Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis.

In this study, we investigated the efficacy of new bifunctional peptide inhibitors (BPIs) in suppressing experimental autoimmune encephalomyelitis (EAE) in an animal model. BPI [e.g. proteolipid protein-cyclo(1,8)-CPRGGSVC-NH2 (PLP-cIBR)] is a conjugate between the PLP139-151 peptide derived from proteolipid protein (PLP) and the cIBR7 peptide derived from domain-1 (D1) of intercellular adhesion molecule-1 (ICAM-1). PLP-cIBR is designed to bind to major histocompatibility complex (MHC)-II and leucocyte function-associated antigen-1 (LFA-1) simultaneously to inhibit the formation of the immunological synapse and alter the differentiation and activation of a subpopulation of T cells, thus inducing immunotolerance. The results show that PLP-cIBR is highly potent in ameliorating EAE, even at low concentrations and less frequent injections. Mice treated with PLP-cIBR had a higher secretion of cytokines related to regulatory and/or suppressor cells compared to phosphate-buffered saline (PBS)-treated mice. In contrast, T helper type 1 (Th1) cytokines were higher in mice treated with PBS compared to PLP-cIBR, suggesting that it suppressed Th1 proliferation. Also, we observed significantly less demyelination in PLP-cIBR-treated mice compared to the control, further indicating that PLP-cIBR promoted protection against demyelination.

Secondary structure of a dynamic type I'beta-hairpin peptide.

A spontaneously folding beta-hairpin peptide (Lys-Lys-Tyr-Thr-Val-Ser-Ile-Asn-Gly-Lys-Lys-Ile-Thr-Val-Ser-Ile) and related cyclic (cyclo-Gly-Lys-Tyr-Ile-Asn-Gly-Lys-Ile-Ile-Asn) and linear (Ser-Ile-Asn-Gly-Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D (1)H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The beta-hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 - i + 2 and i + 2 -i + 3 interaction, confirming the expected structure. Using NMR alpha-proton (H(alpha)) chemical shifts, the extents of folding of the beta-hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 degrees C), which was taken to be maximally folded. Substitution of iso-aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained beta-turns may have adverse effects on secondary structure. N-terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Synthesis and conformational analysis of a coumarinic acid-based cyclic prodrug of an opioid peptide with modified sensitivity to esterase-catalyzed bioconversion.

The coumarinic acid-based cyclic DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) prodrug 1a exhibited more favorable physicochemical properties than did DADLE for permeation across the intestinal mucosa. However, prodrug 1a, whose bioconversion to DADLE was slow, was subject to extensive biliary clearance when administered to rats in vivo. To increase the rate of esterase-catalyzed bioconversion of prodrug 1a, thus decreasing its biliary clearance, the oxymethyl-modified prodrug 1, in which an aldehyde equivalent is inserted between the phenolic group of the promoiety and the carboxylic acid group of the peptide, was synthesized from benzofuran-2-carboxylic acid 16 via a nine-step procedure. Briefly, phenacyl-protected 3-(2-hydroxyphenyl)-propynoic acid 17 was coupled with Boc-d-Leu-OCH(2)I 5 to give the intermediate 18, which was further elaborated and conjugated with tetrapeptide 4 to give linear precursor 2. Precursor 2 was then deprotected and cyclized to obtain compound 1 using a high dilution technique. In an attempt to investigate the effect of the physicochemical properties and the conformation of prodrug 1 on its permeation characteristics, we calculated its physicochemical parameters and determined its solution conformation using spectroscopic techniques (CD and NMR) and molecular dynamics simulations. Prodrug 1 showed a cLogP value and a molecular size similar to that of prodrug 1a. The deconvoluted CD spectra indicated that prodrug 1 has more random component (71%) than prodrug 1a (42%). 2D-NMR studies of prodrug 1 showed no signals for amide-amide hydrogen interactions and few ROE cross-peaks in ROESY spectra. Using distance restraints constructed from ROESY spectra, molecular dynamics simulations of prodrug 1 generated five conformation families. One family satisfied most of the distance restraints and all of the dihedral angles measured by NMR coupling constants. In summary, prodrug 1 showed favorable physicochemical properties for permeation of the intestinal mucosa. Prodrug 1 adopted a more random conformation in solution than prodrug 1a. These differences in solution conformation could affect the permeation of the prodrugs across the intestinal mucosa by passive diffusion and/or their ability to interact with efflux transporter(s) that would limit their transcellular permeation.

Binding and internalization of an LFA-1-derived cyclic peptide by ICAM receptors on activated lymphocyte: a potential ligand for drug targeting to ICAM-1-expressing cells.

PURPOSE: The interaction of cell-adhesion molecules LFA-1/ICAM-1 is critical for many inflammatory and immune responses. Blockades of this interaction using antibodies or peptide analogs are being developed as therapeutic approaches for inflammatory and autoimmune diseases. The aim of this study is to examine the binding and internalization mechanisms of LFA-1 peptide [cLAB.L or cyclo-(1,12)-PenITDGEATDSGC] mediated by ICAM receptors on the surface of lymphocytes. METHODS: The binding and internalization of cLAB.L were evaluated using fluorescence-labeled cLAB.L on activated Molt-3 cells, measured by flow cytometry. Confocal fluorescence microscopy was also used to image the distribution of peptide binding and internalization. RESULTS: The binding of FITC-cLAB.L exhibited bimodal cell distribution and was enhanced by Ca2+ and Mg2+. Marked differences in peptide binding were found between 37 and 4 degrees C, as well as between activated and non-activated cells. Unlabeled peptide, low temperature, and the absence of cell activation suppress the peptide binding. The presence of peptide in the cytoplasm was detected in 37 but not 4 degrees C binding. Peptide cLAB.L inhibited the binding of monoclonal antibodies to domain D1 of ICAM-1 and domain D1 of ICAM-3. CONCLUSIONS: Peptide cLAB.L can bind to the D1-domain of ICAM-1 and, to a lesser extent, to ICAM-3 on activated T-cells. Peptide binding indicates responses to the multiple and dynamic states of activated receptor ICAMs, this peptide may also be internalized by ICAM receptors on T-cells. This work suggests that cLAB.L has a therapeutic potential to target drugs to ICAM-1 expressing cells including autoreactive lymphocytes and inflamed tissues.

Improving the selectivity of HAV-peptides in modulating E-cadherin-E-cadherin interactions in the intercellular junction of MDCK cell monolayers.

PURPOSE: The objective of this work is to understand the sequence specificity of HAV peptides and to improve their selectivity in regulating E-cadherin-E-cadherin interactions in the intercellular junctions. METHODS: Peptide 1 was modified using an alanine scanning method to give peptides 2-6. The ability of these peptides to modulate intercellular junctions was evaluated using Madin-Darby Canine Kidney (MDCK) cell monolayers on Transwell membranes from either the apical (AP) or the basolateral (BL) side. Modulation of the intercellular junctions was measured by the ability to lower the transepithelial electrical resistance (TEER) of MDCK monolayers and by the increase in mannitol flux. Molecular docking experiments were performed to model the binding properties of these peptides to the EC1 domain of E-cadherin. RESULTS: Peptides 5 (Ac-SHAVAS-NH2) and 6 (Ac-SHAVSA-NH2) were found to be more effective than the parent peptide 1 in decreasing the resistance of the cell monolayer. Furthermore, comparative studies with the control and the weak inhibitor peptide 2 indicate that peptide 5 displayed a significant increase in mannitol flux. Molecular docking of peptides 1, 2 and 5 to the EC1 domain suggests that peptide 5 has the lowest binding energy. CONCLUSIONS: HAV peptides have the ability to modulate E-cadherin-E-cadherin interactions in the intercellular junctions of the MDCK cell monolayer, thus indirectly increasing the permeability of the tight junctions. This observation indicates that residues flanking the HAV sequence are important in the binding selectivity of HAV peptides to E-cadherin. Molecular docking can further aid in the design of peptides with better selectivity to the EC1 domain of E-cadherin.

Inhibition of the adherence of T-lymphocytes to epithelial cells by a cyclic peptide derived from inserted domain of lymphocyte function-associated antigen-1.

Tissue inflammation is characterized by aggravated leukocyte infiltration into the sites of inflammation. The mechanism requires the interactions of leukocyte adhesion-molecules and their ligands in the inflamed tissues. In this study, we demonstrate that a cyclic peptide cLAB.L [cyclol, 12-PenlTDGEATDSGC], derived from the "inserted" or I-domain of LFA-1 is able to inhibit the adherence of T-lymphocytes to the epithelial cell monolayers. This inhibition has been thought to involve the disruption of LFA-1/ICAM-1 interaction. The heterotypic adhesion of phorbol ester-activated Molt-3 cells and IFN-gamma-induced Caco-2 monolayers was inhibited upon treatment of the monolayers with monoclonal antibodies (MAbs) to adhesion molecules or with cLAB.L peptide. The adhesion can be inhibited by MAbs to ICAM-1, ICAM-2, and VCAM-1, and cLAB.L peptide in a concentration-dependent manner. However, none of the individual uses of these molecules led to a total inhibition. The inhibitory activity of cLAB.L is greatly reduced by low temperature and the absence of cell activation. Treatment of cLAB.L peptide may trigger an early event of apoptosis on activated but not on non-activated Molt-3 cells; no indication of peptide-induced apoptosis was found on Caco-2 cells. Taken together, data from this work suggest that cLAB.L may have applications to direct cell-targeted delivery during tissue inflammation.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

Synergistic inhibitory activity of alpha- and beta-LFA-1 peptides on LFA-1/ICAM-1 interaction.

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the alpha-subunit and the I-like domain of the beta-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.

Linear and cyclic LFA-1 and ICAM-1 peptides inhibit T cell adhesion and function.

Short peptides derived from functional proteins have been used in several instances to inhibit activity of the parent proteins. In some cases, stability and efficacy were found to be increased by cyclization of these peptides. Inhibition of interaction of the two cell adhesion counter receptors leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1 is being studied as a method for modulating autoimmune diseases such as rheumatoid arthritis and for facilitating organ transplantation. Here, several 10-amino acid peptides derived from the contact domains of LFA-1 and ICAM-1 were evaluated for their ability to interfere with intercellular adhesion by T cells and to inhibit a more biologic, mixed lymphocyte reaction. Both linear and cyclic forms of the peptides were effective at inhibiting intercellular adhesion. Cyclic forms were effective at inhibiting T cell activation and proliferation in the mixed lymphocyte reaction.

Enhancement of transport of D-melphalan analogue by conjugation with L-glutamate across bovine brain microvessel endothelial cell monolayers.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture.

BACKGROUND: The counter receptors intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are lymphocyte cell surface adhesion proteins the interaction of which can provide signals for T cell activation. This binding event is important in T cell function, migration, and general immune system regulation. The ability to inhibit this interaction with monoclonal antibodies has proved to be therapeutically useful for several allograft rejection and autoimmune disease models. METHODS: Short peptides representing counter-receptor contact domains of LFA-1 and ICAM-1 were examined for their ability to inhibit T cell adhesion and T cell function. RESULTS: Peptides encompassing amino acids Q1-C21 and D26-K50 of ICAM-1, I237-I261 and G441-G466 of the LFA-1 alpha-subunit, and D134-Q159 of the LFA-1 beta-subunit inhibited LFA-1/ICAM-1-dependent adhesion in a phorbol-12,13-dibutyrate-induced model of tonsil T cell homotypic adhesion. This inhibition was specific to the peptide sequence and occurred without stimulation of T cell proliferation. The peptides also were effective in preventing T cell function using a one-way mixed lymphocyte reaction model for bone marrow transplantation. CONCLUSIONS: Our data suggest that these peptides or their derivatives may be useful as therapeutic modulators of LFA-1/ICAM-1 interaction during organ transplants.

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker.

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.

Binding and internalization of an ICAM-1 peptide by the surface receptors of T cells.

The objective of this work was to evaluate the binding characteristics of a cyclic peptide, cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH (cIBR), to Molt-3 T cells. This cIBR peptide is derived from sequence numbers 11-20 of intercellular adhesion molecule-1 (ICAM-1). Binding studies were performed using a fluorescence-labeled peptide (FITC-cIBR) in which the fluorescence marker fluorescein 5-isothiocyanate (FITC) was conjugated to the N-terminal of the cIBR peptide. The binding affinity of the FITC-cIBR peptide to Molt-3 T cells was evaluated using a FACScan flow cytometer. The binding specificity of the FITC-cIBR peptide was also confirmed by inhibition of binding using unlabeled peptide (cIBR). The results show that FITC-cIBR binds to two populations of T cells with different affinities; population 1 has high cell numbers (75%) but low affinity, and population 2 has high binding affinity but low cell numbers (25%). Binding to both populations was saturable and could be inhibited by the unlabeled peptide (cIBR), suggesting a receptor-mediated binding process. In addition to binding, receptor-mediated internalization was also observed for population 2; this was confirmed by confocal microscopy and temperature-dependence studies at 37 degrees C and 4 degrees C. The binding and internalization of this peptide may be carried out by surface receptors on Molt-3 T cells such as LFA-1. In the future, the binding and internalization of cIBR peptide can be utilized as a method of targeted drug delivery to leukocytes for the treatment of leukocyte-related diseases.

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.