Epitope‐Resolved Detection of Peanut‐Specific IgE Antibodies by Surface Plasmon Resonance Imaging

Peanut allergy can be life‐threatening and is mediated by allergen‐specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope‐resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ?‐chain‐specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross‐reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 μL each) in a 45 min assay. Excellent correlation of Ara h2‐specific IgE values was found between ImmunoCAP assays and the new SPRi method.     

Molecular Allergen-Specific IgE Recognition Profiles and Cumulative Specific IgE Levels Associated with Phenotypes of Cat Allergy

Cat allergy is a major trigger factor for respiratory reactions (asthma and rhinitis) in patients with immunoglobulin E (IgE) sensitization. In this study, we used a comprehensive panel of purified cat allergen molecules (rFel d 1, nFel d 2, rFel d 3, rFel d 4, rFel d 7, and rFel d 8) that were obtained by recombinant expression in Escherichia coli or by purification as natural proteins to study possible associations with different phenotypes of cat allergy (i.e., rhinitis, conjunctivitis, asthma, and dermatitis) by analyzing molecular IgE recognition profiles in a representative cohort of clinically well-characterized adult cat allergic subjects (n = 84). IgE levels specific to each of the allergen molecules and to natural cat allergen extract were quantified by ImmunoCAP measurements. Cumulative IgE levels specific to the cat allergen molecules correlated significantly with IgE levels specific to the cat allergen extract, indicating that the panel of allergen molecules resembled IgE epitopes of the natural allergen source. rFel d 1 represented the major cat allergen, which was recognized by 97.2% of cat allergic patients; however, rFel d 3, rFel d 4, and rFel d 7 each showed IgE reactivity in more than 50% of cat allergic patients, indicating the importance of additional allergens in cat allergy. Patients with cat-related skin symptoms showed a trend toward higher IgE levels and/or frequencies of sensitization to each of the tested allergen molecules compared with patients suffering only from rhinitis or asthma, while there were no such differences between patients with rhinitis and asthma. The IgE levels specific to allergen molecules, the IgE levels specific to cat allergen extract, and the IgE levels specific to rFel d 1 were significantly higher in patients with four different symptoms compared with patients with 1–2 symptoms. This difference was more pronounced for the sum of IgE levels specific to the allergen molecules and to cat extract than for IgE levels specific for rFel d 1 alone. Our study indicates that, in addition to rFel d 1, rFel d 3, rFel d 4, and rFel d 7 must be considered as important cat allergens. Furthermore, the cumulative sum of IgE levels specific to cat allergen molecules seems to be a biomarker for identifying patients with complex phenotypes of cat allergy. These findings are important for the diagnosis of IgE sensitization to cats and for the design of allergen-specific immunotherapies for the treatment and prevention of cat allergy.     

Multimerization of Peptide Mimotopes for Blocking of Factor VIII Neutralizing Antibodies

About 30 % of patients with severe hemophilia A develop neutralizing antibodies (inhibitors) to coagulation factor VIII (FVIII) upon treatment with exogenous factor preparations. Two peptides, C6 (NPVENMMDRDSQ) and H10 (QSPWQTWFTRAL), that mimic putative inhibitor epitopes (mimotopes), were previously selected by phage display screening of plasma samples from patients with inhibitors. Synthetic peptide mimotopes inhibited IgG binding to FVIII (IC₅₀: 30–50 μM ). This effect was increased by an equimolar combination of both mimotopes. Mimotopes were fused to the C‐terminal multimerization domain of the C4bp α‐chain and expressed as multimers in 293T cells. Multimerized mimotopes showed improved binding to anti‐FVIII IgG and prolonged in vitro half‐life relative to synthetic peptides. The two mimotopes were combined in heteromultimers by co‐transfection of 293T cells with respective vectors, resulting in bi‐specific molecules that almost completely blocked polyclonal antibody binding to FVIII (IC₅₀: 2–3 μM ). This strategy is capable of functionally improving synthetic peptides by multimerization and could provide a basis for novel therapeutic approaches for patients with hemophilia A and inhibitors.     

Fine mapping and structural analysis of immunodominant IgE allergenic epitopes in chicken egg ovalbumin

Ovalbumin is a major allergen in hen egg white that causes IgE-mediatedfood allergic reactions in children. In this study, the immunodominantIgE-binding epitopes of ovalbumin were mapped using arrays ofoverlapping peptides synthesized on activated cellulose membranes.Pooled human sera from 18 patients with egg allergy were usedto probe the membrane. Five distinct regions were found to containdominant allergic IgE epitopes, these being L38T49, D95A102,E191V200, V243E248 and G251N260. The critical amino acids involvedin IgE antibody binding were also determined. These epitopeswere composed primarily of hydrophobic amino acids, followedby polar and charged residues and being comprised of ß-sheetand ß-turn structures. One epitope, D95A102, consistedof a single -helix. These results provide useful informationon the functional role of amino acid residues to evaluate thestructure–function relationships and structural propertiesof allergic epitopes in ovalbumin. They also provide a strategicapproach for engineering ovalbumin to reduce its allergenicity. Received January 9, 2003; revised April 23, 2003; accepted August 28, 2003.     

刀额新对虾主要变应原36kD和68kD蛋白检测虾过敏症的应用

目的评价刀额新对虾主要变应原36kD和68kD蛋白在临床检测虾过敏症特异性IgE方面的应用。方法以纯化的刀额新对虾主要变应原36kD和68kD蛋白为抗原包被酶标反应板,采用间接ELISA法检测1301份人血清样品中的特异性IgE,对其中血清特异性IgE阳性者进行虾类食物过敏反应的回顾性调查。结果1301份血清中,特异性IgE阳性的有127份,阳性率为9.76%(127/1301)。回顾性调查表明,特异性IgE阳性个体均有虾类食物过敏史,与血清学检测结果相一致。结论刀额新对虾主要变应原36kD和68kD蛋白针对血清特异性IgE的检测具有较高的特异性和敏感性,适用于研制临床检测虾过敏症的ELISA试剂。     

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

The metal ion binding properties of two fluorescent analogues of trichogin GA IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time‐resolved optical spectroscopy, and molecular mechanics calculations. Binding of Ca^II and Gd^III to the peptides investigated was shown to promote a structural transition from highly helical conformations to folded structures characterized by formation of a loop that embedded the metal ion. Time‐resolved spectroscopy revealed that peptide dynamics is also remarkably affected by ion binding: peptide‐backbone motions slowed down to the microsecond time scale. Finally, molecular mechanics calculations emphasized the role of the central Gly5‐Gly6 motif, which allowed for the twisting of the peptide segment that gave rise to the formation of the binding cavity.     

Tailor‐Made Designer Helical Peptides that Induce Mitochondrion‐Mediated Cell Death without Necrosis

Managing protein–protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side‐chain‐crosslinked helical peptides based on the binding domain of a pro‐apoptotic protein (Bad) that induces programed cell death. The peptide showed high helical content and bound to its target, Bcl‐X_L, more strongly than its non‐crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl‐X_L localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70 % of HeLa cells died by the apoptosis pathway (without evidence of necrosis).     

An Exploratory Pilot Study of Genetic Marker for IgE-Mediated Allergic Diseases with Expressions of FcεR1α and Cε

The high affinity immunoglobulin E (IgE) receptor-FcεR1 is mainly expressed on the surface of effector cells. Cross-linking of IgE Abs bound to FcεR1 by multi-valent antigens can induce the activation of these cells and the secretion of inflammatory mediators. Since FcεR1 plays a central role in the induction and maintenance of allergic responses, this study aimed to investigate the association of FcεR1 with the allergic phenotype of Cε expression and cytokine and histamine release from peripheral leukocytes. Peripheral leukocytes from 67 allergic and 50 non-allergic subjects were used for genotyping analysis. Peripheral mononuclear cells (PBMCs) were used for Cε expression and ELISpot analysis, while polymorphonuclear cells (PMNs) were used for histamine release. The association between genotype polymorphism of the FcεR1α promoter region (rs2427827 and rs2251746) and allergic features of Cε expression and histamine were analyzed, and their effects on leukocytes function were compared with wild type. The genotype polymorphisms of FcεR1α promoter region with CT and TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with single nucleotide polymorphism (SNP) of FcεR1α promoter region had high levels of total IgE, mite-specific Der p 2 (Group 2 allergen of Dermatophagoides pteronyssinus)-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with SNPs of the FcεR1α promoter region. Despite the increased Cε mRNA expression in PBMCs and histamine release from PMNs and the up-regulated mRNA expression of interleukin (IL)-6 and IL-8 secretions after Der p2 stimulation, there was no statistically significant difference between SNPs of the FcεR1α promoter region and the wild type. SNPs of FcεR1α promoter region were associated with IgE expression, IgE producing B cells, and increased Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by Dermatophagoides pteronyssinus.     

Fluorine‐Containing 6,7‐Dialkoxybiaryl‐Based Inhibitors for Phosphodiesterase 10 A: Synthesis and in vitro Evaluation of Inhibitory Potency,Selectivity, and Metabolism

Based on the potent phosphodiesterase 10 A (PDE10A) inhibitor PQ‐10, we synthesized 32 derivatives to determine relationships between their molecular structure and binding properties. Their roles as potential positron emission tomography (PET) ligands were evaluated, as well as their inhibitory potency toward PDE10A and other PDEs, and their metabolic stability was determined in vitro. According to our findings, halo‐alkyl substituents at position 2 of the quinazoline moiety and/or halo‐alkyloxy substituents at positions 6 or 7 affect not only the compounds′ affinity, but also their selectivity toward PDE10A. As a result of substituting the methoxy group for a monofluoroethoxy or difluoroethoxy group at position 6 of the quinazoline ring, the selectivity for PDE10A over PDE3A increased. The same result was obtained by 6,7‐difluoride substitution on the quinoxaline moiety. Finally, fluorinated compounds (R)‐7‐(fluoromethoxy)‐6‐methoxy‐4‐(3‐(quinoxaline‐2‐yloxy)pyrrolidine‐1‐yl)quinazoline ( 16 a ), 19 a – d , (R)‐tert‐butyl‐3‐(6‐fluoroquinoxalin‐2‐yloxy)pyrrolidine‐1‐carboxylate ( 29 ), and 35 (IC₅₀ PDE10A 11–65 nM ) showed the highest inhibitory potential. Further, fluoroethoxy substitution at position 7 of the quinazoline ring improved metabolic stability over that of the lead structure PQ‐10.     

Preparation and Characterization of Energetic Nanocomposites of Organic Gel – Inorganic Oxidizers

Two types of polymeric sols, resorcinol‐formaldehyde (RF) and resorcinol‐furfural (RFur), were mixed in a water‐containing medium with aqueous solutions of inorganic salts: NH₄ClO₄, Mg(ClO₄)₂, and NH₄NO₃. After gelation and an ambient pressure drying, hybrid nanocomposites with properties of energetic materials were obtained. It was stated that salt concentration and addition of a second solvent (e.g., methanol or N,N‐dimethylformamide) to the mixture of reagents have crucial meaning for gel formation. In the case when only water was used, the mixture of organic sol/inorganic salt did not transfer from sol to gel, and precipitates were formed. Conventional drying of wet gelled composites leads to rigid material called xerogels. The RF xerogels are red and RFur xerogels are black. Typically, xerogels are transparent at low salt concentration (below 30%). The microstructure, morphology, and some other properties of chosen composites were studied by means of HR SEM, AFM, XRD, DTA/TG, and N₂ adsorption isotherm techniques. SEM observation revealed that sizes of the oxidizer particles vary from less than 100 nm to ca. 1000 nm. XRD analyses also confirmed the presence of nanometer‐sized crystals of oxidizers in some formulations. The specific surface area of polymeric matrix/oxidizer composites was found to be in the range from 0.002 to 0.3 m² g⁻¹. After removing the salt from the composites (by extraction with boiling water), the specific surface area grows even up to 210 m² g⁻¹. TG/DTA analyses showed that the tested composites decompose as typical energetic materials. If pre‐heated and exposed to flame, some of them (especially RF/Mg(ClO₄)₂ composites) undergo violent deflagration with loud sound and flash effect.     

Anti‐HIV‐1 Peptide Derivatives Based on the HIV‐1 Co‐receptor CXCR4

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.     

A New Methodology for Incorporating Chiral Linkers into Stapled Peptides

Stapled peptides have arisen as a new class of chemical probe and potential therapeutic agents for modulating protein–protein interactions. Here, we report the first two‐component i,i+7 stapling methodology that makes use of two orthogonal, on‐resin stapling reactions to incorporate linkers bearing a chiral centre into a p53‐derived stapled peptide. Post‐stapling modifications to the chain were performed on‐resin and enabled rapid access to various peptide derivatives from a single staple. The stapled peptides have increased helicity, protease stability and in vitro binding affinities to MDM2 compared to the equivalent unstapled peptide. This approach can be used to generate a library of diverse stapled peptides with different properties starting from a single stapled peptide, with scope for much greater functional diversity than that provided by existing stapling methodologies.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

Peptide Architecture: Adding an α‐Helix to the PYY Lysine Side Chain Provides Nanomolar Binding and Body‐Weight‐Lowering Effects

The gut hormone PYY3‐36 influences food intake and body weight via interaction with hypothalamic presynaptic Y2 receptors (Y2R). Novel Y2R‐selective analogues of PYY3‐36 are therefore potential drug candidates for the treatment of obesity. It has been hypothesized that PYY3‐36 and possibly also the related PP‐fold peptides, NPY and PP, bind to the membrane via their amphipathic α‐helix prior to receptor interaction. The PYY3‐36 amphipathic α‐helix causes the peptide to associate with the membrane, making it essential for Y receptor potency as it potentially guides the C‐terminal pentapeptide into the correct conformation for receptor activation. Based on this hypothesis, the importance of the amphipathic nature of PYY3‐36, as well as the ability of amphipathic α‐helices to interact in solution to form di‐ and tetramers, we redesigned the peptide architecture by addition of an amphipathic α‐helix via the Lys 4 side chain of PYY3‐36. Two different amphipathic sequences were introduced; first, PYY17‐31, the native α‐helix of PYY, and secondly, its retro counterpart, PYY31‐17, which is also predicted to form an α‐helix. Moreover, several different turn motifs between the branching point and the additional α‐helix were tested. Several novel peptides with nanomolar Y2R binding affinities, as well as increased Y receptor selectivity, were identified. CD experiments showed the modifications to be well accepted, and an increase in mean ellipticity (ME) signifying an increased degree of α‐helicity was observed. Receptor binding experiments indicated that the direction of the additional α‐helix is less important, in contrast to the turn motifs, which greatly affect the Y1R binding and thus determine the Y1R activity. Conversely, the structure–activity relationships from in vivo data showed that the peptide containing the retro‐sequence was inactive, even though the binding data demonstrated high affinity and selectivity. This demonstrates that radical redesign of peptide architecture can provide nanomolar binding with improved subtype selectivity and with in vivo efficacy.     

Grafting of sulfonated monomer onto an amino‐silane functionalized 2‐aminoterephthalate metal − organic framework via surface‐initiated redox polymerization: proton‐conducting solid electrolytes

A post‐polymerization method for metal–organic frameworks (MOFs) has been developed to produce super‐acidic solid nanoparticles. Thus, the NH₂MIL‐53(Al) MOF was functionalized with (3‐aminopropyl)triethoxysilane (APTES) from amine groups to yield active site anchored MOF nanoparticles. Then, sulfonated polymer/MOF hybrid nanoparticles were prepared by redox polymerization of 2‐acrylamido‐2‐methyl‐1‐propane sulfonic acid (MOF‐g‐PAMPS), initiated onto the surfaces of aminopropyl‐functionalized NH₂MIL‐53(Al) nanoparticles. The synthesis and modification of NH₂MIL‐53(Al) nanoparticles were characterized by Fourier transform infrared (FTIR) spectroscopy and TGA. FTIR and TGA results indicated that APTES modifier agent and AMPS monomer were successfully grafted onto the MOF nanoparticles. The grafting efficiency of PAMPS polymer onto the MOF nanoparticles was estimated from TGA thermograms to be 33%. Also, sulfonated polymer/MOF hybrid nanoparticles showed a proton conductivity as high as 4.9 × 10^?5 S cm^?1. Nitrogen adsorption of modified NH₂MIL‐53(Al) showed also a decrease in pore volume. The morphology and crystalline structure of MOF nanoparticles before and after the modification processes were studied by SEM and XRD, respectively. © 2015 Society of Chemical Industry     

Synthesis and Evaluation of a Series of Long‐Acting Glucagon‐Like Peptide‐1 (GLP‐1) Pentasaccharide Conjugates for the Treatment of Type 2 Diabetes

The present study details the development of a family of novel D ‐Ala⁸ glucagon‐like peptide‐1 (GLP‐1) peptide conjugates by site specific conjugation to an antithrombin III (ATIII) binding carrier pentasaccharide through tetraethylene glycol linkers. All conjugates were found to possess potent insulin‐releasing activity. Peptides with short linkers (<25 atoms) conjugated at Lys³⁴ and Lys³⁷ displayed strong GLP‐1 receptor (GLP‐1‐R) binding affinity. All D ‐Ala⁸GLP‐1 conjugates exhibited prominent glucose‐lowering action. Biological activity of the Lys³⁷ short‐linker peptide was evident up to 72 h post‐injection. In agreement, the pharmacokinetic profile of this conjugate (t_1/2, 11 h) was superior to that of the GLP‐1‐R agonist, exenatide. Once‐daily injection of the Lys³⁷ short‐linker peptide in ob/ob mice for 21 days significantly decreased food intake and improved HbA_1c and glucose tolerance. Islet size was decreased, with no discernible change in islet number. The beneficial effects of the Lys³⁷ short‐linker peptide were similar to or better than either exenatide or liraglutide, another GLP‐1‐R agonist. In conclusion, GLP‐1 peptides conjugated to an ATIII binding carrier pentasaccharide have a substantially prolonged bioactive profile compatible for possible once‐weekly treatment of type 2 diabetes in humans.     

Fabrication of BaZr0.1Ce0.7Y0.2O3 – δ ‐Based Proton‐Conducting Solid Oxide Fuel Cells Co‐Fired at 1,150 °C

Dense proton‐conducting BaZr_0.1Ce_0.7Y_0.2O_{3 – δ} (BZCY) electrolyte membranes were successfully fabricated on NiO–BZCY anode substrates at a low temperature of 1,150 °C via a combined co‐press and co‐firing process. To fabricate full cells, the LaSr₃Co_1.5Fe_1.5O_{10 – δ}–BZCY composite cathode layer was fixed to the electrolyte membrane by two means of one‐step co‐firing and two‐step co‐firing, respectively. The SEM results revealed that the cathode layer bonded more closely to the electrolyte membrane via the one‐step co‐firing process. Correspondingly, determined from the electrochemical impedance spectroscopy measured under open current conditions, the electrode polarisation and Ohmic resistances of the one‐step co‐fired cell were dramatically lower than the other one for its excellent interface adhesion. With humidified hydrogen (2% H₂O) as the fuel and static air as the oxidant, the maximum power density of the one‐step co‐fired single cell achieved 328 mW cm^–2 at 700 °C, showing a much better performance than that of the two‐step co‐fired single cell, which was 264 mW cm^–2 at 700 °C.     

Identification of Receptor‐Interacting Regions of Vitellogenin within Evolutionarily Conserved β‐Sheet Structures by Using a Peptide Array

Vitellogenesis, a key process in oviparous animals, is characterized by enhanced synthesis of the lipoprotein vitellogenin, which serves as the major yolk‐protein precursor. In most oviparous animals, and specifically in crustaceans, vitellogenin is mainly synthesized in the hepatopancreas, secreted to the hemolymph, and taken up into the ovary by receptor‐mediated endocytosis. In the present study, localization of the vitellogenin receptor and its interaction with vitellogenin were investigated in the freshwater prawn Macrobrachium rosenbergii. The receptor was immuno‐histochemically localized to the cell periphery and around yolk vesicles. A receptor blot assay revealed that the vitellogenin receptor interacts with most known vitellogenin subunits, the most prominent being the 79 kDa subunit. The receptor was, moreover, able to interact with trypsin‐digested vitellogenin peptides. By combining a novel peptide‐array approach with tandem mass spectrometry, eleven vitellogenin‐derived peptides that interacted with the receptor were identified. A 3D model of vitellogenin indicated that four of the identified peptides are N‐terminally localized. One of the peptides is homologous to the receptor‐recognized site of vertebrate vitellogenin, and assumes a conserved β‐sheet structure. These findings suggest that this specific β‐sheet region in the vitellogenin N‐terminal lipoprotein domain is the receptor‐interacting site, with the rest of the protein serving to enhance affinity for the receptor. The conservation of the receptor recognition site in invertebrate and vertebrate vitellogenin might have vast implications for oviparous species reproduction, development, immunity, and pest management.     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.