A novel interleukin‐13 receptor alpha 2‐targeted hybrid peptide for effective glioblastoma therapy

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic‐type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin‐13 receptor alpha 2 (IL‐13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL‐13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL‐13Rα2–lytic hybrid peptide. The IL‐13Rα2–lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL‐13Rα2–lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin‐13 and induced rapid ATP dynamics in GB cells. Anti‐tumor activity of the IL‐13Rα2–lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL‐13Rα2–lytic hybrid peptide might be a potent therapeutic option for patients with GB.     

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid‐binding peptide

Abstract: A two‐step targeting strategy was used to identify improved laccases for bleaching carotenoid‐containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides’ binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid‐binding properties of the peptide YGYLPSR and subsequently cloned and expressed C‐terminal variants of laccase from Stachybotrys chartarum containing carotenoid‐binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide–laccase fusions demonstrate enhanced catalytic properties on stained fabrics.     

Structure–function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein

Abstract: Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173–178‐residue stretch of the human 37LRP sequence, has been identified as the laminin‐1‐binding site. Peptides from 37LRP of species that contain this palindrome‐bind laminin‐1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15‐residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin‐1‐binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin‐1‐binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin‐1‐binding affinity of the peptides influences their inhibitory activity.     

Plasmodium falciparum EBA‐140 kDa protein peptides that bind to human red blood cells

Abstract: The erythrocyte‐binding antigen 140 (EBA140) sequence was chemically synthesized in 61 20‐mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte‐binding assays. Peptides 26135, 26144, 26147, 26160, 26170 and 26177 presented high erythrocyte‐binding activity, with affinity constants ranging from 350 to 750 nm . Critical erythrocyte‐binding residues were determined by competition‐binding assays with glycine analogous peptides. Cross‐linking assays with SDS‐PAGE from high erythrocyte membrane protein binding peptides showed that all these peptides bound specifically to 25, 52 and 75 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide‐binding assays using enzyme‐treated erythrocytes, showing that these protein receptors are susceptible to structural changes provoked by enzyme treatment (neuraminidase, trypsin or chymotrypsin). Inhibition invasion assays in ‘in vitro’ cultures showed that all specific high binding sequences were able to inhibit invasion by 11–69% at 200 μm concentration.     

A novel peptide isolated from phage display peptides library recognized by an antibody against connective tissue growth factor (CTGF)

The aim of this study was to isolate a peptide binding to an antibody against CTGF C-terminal domain from the peptide library and to evaluate its immunological and biological activities. A phage display 12-mer peptide library was screened using anti-CTGF/C antibody as the target. Ten of the positive clones were sequenced after three rounds bio-panning. The DNA encoding peptide ZD521 was cloned and expressed as the fusion protein(TrxA-ZD521). The specificity of ZD521 to anti-CTGF/C antibody was determined by competitive inhibition assay. Mice were immunized with purified fusion protein(TrxA-ZD521) and the anti-peptide or anti-CTGF response of antiserum was also tested by enzyme-linked immunoabsorbent assay (ELISA) and Western blot. The inhibition effect of anti-serum on proliferation of kidney mesangial cells was evaluated by MTT. A peptide ZD521(GEPQTKLFSFPL) that could specifically recognize anti-CTGF/C antibody was isolated. No sequence homology was found between ZD521 and CTGF/C. The purified TrxA-ZD521 could specifically bind to anti-CTGF/C antibody and block the binding of anti-CTGF/C antibody to CTGF/C and native CTGF(mesangial cell lysate). Moreover, the antiserum from mice immunized with TrxA-ZD521 could also bind to CTGF/C recombinant protein and native CTGF, as well as significantly inhibit the proliferation of kidney mesangial cells induced by CTGF/C. Therefore, ZD521 might be a conformational epitope of CTGF which is potentially useful to be developed as a vaccine for prevention and treatment of fibrosis disorders.     

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.     

Poly‐l‐proline type II peptide mimics as probes of the active site occupancy requirements of cGMP‐dependent protein kinase

Abstract: Based on the X‐ray crystal structure of cAMP‐dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X‐ray crystal structure of cyclin‐dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly‐l ‐proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP‐dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P ? 1, P ? 2, and P ? 3 residues of substrate peptides bind in the PPII conformation (φ approximately ?75°, ψ approximately 145°). In addition, the inhibition data also suggest that the P ? 1, P ? 2, and P ? 3 residues in substrate peptides bind with a gauche(?) χ1 angle.     

Helical Peptides Derived from Lactoferrin Bind Hepatitis C Virus Envelope Protein E2

Hepatitis C virus is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. Hepatitis C virus envelope 2 glycoprotein (E2) binds several cell‐surface molecules that act as receptor candidates mediating hepatitis C virus entry into hepatocytes. Peptides derived from human lactoferrin have been shown to bind hepatitis C virus‐E2 protein thereby preventing hepatitis C virus entry in cultured hepatocytes. In this study, starting from a 33‐residue human lactoferrin‐derived peptide, a number of biotin‐linked α‐peptides were synthesized and investigated for their E2 protein binding activity. E2 protein from hepatitis C virus genotype 1b was expressed in 293 human embrionic kidney cells and purified using affinity chromatography. A biotin‐streptavidin based binding assay was developed to determine the binding affinity of the synthetic peptides for E2 protein. Two of the peptides bound E2 specifically with submicromolar to low micromolar affinity [equilibrium dissociation constant (K_d) of 0.569 and 28.8 μm] . Further, these two peptides had the highest helical content in solution as observed by circular dichroism spectroscopy, suggesting that binding affinity increases with increase in helicity. These results have provided new lead peptides for future investigations of hepatitis C virus entry inhibitors that may provide an interesting approach to prevent hepatitis C virus infectivity.     

Characterization of Peptide 20–30 of Follicle Stimulating Hormone Receptor as an Antagonist of Receptor Activity: Significance of Charged Residues

We had previously reported the region (20–30) from follicle stimulating hormone receptor as being an immunodominant epitope and the smallest reported peptide capable of inhibiting hormone binding. We now report it to be an effective antagonist of ligand‐induced cAMP signalling as well. The region (20–30) of follicle stimulating hormone receptor has three charged residues, namely, E²², D²⁶ and R²⁹ that are specific to follicle stimulating hormone receptor and are conserved in mammals. This study aimed to verify whether the charged residues contribute to the activity of the follicle stimulating hormone receptor peptide (20–30). This was done using analogs of follicle stimulating hormone receptor peptide (20–30), each having an alanine substitution for a corresponding charged residue. The analog peptides displayed a loss of activity and could not inhibit hormone binding or the subsequent signal transduction. The ability of follicle stimulating hormone receptor peptide (20–30) to bind antipeptide antibodies against follicle stimulating hormone receptor peptide (9–30) was either decreased or abolished with the alanine substituted analog peptides of follicle stimulating hormone receptor peptide (20–30). The loss of function led us to verify whether there was a conformational change as well. CD spectral analysis did not reveal a significant change. These observations indicate that the charged aminoacids present in follicle stimulating hormone receptor peptide (20–30) are crucial for the observed follicle stimulating hormone antagonistic activity. This information could form the basis for the design of novel compounds capable of functioning as follicle stimulating hormone antagonists.     

Synthesis,conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*

Abstract: Biotin‐avidin immobilization has been routinely used as a tool to study peptide–receptor and peptide–antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides’ receptor, and to analyse ligand‐receptor binding. Insulin‐like peptide 3 (INSL3) is a peptide hormone which contains A‐ and B‐chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein‐coupled receptor, we chemically synthesized N^α‐mono‐biotinylated human INSL3 (B‐hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with ³³P‐labelled relaxin H2 (B₃₃). The modified B‐hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B‐hINSL3 contains a higher α‐helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N‐terminal region of the A‐chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N‐terminus of the A‐chain promoted conformational stability which, in turn, permitted better receptor activation.     

Identification of oligopeptide binding to colon cancer cells separated from patients using laser capture microdissection

The development of intravascular conjugates that efficiently deliver genes or drugs to tumors is limited by the lack of efficacious targeting ligands. Small targeting peptides, such as those iterated by phage display technology, offer enormous potential for these applications. The majority of reports published to date have focused on the identification of peptides isolated for their ability to bind to human cancer cell lines in vitro, and have failed to account for the loss of polarization and de-differentiation of such cells from their in vivo state. Here, we report a novel approach for the identification of peptides capable of binding specifically to cancer cells derived from clinically resected human colon cancer. In this strategy, laser capture microdissection (LCM) is performed on a surgically resected colon cancer specimen to separate only cancer cells from the specimen. Subsequently, biopanning was performed on the LCM-selected colon cancer cells to identify peptide sequences that bound specifically to them. A peptide containing the SPT motif was selected as the most promising consensus sequence binding specifically to the LCM-selected colon cancer cells. Phage clones displaying the SPT motif demonstrated 9-fold higher binding to colon cancer cells derived from a patient than insertless phage (p < 0.05), while, recovery of the SPT phage from the colon cancer cell lines DLD-1 and HCT-15 was 7-fold higher than that of the control insertless phage (p < 0.05). The binding of SPT phage to colon cancer cells from the patient was confirmed by immunofluorescence. Additionally, a synthesized SPT-containing peptide (SPTKSNS) showed binding activity in the absence of mitogenic effects on colon cancer cells in vitro. In summary, we have introduced LCM into a biopanning procedure and identified a small peptide that binds preferentially to colon cancer cells derived from a clinically resected sample. This procedure could be applicable for the design of customized cancer cell targeting methodologies using clinical biopsy samples from human subjects.     

Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

X-ray crystal structure of bone marrow kinase in the x chromosome: a Tec family kinase

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton’s tyrosine kinase and IL‐2‐inducible T‐cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X‐ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 Å resolution and PP2 at 1.9 Å resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well‐ordered protein conformation that includes an open/extended activation loop and a stabilized DFG‐motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG‐motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.     

Functionalization of carbon nanomaterials by evolutionary molecular engineering: potential application in drug delivery systems

By virtue of the progress made in evolutionary molecular engineering, peptide aptamers that specifically recognize target molecules are now routinely created using a peptide phage display system. The system was originally developed for isolating peptides that specifically recognized biomacromolecules (e.g. proteinous receptors), but are now also being used to acquire peptide motifs that bind to inorganic materials, such as semiconductors, metals and carbon nanomaterials. We have created the peptide aptamer against carbon nanohorns, a vesicular carbon nanomaterial whose size is 80-100 nm in diameter. By combining the peptide motif that has affinity to the surfaces of carbon nanohorns with peptide aptamers that can target specific organs, we can functionalize the carbon nanomaterial to provide novel types of carriers for drug delivery systems.     

Functionalization of carbon nanomaterials by evolutionary molecular engineering: Potential application in drug delivery systems

By virtue of the progress made in evolutionary molecular engineering, peptide aptamers that specifically recognize target molecules are now routinely created using a peptide phage display system. The system was originally developed for isolating peptides that specifically recognized biomacromolecules (e.g. proteinous receptors), but are now also being used to acquire peptide motifs that bind to inorganic materials, such as semiconductors, metals and carbon nanomaterials. We have created the peptide aptamer against carbon nanohorns, a vesicular carbon nanomaterial whose size is 80–100 nm in diameter. By combining the peptide motif that has affinity to the surfaces of carbon nanohorns with peptide aptamers that can target specific organs, we can functionalize the carbon nanomaterial to provide novel types of carriers for drug delivery systems.     

The Design,Synthesis and Potential Utility of Fluorescence Probes that Target DFG‐out Conformation of p38α for High Throughput Screening Binding Assay

The design, synthesis and utility of fluorescence probes that bind to the DFG‐out conformation of p38α kinase are described. Probes that demonstrate good affinity for p38α, have been identified and one of the probes, PF‐04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non‐classical p38α inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non‐classical kinase inhibitors to the unactive form of the enzyme.     

Evaluation of an LC8-binding peptide for the attachment of artificial cargo to dynein

The limited cytoplasmic mobility of nonviral gene carriers is likely to contribute to their low transfection efficiency. This limitation could be overcome by mimicking the viral strategy of recruiting the dynein motor complex for efficient transport toward the host cell nucleus. A promising approach for attaching artificial cargo to dynein is through an adaptor peptide that binds the 8 kDa light chain (LC8) found in the cargo-binding region of the dynein complex. Several viral proteins that bind LC8 have in common an LC8-binding motif defined by (K/R)XTQT. Short peptides containing this motif have also been shown to bind recombinant LC8 in vitro. However, since the majority of intracellular LC8 exists outside of the dynein complex, it remains unclear whether peptides displaying this LC8-binding motif can access and bind to dynein-associated LC8. In this study, we employed biochemical analysis to investigate the feasibility of attaching artificial cargo to the dynein motor complex using a peptide displaying the well-characterized LC8-binding motif. We report that free intracellular LC8 bound specifically to an LC8-binding (TQT) peptide and not to a control peptide with a mutated LC8-binding motif. However, a similar binding interaction between the TQT peptide and intracellular dynein was not detected. To determine whether dynein binding of the TQT peptide was prevented by competition with free intracellular LC8 or due to the inability of the peptide to access its LC8 binding site in the dynein complex, the TQT peptide was evaluated for its ability to bind either purified LC8 or purified dynein. Our results demonstrate that, while the TQT peptide readily binds free LC8, it cannot bind to dynein-associated LC8. The results emphasize the need to identify functional dynein-binding peptides and highlight the importance of designing peptides that bind to the intact dynein motor complex.     

In silico screening and surface plasma resonance‐based verification of programmed death 1‐targeted peptides

Programmed death 1 (PD‐1) is a key immune checkpoint molecule. When it binds to programmed death‐ligand 1 (PD‐L1), it can negatively regulate the immune response. Therefore, blockade of the PD‐1/PD‐L1 interaction could unleash the power of immune system. Though successes achieved by anti‐PD‐1/PD‐L1 antibody drugs in clinical for various cancers, many intrinsic limitations of the high molecular weight drugs require alternatives such as peptide drugs and chemical compounds. In this study, we described a novel in silico approach which was used to screen peptides from PDB database and aimed to identify peptides that have potential to bind the PD‐L1 binding area of PD‐1 molecule. Based on the docking poses, eight peptides were synthesized and measured for their binding abilities by surface plasma resonance technique. The K_D values of the synthesized peptides ranged from 10.0 to 133.0 μM. Furthermore, the binding mechanism between PD‐1 and the peptides was studied. In conclusion, we established a fast and reliable screening method for peptide discovery, which could be applied for identifying peptide inhibitors of various targets. The synthesized peptides could be served as starting points for designing PD‐1 drug for cancer immunotherapy.     

噬菌体肽库展示的应用及其最新进展

噬菌体肽库展示技术是近年来发展起来的用丝状噬菌体展示外源肽的一项新技术 ,被广泛应用于研究蛋白质与蛋白质之间的相互作用 ,新药的开发等各个领域。该文就该技术的原理、发展历史以及目前的常见应用作一简要概述     

Identification of small peptides that interact specifically with a small organic dye

Using a “one-bead one-peptide” combinatorial peptide library method, we have been able to identify peptide ligands that interact specifically with various macromolecular targets such as monoclonal antibodies, streptavidin, avidin, MHC-Class I molecules, proteases, growth factor receptors, and gpIIb/IIIa integrin. In this paper, we test the hypothesis that small peptides that interact specifically with a small organic molecule can also be identified using this combinatorial peptide method. Using a small organic dye molecule, indigo carmine, as a color probe to screen a random L-heptapeptide and two D-hexa and octapeptide libraries, we were able to identify a specific peptide binding motif. Potential applications of this technology are described. © 1994 Wiley-Less, Inc.