Me2-Dmt-Tic-Ala-OH (15) retained high delta receptor affinities and delta selectivities and acquired enhanced potency in pharmacological bioassays on MVD greater than that of other peptide or non-peptide delta antagonists.

E. REALI, R. GUERRINI*, B. GIORI, M. BORGHI, M. MARASTONI*, R. TOMATIS*, S. TRANIELLO,M. G. MASUCCI† & R. GAVIOLIDepartment of Biochemistry and Molecular Biology and *Department of PharmaceuticalSciences, University of Ferrara, Italy and † Microbiology and Tumour Biology Centre, Karolinska Institute, Stockholm, Sweden(Accepted for publication 19 March 1996)SUMMARYCytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by theirability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTLresponses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation withthe autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivitiesdirected to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBVnuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulationof lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency ofCTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented byHLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstratedthat clones activated under these conditions are as efficient as clones activated by conventional LCLstimulations. Induction of memory CTL responses required low surface density of MHC:peptidecomplexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentra-tions of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealedthe presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific responseupon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulationwith synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and maybe particularly helpful for the selective expansion of subdominant CTL specificities.Keywords cytotoxic T lymphocytes immunogenic peptides HLA-A11 Epstein–Barr virusINTRODUCTIONCytotoxic T lymphocytes (CTL) play an important role in thecontrol of viral, bacterial and parasitic infections, and can recog-nize and destroy tumour cells [1–6]. The molecular targets of theirattack are peptide fragments, derived from processed antigenicproteins, selected for presentation at the surface of target cells bytheir ability to bind to the antigen-presenting groove of MHC classI molecules [7,8].Vaccination with synthetic peptides or with peptide-pulsedcells was shown to induce specific CTL against immunogenicviral or tumour-derived epitopes in vivo [9–12]. In addition,passive transfer of these CTL has been successfully employed inthe management of these diseases [2,4,13].Autologous virus-infected or malignant cells are commonlyused for in vitro induction and expansion of virus- or tumour-specific CTL. These protocols of stimulation lead to the activationof polyclonal CTL responses where only a fraction of the effectorsis likely to bear the desired specificities. In addition, most CTL inpolyclonal cultures are directed to immunodominant epitopes,while reactivities to subdominant epitopes are often lost duringin vitro propagation [14,15]. This is a potential drawback of thesestrategies, since immunodominant epitopes are often selected formutations in vivo, hampering their effectiveness as therapeutictargets [16–21]. Hence, alternative protocols of T cell activationwould allow triggering of CTL responses with unique specificities,and the selective activation of responses to subdominant epitopeswhich are not detected in polyclonal CTL cultures obtained byconventional stimulations.In this study we have exploited the presence of Epstein–Barrvirus (EBV)-specific memory CTL precursors in blood lympho-cytes from EBV

In this study, we define calmodulin binding sites of skeletal, cardiac, and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR [Menegazzi, P., et al. (1994) Biochemistry 33, 9078-9084] were synthesized, and their interaction with calmodulin was monitored by fluorescent techniques. The central portions of the skeletal, cardiac, and brain RYR protomers display one high (CaM1; Kd ranging between 2.7 and 10.2 nM) and one low affinity (CaM2; Kd ranging between 116 and 142 nM) calmodulin binding site. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) was identified a few residues upstream from the putative transmembrane segment M1 or M5. Its affinity for calmodulin varied between the RYR isoforms: the cardiac RYR CaM3 displays a high affinity (9.09 +/- 1.0 nM, n = 5), while the skeletal and brain RYR CaM3 have low affinity, the lowest affinity being displayed by the brain isoform (234 +/- 39 nM, n = 3). The RYRs calmodulin binding site CaM1 encompasses the sequence Arg-His-Arg-Val(Ile)-Ser-Leu, which is phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. Phosphorylation of RYR PM1 peptides occurs on the Ser, corresponding to amino acid number 2919, 3020, and 3055 of the brain, cardiac, and skeletal RYR protomers, respectively. We found that phosphorylation of the RYR PM1 peptides was inhibited by calmodulin binding and that the formation of the PM1 peptide-calmodulin complex was inhibited by peptide phosphorylation. These data indicate that the effect of calmodulin binding to RYR CaM1 may be regulated by the phosphorylation state of the Ser residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu.

MHC class I antigens bind peptides derived from endogenous proteins and present them to cytotoxic T lymphocytes. This binding is selective and shows high allele specificity. Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Peptide analogues, derived from previously identified high affinity peptides and carrying amino acid substitutions in position 2, were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we were able to discriminate among apparent and true binders. Only analogues carrying in position 2 the amino acids valine, threonine and isoleucine formed stable complexes with HLA-A11 with a half life > or = 72 hours.

The surprising change of selectivity induced by the change of chirality in peptides containing the tetrahydro-3-isoquinoline carboxylic acid (Tic) in second position, interpreted as a conformational preference induced on the Tyr-Xaa-Phe domain, can instead be attributed to the Tyr-Tic message domain. The relative spatial disposition of the aromatic rings of δ-selective non peptidic opiates is compatible with a message domain, in opioid peptides, of only two residues. This hypothesis was tested through the synthesis of Tyr-L-Tic-NH 2 , Tyr-D-Tic-NH 2 , Tyr-L-Tic-Ala-NH 2 , Tyr-L-Tic-Ala-OH and Tyr-D-Tic-Ala-NH 2 . Peptides containing Tyr-L-Tic- behave as very selective δ antagonists and those containing Tyr-DTic- as non selective agonists. This is the first case of opioid peptides containing a two-residue message domain and of opioid dipeptides with substantial opioid activity.