Alanine scan of an immunosuppressive peptide (CP): analysis of structure-function relationships.

Core peptide is a hydrophobic peptide, the sequence of which is derived from the T-cell antigen receptor alpha-chain transmembrane region. Previous studies have shown that core peptide can inhibit T-cell-mediated immune responses both in vitro and in vivo. Here, we report the role each constituent amino acid plays within core peptide using an alanine scan and the amino acid effect on function using a biological antigen presentation assay. The biophysical behaviour of these analogues in model membranes was analysed using surface plasmon resonance studies and then binding correlated with T-cell function. Removal of any single hydrophobic amino acid between the two charged amino acids in core peptide (R, K) resulted in lower binding. Changing the overall net charge of core peptide, by removing either of the positively charged residues (R or K), had varying effects on peptide binding and IL-2 production. There was a direct correlation (ρ = 0.718) between peptide binding to model membranes and peptide ability to inhibit IL-2. Except for IL-2 inhibition, production of other T-cell cytokines such as GM-CSF, IFN-γ, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and T-cell antigen receptor alpha-chain was not detected using a fluorescent bead immunoassay. This study provides important structure-function relationships essential for further drug design.

The potential of liposomal drug delivery for the treatment of inflammatory arthritis.

OBJECTIVE: To review the use of liposomes as a delivery agent in inflammatory arthritis. METHODS: The literature on liposomes and liposomal drug delivery for the treatment of inflammatory arthritis was reviewed. A PubMed search of articles in the English-language journals from 1965 to 2007 was performed. The index words used were as follows: "rheumatoid arthritis," "liposomes," and "targeted delivery." Papers identified were reviewed, abstracted, and summarized. RESULTS: Liposomes have the capacity to be used as delivery and targeting agents for the administration of antirheumatic drugs at lower doses with reduced toxicity. In other areas of medicine, the pace of progress has been rapid. In the case of infectious diseases and cancer, liposomal drug delivery has progressed and developed into commercially viable therapeutic options for the treatment of fungal infections (amphotericin B), or metastatic breast cancer and Kaposi sarcoma (doxorubicin, daunorubicin), respectively. In arthritis, the efficacy of prednisolone-loaded long-circulating liposomes is currently being evaluated in a phase II clinical trial. Liposome's application to arthritis is still in its infancy but appears promising as new patents are filed. With improvements in liposomal formulation and targeted synovial delivery, liposomes offer increased therapeutic activity and improvement in the risk-benefit ratio. CONCLUSION: Recent research into synovial targets and improved liposomal formulations continues to improve our capacity to use liposomes for targeted delivery. With time, this approach has the potential to improve drug delivery and reduce systemic complications.

Therapeutic application of transmembrane T and natural killer cell receptor peptides.

Autoimmune diseases primarily mediated by T-cells effect a significant proportion of the population and include common and distressing conditions such as diabetes, multiple clerosis, inflammatory bowel disease, skin diseases and arthritis. Current treatments are restrictive in terms of range of options and side-effect profiles and new drugs and new approaches are always eagerly sought. With the T-cell antigen receptor (TCR) as a model system we have identified a new approach to inhibit T-cell activation. By means of peptides derived from the transmembrane TCR-alpha chain region we have shown that T-cells, the major effector cells of disease, can be inhibited in vitro and the immune responses leading to disease ameliorated in animal models. The exact molecular mechanism of peptide action is still uncertain and assumed to involve a disturbance in transmembrane protein-protein interactions mediated by amino acid charges that disrupt normal signaling pathways. This chapter summarizes the results to date ofTCR core peptide (CP); the most effective peptide noted so far, in terms of function, behavior in membranes and future development and application as a therapeutic agent. The lessons learned from this model can be applied to other multi-subunit receptors that serve critical cellular functions and open new doors for drug design, development and application.

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes.

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC). Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.

Immunoreceptor transmembrane peptides and their effect on natural killer (NK) cell cytotoxicity.

Short peptides derived from the transmembrane sequence of NK activating receptors and associated molecules were tested in vitro for inhibition of NK cell cytotoxicity using a standard (51)Cr release assay in the absence or presence of peptides. NKL23 cell line was used as the NK effector and the target was the NKL23 sensitive 721.221 cell line. NKp46, NKp30, NKG2D and CD3-zeta peptides inhibited NK activity at higher concentration (100 microM) compared to controls by 6-13% (p<0.05). Modification of one non-effective peptide (NKP44) significantly enhanced inhibition by 30%, 17% and 11% at 100 microM, 50 microM and 10 microM respectively compared to controls. A T-cell antigen receptor-alpha chain transmembrane sequence derived peptide (CP) significantly inhibited NKL cell activation by 20-30% (p<0.05) at 50 microM and 100 microM concentrations compared to the control. The structural similarities between these immuno-receptors, and in particular the need for transmembrane electrostatic interactions for receptor function, provides the basis for current and future targeted therapeutic strategies.

Hydrophobic transmembrane-peptide lipid conjugations enhance membrane binding and functional activity in T-cells.

Application of different delivery methods for therapeutic peptides has gained much attention in recent years. In this paper we conjugated a transmembrane hydrophobic peptide (core peptide; CP) derived from the T-cell antigen receptor alpha-chain sequence with either one (LP1), two (LP2), or three (LP3) palmitic acids through a Tris linkage. The effect of these lipopeptides (LPs) were compared to CP's activity both in vitro and in model membrane binding experiments using surface plasmon resonance. The influence of charged amino acids, arginine and lysine, within the CP sequence was examined by synthesizing analogues where arginine and lysine were replaced by the neutral amino acid alanine and these analogues were subsequently Tris-lipid conjugated with either one (XP1), two (XP2), or three (XP3) palmitic acids through a Tris linkage. The results indicated that the amount of irreversible binding for LPs were all greater than that of the underivatized CP in model membranes. None of the LPs could be dissociated from the liposome membranes, even after prolonged washing. Binding results for the neutral conjugates showed that only the XP1 bound to model membranes. This binding was 20% as efficient compared to LP1. In biological assays it was found that LP1 and XP1 were toxic to cells. LP3 inhibited IL-2 production more effectively than CP. Control lipopeptides (XP2, XP3) did not inhibit IL-2 production. These results demonstrate that the number of lipids conjugated to peptide, and the charged amino acids of CP, are both essential factors for peptide function and activity that can be enhanced by lipidation.

T cell antigen receptor peptide-lipid membrane interactions using surface plasmon resonance.

This study examines the binding properties of a new class of immunomodulating peptides derived from the transmembrane region of the T cell antigen receptor, on model membranes using surface plasmon resonance. The di-basic "core" peptide was found to bind to both zwitterionic and anionic model membranes as well as to a T cell membrane preparation. By contrast, switching one or both of the basic residues to acidic residues led to a complete loss of binding to model membranes. In addition, the position of the charged amino acids in the sequence, the number of hydrophobic amino acids between the charged residues, and substitution of one or both basic to neutral amino acids were found to effect binding. These results when compared with in vitro T cell stimulation assays and in vivo adjuvant-induced arthritis models, showed very close correlation and confirmed the findings that amino acid charge and location may have a role in peptide activity. These initial biophysical peptide-membrane interactions are critically important and correlate well with the subsequent cellular expression and biological effect of these hydrophobic peptides. Targeting and understanding the biophysical interactions between peptides and membranes at their site of action is paramount to the description of cell function and drug design.