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Chemically modified peptide ligands were identified from α-helix peptide phage libraries with different types of staple linkers. The hDM2-protein was used as a representative target of protein–protein interactions to screen ligands for p53 binding sites in hDM2. Two types of staple linkers were used for the chemical modification of the peptide phage display libraries before affinity selection. The identified stapled peptides could bind to hDM2 competitively with the p53 peptide. The stapled peptide phage libraries developed in this study will improve the discovery of protein–protein interaction inhibitors through the synergistic effect of peptide units and staple linkers.

We recently developed a self-assembling peptide, E1Y9, that self-assembles into nanofibers and forms a hydrogel in the presence of Ca(2+) . E1Y9 derivatives conjugated with functional peptide sequences derived from extracellular matrices (ECMs) reportedly self-assemble into peptide nanofibers that enhance cell adhesion and differentiation. In this study, E1Y9/E1Y9-IKVAV-mixed hydrogels were constructed to serve as artificial ECMs that promote cell differentiation. E1Y9 and E1Y9-IKVAV co-assembled into networked nanofibers, and hydrogels with disk and string shapes were formed in response to Ca(2+) treatment. The neuronal differentiation of PC12 cells was facilitated on hydrogels of both shapes that contained the IKVAV motifs. Moreover, long neurites extended along the long axis of the string-shaped gel, suggesting that the structure of hydrogels of this shape can affect cellular orientation. Thus, E1Y9 hydrogels can potentially be used as artificial ECMs with desirable bioactivities and shapes that could be useful in tissue engineering applications. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 476-483, 2016.

Enzyme inhibitory peptides with a loop structure stabilized using an antiparallel β-sheet scaffold (β-loop peptide) were obtained from a designed peptide phage library. Human dihydrofolate reductase (hDHFR) was used as the target enzyme. The obtained β-loop peptides were competitive inhibitors of hDHFR with micromolar inhibition constants and dissociation constants.

Background Lectins are carbohydrate binding proteins and related to various biological events and diseases including virus infection and cancer metastasis. In particular, galactose-binding lectins have attracted attention as targets for drug delivery and cancer markers. We, previously, demonstrated that sugar-modified peptides (glycopeptides) were useful ligands for the detection and characterization of lectins compared to the sugar unit alone. Gold nanoparticles (GNPs) conjugated with mannose-modified glycopeptides were useful in detection of concanavalin A, a mannose binding lectin. Objectives The main objective of this study was to expand our glycopeptide-GNP conjugates for detection and imaging of galactose-binding lectins. Methods Four galactose-modified peptides (glycopeptides) were synthesized by Fmoc-based solid peptide synthesis method. Synthesized glycopeptides were conjugated with PEG-coated GNPs using thiol-maleimide chemistry. The interaction between glycopeptide-GNPs (GP/GNPs) (0.5 nM) and RCA120, a galactose binding lectin, (0.5-1000 nM) was evaluated by mesuring absorption spectra of GNPs. The inhibition experiment in the interaction between GP/GNPs (0.5 nM) and RCA120 (100 nM) was performed in the presence of 60 mM α- methyl mannose or 60 mM lactose. HepG2 and MCF7 cells were placed on 22×22 mm cover slip in 6 well cell culture plates (2×105 cells / well) and cultured overnight at 37°C under 5% CO2 condition. 1 mL of GP/GNPs (0.2 nM) were added in each well and incubated for 18 h at 37°C under 5% CO2 condition. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde solution. The cover slips were coated with 90% glycerol and sealed to slide glass. Dark-field images based on elastic light scattering were taken using a Nikon microscope (TieU) with an immersion dark field condenser. Results In the titration experiment of RCA120, GP/GNPs showed a decrease of absorbance according to the addition of RCA120, suggesting that the aggregation of GP/GNPs is induced through the binding to RCA120. The EC50 values of AA(Gal)/GNP, WF(Gal)/GNP, TS(Gal)/GNP and ED(Gal)/ GNP were estimated as 66.2 nM, 43.2 nM, 38.6 nM and 104.4 nM, respectively. TS(Gal)/GNP showed the lowest EC50 value among GP/GNPs. RCA120 has several binding sites for the galactose, and there are hydrophilic amino acids (Thr24, Glu26, Gln35, Asn42 and Asp44) around one of galactose binding sites. This result indicates that the hydrogen bonds between these amino acids and Thr/Ser residues of TS(Gal) contribute to the efficient aggregation of TS(Gal)/GNP. Next, inhibition experiments in the aggregation of WF(Gal)/GNP with RCA120 revealed that lactose inhibits the WF(Gal)/GNP binding with RCA120, but α-methyl mannose does not, and that WF(Gal)/GNP selectively interacts with RCA120 and forms the aggregate. Finally, a galactose binding protein on the surface of HepG2 cells was successfully visualized by using GP/GNPs as optical probes. Conclusion Our results demonstrated that GP/GNPs could detect RCA120 by the selective binding and the aggregation formation. Furthermore, a galactose binding protein on the surface of HepG2 cells is successfully visualized using WF(Gal)/GNP as an optical probe. Thus, GNPs conjugated with glycopeptides will be useful probes for the selective detection and imaging of lectins.

There is an increasing need for the development of functional artificial extracellular matrices (ECMs) for tissue engineering. Recently, we have successfully designed a self-assembling peptide, named E1Y9, to construct functional ECMs. We describe here an enhancement of abilities of E1Y9 materials to promote cell adhesion and differentiation, using functional peptide sequences derived from natural extracellular matrix proteins. We designed functionalized self-assembling peptides, RGDS-conjugated E1Y9 (E1Y9-RGDS) and IKVAV-conjugated E1Y9 (E1Y9-IKVAV). E1Y9-RGDS and E1Y9-IKVAV formed peptide nanofibers in a similar manner to E1Y9, with β-sheet secondary structures. Surfaces coated with peptide nanofibers displayed the higher bioactivities of E1Y9-RGDS for cell adhesion and E1Y9-IKVAV for cell differentiation than those of E1Y9, with the activities being dependent on the concentrations of the functional peptides. These functionalized peptides will be useful for the construction of functional ECMs in cell and tissue engineering. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 731–737, 2013.

A monosaccharide-modified β-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The β-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics.

Structure-activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an alpha-helical conformation, Glu-Lys pairs were introduced into the i and i+4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an alpha-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.

Treatment of N-arylsulfonylaziridines bearing α,β-unsaturated esters with alcohols, thiols or weak acids such as AcOH in the presence of catalytic amount of Lewis acids affords regio- and stereoselectively ring-opened products, such as δ-aminated γ-alkoxy-(alkylthio or acetoxy)-α,β-enoates. In addition, the regio- and stereoselective ring-opening reactions can be performed on solid supports and applied to stereoselective synthesis of (E)-alkene dipeptide isosteres.

The chemokine receptor, CXCR4, is a GPCR that transduces signals of its endogenous ligand, CXCL12 (stromal cell-derived factor-1, SDF-1). The CXCL12-CXCR4 system plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, etc. This system has recently been proven to be involved in several problematic diseases, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA) and pulmonary fibrosis. Thus, CXCR4 is thought to be an important therapeutic target to overcome the above diseases. Fourteen-mer peptides, T140 and its analogs, were previously found to be specific CXCR4 antagonists that were characterized as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-RA agents. Based on our knowledge of pharmacophores of T140, CXCR4 antagonists, such as FC131, were previously found by the efficient utilization of cyclic pentapeptide libraries. This review article focuses on our recent research on the development of low molecular weight CXCR4 antagonists including FC131 analogs, in which structural tuning of the cyclic peptide ring and chemical modifications were performed for an increase in potency and a reduction of the peptide character.

The chemokine receptor CXCR4 possesses multiple critical functions in normal and pathologic physiology. CXCR4 is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). The interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), and pulmonary fibrosis. It is conjectured that this interaction may be a critical therapeutic target in all of these diseases, and several CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogues, were previously developed in our laboratory as specific CXCR4 antagonists that were identified as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents, and anti-RA agents. Cyclic pentapeptides, such as FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)], were also previously found as CXCR4 antagonist leads based on pharmacophores of T140. This review article describes the elucidation of multiple functions of CXCR4 antagonists and the development of a number of low-molecular weight CXCR4 antagonists involving FC131 analogues and other compounds with different scaffolds including linear-type structures.

Protein phosphorylation is ubiquitously involved in living cells, and it is one of the key events controlling protein-protein surface interactions, which are essential in signal transduction cascades. We now report that the small molecular receptors bearing binuclear Zn(II)-Dpa can strongly bind to a bis-phosphorylated peptide in a cross-linking manner under neutral aqueous conditions when the distance between the two Zn(II) centers can appropriately fit in that of the two phosphate groups of the phosphorylated peptide. The binding property was quantitatively determined by ITC (isothermal titration calorimetry), induced CD (circular dichroism), and NMR. On the basis of these findings, we demonstrated that these types of small molecules were able to effectively disrupt the phosphoprotein-protein interaction in a phosphorylated CTD peptide and the Pin1 WW domain, a phosphoprotein binding domain, at a micromolar level. The strategy based on a small molecular disruptor that directly interacts with phosphoprotein is unique and should be promising in developing a designer inhibitor for phosphoprotein-protein interaction.

Several low molecular weight nonpeptide compounds having the dipicolylamine-zinc(II) complex structure were identified as potent and selective antagonists of the chemokine receptor CXCR4. These compounds showed strong inhibitory activity against CXCL12 binding to CXCR4, and the top compound exhibited significant anti-HIV activity. Zinc(II)-dipicolylamine unit-containing compounds proved to be useful and attractive lead compounds for chemotherapy of these diseases as nonpeptide CXCR4 antagonists possessing the novel scaffold structure.

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Suzuki-coupling, a representative cross-coupling reaction between small molecules, can proceed on a protein surface in aqueous solution under mild conditions. The modified protein produced by the coupling reaction maintained its native like structure and function. In addition, fluorescent dye appended-protein acts as a fluorescent biosensor.

Designed cyclodextrin-peptide conjugates, which have one or two beta-hairpin peptides, have been synthesized as catalysts for ester hydrolysis. One or two beta-hairpin peptides were located at the primary hydroxyl group side of beta-cyclodextrin so as to arrange two histidine residues that act as a general acid and a general base catalysts and provide the substrate recognition subsite. Kinetic studies revealed that the two-beta-hairpin peptide was more effective than that of the one-beta-hairpin peptide for substrate recognition.

Artificial functional peptides are valuable tools in variousfields of chemical biology. Small peptides, such as anoligohistidine tag (His tag), can be genetically incorporatedinto target proteins and used for purification of recombinantproteins, immobilization of proteins on microplates, andbioimaging of proteins on the surface of living cells with theircomplementary partner molecules, such as Ni

The human immunodeficiency virus type 1 (HIV-1) uses CD4 and the co-receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4-ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4-ED-derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti-HIV-1 activity. The 39-mer extracellular N-terminal region (NT) was divided into three fragments with 10-mer overlapping sites (N1-N3), and these linear peptides were synthesized. Peptide N1 contains Met 1-Asp 20 and shows significant anti-HIV-1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2, which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti-HIV-1 activity than their linear peptide counterparts, L1 and L2. The cytotoxicities of C1 and C2 are lower than those of L1 and L2. These results indicate that Met 1-Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti-HIV-1 drug candidates.

Antibiotic resistance is increasingly seen as a serious problem that threatens public health and erodes our capacity to effectively combat disease. So-called non-iron metalloporhyrins have shown promising antibacterial properties against a number of pathogenic bacteria including Staphylococcus aureus. However, little is known about the molecular mechanism(s) of action of these compounds and in particular how they reach the interior of the bacterial cells. A popular hypothesis indicates that non-iron metalloporphyrins infiltrate into bacterial cells like a "Trojan horse" using heme transport systems. Iron-regulated surface determinant (Isd) is the best characterized heme transport system of S. aureus. Herein we studied the molecular mechanism by which the extracellular heme-receptor IsdH-NEAT3 of Isd recognizes antimicrobial metalloporphyrins. We found that potent antibacterial porphyrins Ga(III)-protoporphyrin IX (PPIX) and Mn(III)-PPIX closely mimicked the properties of the natural ligand heme, namely (i) stable binding to IsdH-NEAT3 with comparable affinities for the receptor, (ii) nearly undistinghuishable three-dimensional structure when complexed with IsdH-NEAT3, and (iii) similar transfer properties to a second receptor IsdA. On the contrary, weaker antibacterial porphyrins Mg(II)-PPIX, Zn(II)-PPIX, and Cu(II)-PPIX were not captured effectively by IsdH-NEAT3 under our experimental conditions and displayed lower affinities. Moreover, reduction of Fe(III)-PPIX to Fe(II)-PPIX with dithionite abrogated stable binding to receptor. These data revealed a clear connection between oxidation state of metal and effective attachment to IsdH-NEAT3. Also, the strong correlation between binding affinity and reported antimicrobial potency suggested that the Isd system may be used by these antibacterial compounds to gain access to the interior of the cells. We hope these results will increase our understanding of Isd system of S. aureus and highlight its biomedical potential to deliver new and more efficient antibacterial treatments.

A designed cyclodextrin peptide hybrid, which has multiple functional groups on its alpha-helix peptide backbone, has been synthesized as a catalyst for ester hydrolysis. Kinetic study revealed that the carboxylate group plays a key role in this system.

A highly potent CXCR4 antagonist 2 [cyclo (-D-Tyr1-Arg2-Arg3-Nal4-Gly5-)] has previously been identified by screening cyclic pentapeptide libraries that were designed based on pharmacophore residues of a 14-residue peptidic CXCR4 antagonist 1. In the present study, D-Tyr and Arg in peptide 2 were replaced by a bicyclic aromatic amino acid and a cationic amino acid, respectively, and their binding activity for CXCR4 was evaluated for identification of the novel pharmacophore.

Development of CXCR4-specific ligands is an important issue in chemotherapy of HIV infection, cancer metastasis, and rheumatoid arthritis, and numerous potential ligands have been developed to date. However, it is difficult to assess their binding mode and specificity because of uncertainties in the structure of the CXCR4-ligand complexes. To address this problem, we have synthesized fluorophore labeled Ac-TZ14011, which is derived from T140, a powerful CXCR4 antagonist. Binding of Ac-TZ14011 to CXCR4 on the cell membrane was observed by fluorescence microscope, and analysis of the binding data produced IC 50 values of several ligands comparable to those obtained in RI-based assays. This fluorescence-based assay is applicable to explore new pharmacophores of CXCR4-specific ligands with high-throughput screening and also to screening of the other GPCR binding ligands.

Structure–activity relationship studies on CXCR4 antagonists, which were previously found by using cyclic pentapeptide libraries, were performed to optimize side-chain functional groups, involving conformationally constrained analogues. In addition, a new lead of cyclic pentapeptides with the introduction of a novel pharmacophore was developed.

A linear type of several low molecular weight CXCR4 antagonists were developed based on T140 analogs, which were previously found to be strong CXCR4 antagonists that block X4-HIV-1 entry and have inhibitory activities against cancer metastasis/progression and rheumatoid arthritis.

Chemokine receptors have attracted a good deal of public attention as important therapeutic targets for many diseases and disorders. In this issue of Chemistry & Biology, Kumar and colleagues propose a new concept of synthetic modular modifications to generate unnatural chemokines, which exhibit high receptor selectivity [1].

The effect of a hydrochloric acid solution on the circumoval precipitin (COP) reactivity of Schistosoma japonicum eggs was studied. When eggs, recovered by trypsin digestion from tissues of an 8 week-infected rabbit, were treated with a pepsin HCl solution, they had significantly higher reactivity than the saline-treated eggs (control). A similar effect was obtained with HCl solution (0.008 N), alone. In contrast, a sodium hydroxide solution (0.05 N) decreased the reactivity. Treatment with different concentrations of HCl (0.01 to 6N) for 2 days revealed that the reactivity was highest at 0.1 N, and was diminished by the higher concentrations (3 and 6N). Preservation in an HCl solution (0.01N) yielded a peak reactivity at 2 days. While the exact role of the HCl solution was not studied, this study contributes to the preparation of egg specimens and to our understanding of the low reactivity of eggs.

Useful methods of protein labeling via functional peptide tags have been developed in the field of proteome and chemical biology. New tag-probe pairs based on leucine zipper peptides for labeling target proteins are described. These consist of an α-helical probe peptide with an environmental-sensitive fluorescent dye and two antiparallel α-helical tag peptides, and may be crosslinked, from the Cys residue of the tag peptide to the N(α)-chloroacetyl group of the probe peptide. Binding of the probe peptide to the tag peptides results in movement of the fluorophore from a hydrophilic to a hydrophobic environment inside the leucine zipper structure, causing a dramatic fluorescent change, mediated by the binding of the two peptides. As a spacer between the N(α)-chloroacetyl group and the original probe sequence, a single Gly residue was the most suitable among 0-2 Gly residues. Crosslinking leads to superior fluorescence response, binding affinity, and chemical stability. These ZIP tag-probe pairs are useful for labeling and fluorescent imaging of proteins.