Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

BACKGROUND: The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. METHODS: Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. RESULTS: Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC50 of 1.75 µM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. CONCLUSIONS: Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Hepatitis B virus peptide inhibitors: solution structures and interactions with the viral capsid.

Hepatitis B virus (HBV) infection remains a health problem globally despite the availability of effective vaccines. In the assembly of the infectious virion, both the preS and S regions of the HBV large surface antigen (L-HBsAg) interact synergistically with the viral core antigen (HBcAg). Peptides preS and S based on the L-HBsAg were demonstrated as potential inhibitors to block the viral assembly. Therefore, the objectives of this study were to determine the solution structures of these peptides and study their interactions with HBcAg. The solution structures of these peptides were solved using (1)H, (13)C, and (15)N NMR spectroscopy. Peptide preS has several structured regions of ß-turns at Ser7-Pro8-Pro9, Arg11-Thr12-Thr13 and Ser22-Thr23-Thr24 sequences whereas peptide S has only one structured region observed at Ser3-Asn4-His5. Both peptides contain bend-like structures surrounding the turn structures. Docking studies revealed that both peptides interacted with the immunodominant region of HBcAg located at the tip of the viral capsid spikes. Saturation Transfer Difference (STD) NMR experiments identified several aromatic residues in peptides preS and S that interact with HBcAg. This study provides insights into the contact regions of L-HBsAg and HBcAg at atomic resolution which can be used to design antiviral agents that inhibit HBV morphogenesis.

Exploration of neuroprotective effect from Coriandrum sativum L. ethanolic seeds extracts on brain of obese rats.

In this study, the potential neuroprotective ability of coriander seeds (Coriandrum sativum L.) ethanolic extract (CSES) as a neuroprotectant agent in the brains of high-fat diet-induced obese rats was analyzed. The study investigated how CSES impacts oxidative stress markers (i.e., malondialdehyde/MDA, glutathione/GSH and catalase), inflammation marker (i.e., Interleukin-6/IL-6), cellular senescence markers (i.e., senescence-associated ß-galactoside/SA-ß-Gal activity and p16), brain damage marker (i.e., Neuron-specific Enolase/NSE), and neurogenesis markers (i.e., mature Brain-derived Neurotropic Factor/BDNF, pro-BDNF, and mature/pro-BDNF ratio). Male adult Wistar rats were fed a high-fat diet and given CSES once daily, at 100 mg/kg body weight, for 12 weeks. CSES significantly reduced MDA concentration (p = < 0.001), SA-ß-Gal activity (p = 0.010), and increased GSH concentration (p = 0.047) in the brain of obese rats; however, the decrease of IL-6, NSE, and p16 as well as the increase of catalase specific activity and BDNF expression were not significant. Moreover, the mature/pro-BDNF ratio was significantly higher in the brains of non-obese rats, both given the control diet and the high-fat diet compared to the control. Our results suggest that obese rats benefited from consuming CSES, showing improved oxidative stress levels, reduced cellular senescence and increased endogenous antioxidants, making CSES a potential neuroprotective agent.

Various polar tripeptides as asymmetric organocatalyst in direct aldol reactions in aqueous media.

A series of tripeptide organocatalysts containing a secondary amine group and two amino acids with polar side chain units were developed and evaluated in the direct asymmetric intermolecular aldol reaction of 4-nitrobenzaldehyde and cyclohexanone. The effectiveness of short polar peptides as asymmetric catalysts in aldol reactions to attain high yields of enantio- and diastereoselective isomers were investigated. In a comparison, glutamic acid and histidine produced higher % ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric aldol addition reaction in aqueous media.

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays. RESULTS: Twenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment. CONCLUSION: Three compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.

Nanoglue: an alternative way to display cell-internalizing peptide at the spikes of hepatitis B virus core nanoparticles for cell-targeting delivery.

Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.

Structure-based and ligand-based virtual screening of novel methyltransferase inhibitors of the dengue virus.

BACKGROUND: The dengue virus is the most significant arthropod-borne human pathogen, and an increasing number of cases have been reported over the last few decades. Currently neither vaccines nor drugs against the dengue virus are available. NS5 methyltransferase (MTase), which is located on the surface of the dengue virus and assists in viral attachment to the host cell, is a promising antiviral target. In order to search for novel inhibitors of NS5 MTase, we performed a computer-aided virtual screening of more than 5 million commercially available chemical compounds using two approaches: i) structure-based screening using the crystal structure of NS5 MTase and ii) ligand-based screening using active ligands of NS5 MTase. Structure-based screening was performed using the LIDAEUS (LIgand Discovery At Edinburgh UniverSity) program. The ligand-based screening was carried out using the EDULISS (EDinburgh University LIgand Selection System) program. RESULTS: The selection of potential inhibitors of dengue NS5 MTase was based on two criteria: the compounds must bind to NS5 MTase with a higher affinity than that of active NS5 MTase ligands, such as ribavirin triphosphate (RTP) and S-adenosyl-L-homocysteine (SAH); and the compounds must interact with residues that are catalytically important for the function of NS5 MTase. We found several compounds that bind strongly to the RNA cap site and the S-adenosyl-L-methionine (SAM) binding site of NS5 MTase with better binding affinities than that of RTP and SAH. We analyzed the mode of binding for each compound to its binding site, and our results suggest that all compounds bind to their respective binding sites by interacting with, and thus blocking, residues that are vital for maintaining the catalytic activity of NS5 MTase. CONCLUSIONS: We discovered several potential compounds that are active against dengue virus NS5 MTase through virtual screening using structure-based and ligand-based methods. These compounds were predicted to bind into the SAM binding site and the RNA cap site with higher affinities than SAH and RTP. These compounds are commercially available and can be purchased for further biological activity tests.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.

Novel furan-containing peptide-based inhibitors of protein arginine deiminase type IV (PAD4).

Protein arginine deiminase type IV (PAD4) is responsible for the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullinated protein is the autoantigen in rheumatoid arthritis, and therefore, PAD4 is currently a promising therapeutic target for the disease. Recently, we reported the importance of the furan ring in the structure of PAD4 inhibitors. In this study, the furan ring was incorporated into peptides to act as the "warhead" of the inhibitors for PAD4. IC50 studies showed that the furan-containing peptide-based inhibitors were able to inhibit PAD4 to a better extent than the furan-containing small molecules that were previously reported. The best peptide-based inhibitor inhibited PAD4 reversibly and competitively with an IC50 value of 243.2 ± 2.4 µm. NMR spectroscopy and NMR-restrained molecular dynamic simulations revealed that the peptide-based inhibitor had a random structure. Molecular docking studies showed that the peptide-based inhibitor entered the binding site and interacted with the essential amino acids involved in the catalytic activity. The peptide-based inhibitor could be further developed into a therapeutic drug for rheumatoid arthritis.

Structure and Dynamics of Antifreeze Protein--Model Membrane Interactions: A Combined Spectroscopic and Molecular Dynamics Study.

Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.

Modeling stability and flexibility of α-Chymotrypsin in room temperature ionic liquids.

We investigate the structure and dynamics of α-Chymotrypsin in five room temperature ionic liquids (RTILs) sharing a common cation, hydrated with different water percentages (w/w) (weight of water over protein). Results from molecular dynamics simulations are correlated with experimental evidences from studies on the activity of enzymes in RTILs. α-Chymotrypsin protein structure is closer to its native crystallographic structure in RTILs than in aqueous environment. We show that the structural properties of α-Chymotrypsin were affected by the water concentration assayed in a typical bell-shaped profile, which is also frequently reported for organic solvents. The protein structure was more native like at 10-20% of water (w/w) for all RTILs except for [BMIM][Cl]. We found that the fluctuations of the main chain in [BMIM][BF4] and [BMIM][TfO] were not significantly affected by the increasing amount of water. However, we were able to show that the flexible regions were the ones more hydrated, indicating that water is responsible for the flexibility of the protein. The solvation of the enzyme in water-immiscible RTILs, such as [BMIM][PF6] and [BMIM][Tf2N] lead to higher enzyme flexibility at increased water content. Enzyme solvation by [BMIM][Cl] resulted in ion penetration in the core enzyme structure, causing incremented flexibility and destabilization at low water percentages. All RTILs stripped water molecules from the protein surface, following a similar behavior also found in organic solvents. Anions formed structured arrangements around the protein, which allowed non-stripped water molecules to localize on the protein surface.

Solution structures, dynamics, and ice growth inhibitory activity of peptide fragments derived from an antarctic yeast protein.

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.

An improved method for the preparations of nanostructured lipid carriers containing heat-sensitive bioactives.

Heat-sensitive bioactive compounds such as ß-carotene and tocols, are widely used in the pharmaceutical and cosmetic fields. Their chemical stability in delivery systems is one of the major concerns in the production of nanostructured lipid carriers (NLCs). A previously established high-temperature high-pressure homogenisation technique involved in the preparation of NLCs can cause degradation of heat-sensitive compounds. Therefore, a novel preparation process needs to be developed to minimise the degradation of heat-sensitive active compounds during the preparation of NLCs. In this work, modified methods A and B were designed to minimise the degradation of ß-carotene and tocols during the production of NLCs. These methods improved the chemical stability of heat-sensitive bioactive compounds (ß-carotene and tocols) significantly compared to the previously established method. The physical stability of the formulation was maintained throughout study duration.

Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes.

The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes.

Solution structure of a novel T-cell adhesion inhibitor derived from the fragment of ICAM-1 receptor: cyclo(1,8)-Cys-Pro-Arg-Gly-Gly-Ser-Val-Cys.

This study is aimed at elucidating the structure of a novel T-cell adhesion inhibitor, cyclo(1,8)-CPRGGSVC using one- and two-dimensional (2D) (1)H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)-PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule-1 (ICAM-1). Our previous results show that the cyclo(1,8)-CPRGGSVC peptide binds to the LFA-1 I-domain and inhibits heterotypic T-cell adhesion, presumably by blocking the LFA-1/ICAM-1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type-I beta-turn conformation at the Pro2-Arg3-Gly4-Gly5 (PRGG) sequence. The beta-turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM-1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)-CPRGGSVC peptide. Meanwhile, the Gly5-Ser6-Val7-Cys8-Cys1 (GSVCC) sequence forms a "turn-like" random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)-CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA-1 I-domain. The conserved beta-turn conformation of the PRGG motif in ICAM-1, cIBR, and cyclo(1,8)-CPRGGSVC peptides can potentially be used to design peptidomimetics. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 633-641, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Effect of modification of the physicochemical properties of ICAM-1-derived peptides on internalization and intracellular distribution in the human leukemic cell line HL-60.

The objective of this work is to test the hypothesis that increasing the hydrophilicity of DOX-peptide conjugates may modify their entry mechanisms into HL-60 cells from passive diffusion to receptor-mediated uptake. To test this hypothesis, the entry mechanisms and the intracellular disposition of DOX-cIBR7, DOX-PEGcIBR7, FITC-cIBR, and FITC-cIBR7 were evaluated in HL-60 cells. To increase the hydrophilicity of the peptide, the cIBR peptide (cyclo(1,12)Pen-PRGGSVLVTGC) was modified to cIBR7 peptide (cyclo(1,8)CPRGGSVC) by removing the hydrophobic residues at the C-terminus. DOX-cIBR7 conjugate, which has higher hydrophilicity than DOX-cIBR, was synthesized. Second, a hydrophilic linker (11-amino-3,6,9-trioxaundecanate linker) was incorporated between DOX and cIBR7 to generate DOX-PEGcIBR7 with higher hydrophilicity than DOX-cIBR7. As controls, FITC-cIBR and FITC-cIBR7 were used to check for any endocytic uptake process of the peptide. As previously found with DOX-cIBR, DOX-cIBR7, and DOX-PEGcIBR7, conjugates enter the cells via passive diffusion and not via the energy-dependent endocytic process. This result suggests that an increase in hydrophilicity does not influence the entry mechanism of the DOX-peptide conjugates. In contrast to the DOX-cIBR7 conjugate, the FITC-cIBR7 conjugate showed energy-dependent cellular entry into the cells and followed an endocytic pathway similar to that for dextran. Finally, the entry of DOX-cIBR7 and DOX-PEGcIBR into the cell cytosol was shown to be due to the properties of DOX and not to those of the peptide.

Structural modifications of ICAM-1 cyclic peptides to improve the activity to inhibit heterotypic adhesion of T cells.

Lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) interaction plays an important role in the formation of the immunological synapse between T cells and antigen-presenting cells. Blocking of LFA-1/ICAM-1 interactions has been shown to suppress the progression of autoimmune diseases. cIBR peptide (cyclo(1,12)PenPRGGSVLVTGC) inhibits ICAM-1/LFA-1 interaction by binding to the I-domain of LFA-1. To increase the bioactivity of cIBR peptide, we systemically modified the structure of the peptide by (i) replacing the Pen residue at the N-terminus with Cys, (ii) cyclization using amide bond formation between Lys-Glu side chains, and (iii) reducing the peptide size by eliminating the C-terminal residue. We found that the activity of cIBR peptide was not affected by replacing Phe with Cys. Peptide cyclization by forming the Lys-Glu amide bond also increased the activity of cIBR peptide, presumably due to the resistance of the amide bond to the reducing nature of glutathione in plasma. We also found that a reduced derivative of cIBR with eight residues (cyclo(1,8)CPRGGSVC) has a bioactivity similar to that of the larger cIBR peptides. Our findings suggest that, by systemically modifying the structure of cIBR peptide, the biological activity of these derivatives can be optimized for future use to inhibit T-cell adhesion in in vivo models of autoimmune diseases.

Sequence recognition of alpha-LFA-1-derived peptides by ICAM-1 cell receptors: inhibitors of T-cell adhesion.

Blocking the T-cell adhesion signal from intercellular adhesion molecule-1/leukocyte function-associated antigen-1 interactions (Signal-2) can suppress the progression of autoimmune diseases (i.e. type-1 diabetes, psoriasis) and prevent allograph rejection. In this study, we determined the active region(s) of cLAB.L peptide [cyclo(1,12)Pen-ITDGEATDSGC] by synthesizing and evaluating the biologic activity of hexapeptides in inhibiting T-cell adhesion. A new heterotypic T-cell adhesion assay was also developed to provide a model for the T-cell adhesion process during lung inflammation. Two hexapeptides, ITDGEA and DGEATD, were found to be more active than the other linear hexapeptides. The cyclic derivative of ITDGEA [i.e. cyclo(1,6)ITDGEA] has similar activity than the parent linear peptide and has lower activity than cLAB.L peptide. Computational-binding experiments were carried out to explain the possible mechanism of binding of these peptides to intercellular adhesion molecule-1. Both ITDGEA and DGEATD bind the same site on intercellular adhesion molecule-1 and they interact with the Gln34 and Gln73 residues on D1 of intercellular adhesion molecule-1. In the future, more potent derivatives of cyclo(1,6)ITDGEA will be designed by utilizing structural and binding studies of the peptide to intercellular adhesion molecule-1. The heterotypic T-cell adhesion to Calu-3 will also be used as another assay to evaluate the selectivity of the designed peptides.

ICAM-1 peptide inhibitors of T-cell adhesion bind to the allosteric site of LFA-1. An NMR characterization.

We have used nuclear magnetic resonance to characterize the binding site of two intercellular adhesion molecule-1 derived cyclic peptides, cIBC and cIBR, to the I-domain of leukocyte function-associated antigen-1. These peptides inhibit the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction known to play a key role in autoimmune diseases and cancer metastasis. Perturbation of the chemical shifts and intensities of the nuclear magnetic resonance signals corresponding to a number of residues of the I-domain of leukocyte function-associated antigen-1 show that both peptides bind to the I-domain allosteric site, the binding site of I-domain allosteric inhibitors such as lovastatin, and therefore the peptides probably also act as allosteric inhibitors of leukocyte function-associated antigen-1. Molecular models of the interaction of these two cyclic peptides with leukocyte function-associated antigen-1 I-domain show that the binding mode of the three molecules are analogous: the hydrophobic residues of the peptides remain buried and occupy the same positions as the apolar groups of lovastatin, while the peptides regions containing the most polar residues are flexible and primarily exposed to the solvent. These results suggest an allosteric mechanism for the inhibitory effect on T-cell adhesion displayed by both peptides, which exhibit potential as therapeutic agents.

Characterization of binding properties of ICAM-1 peptides to LFA-1: inhibitors of T-cell adhesion.

In the present study, we characterized the binding site of two intercellular adhesion molecule-1-derived cyclic peptides, cIBC and cIBR, to the LFA-1 on the surface of T cells. These peptides had been able to inhibit LFA-1/intercellular adhesion molecule-1 signal by blocking the signal-2 of immune synapse. Both peptides prefer to bind to the closed form of LFA-1 I-domain, indicating that two peptides act as allosteric inhibitors against intercellular adhesion molecule-1. Binding site mapping using monoclonal antibodies proposes that cIBC binds to around residues 266-272 of LFA-1 I-domain where this site is adjacent to the metal ion-dependent adhesion site. On the other hand, cIBR binds to the pocket called L-site where is distant from metal ion-dependent adhesion site. Cross-inhibition mapping between two peptides show that cIBR could inhibit the binding of cIBC but not vice versa, suggesting that cIBR has some properties that allow this peptide bind to more than one site. Structural comparison between cIBC and cIBR reveals that cIBR is more flexible than cIBC, allowing this peptide bind to exposed region, such as cIBC-binding site as well as cramped pocket like L-site. Our findings are important for understanding the selectivity of cIBC and cIBR peptides; thus, they can be conjugated with drugs and transported specifically to the target.