Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Cell Line MeSH
• Cytokines metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Interleukin-17 metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Inflammation Mediators metabolism   MeSH
• Models, Molecular MeSH
• Receptors, Interleukin-17 antagonists & inhibitors   chemistry   metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Protein Stability MeSH
• Carrier Proteins metabolism   MeSH
• Protein Binding MeSH
• Inflammation immunology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.

• MeSH
• Lymphocyte Activation immunology   MeSH
• Cell Line MeSH
• Th17 Cells immunology   metabolism   MeSH
• Dendritic Cells immunology   metabolism   MeSH
• Phagocytes immunology   metabolism   MeSH
• Interleukin-23 Subunit p19 chemistry   metabolism   pharmacology   MeSH
• Interleukin-23 chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Macrophages immunology   metabolism   MeSH
• Models, Molecular MeSH
• Receptors, Interleukin metabolism   MeSH
• Recombinant Proteins MeSH
• Signal Transduction MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Poly(lactic acid) (PLA) has shown much success in the preparation of tissue engineering scaffolds as it can be fabricated with a tailored architecture. However, the PLA surface has drawbacks including the lack of biofunctional motifs which are essential for high affinity to biological cells. Therefore, this study describes a multistep physicochemical approach for the immobilization of d-glucosamine (GlcN), a naturally occurring monosaccharide having many biological functions, on the PLA surface aiming at enhancing the cell proliferation activity. In this approach, poly(acrylic acid) (PAAc) spacer arms are first introduced into the PLA surface via plasma post-irradiation grafting technique. Then, covalent coupling or physical adsorption of GlcN with/on the PAAc spacer is carried out. Factors affecting the grafting yield are controlled to produce a suitable spacer for bioimmobilization. X-ray photon spectroscopic (XPS) analyses confirm the immobilization of GlcN on the PLA surface. The XPS results reveal also that increasing the yield of grafted PAAc spacer on the PLA surface increases the amount of covalently immobilized GlcN, but actually inhibits the immobilization process using the physical adsorption method. Contact angle measurements and atomic force microscopy (AFM) show a substantial increase of surface energy and roughness of PLA surface, respectively, upon the multistep modification procedure. The cytocompatibility of the modified surfaces is assessed using a mouse embryonic fibroblast (MEF) cell line. Observation from the cell culture basically demonstrates the potential of GlcN immobilization in improving the cytocompatibility of the PLA surface. Moreover, the covalent immobilization of GlcN seems to produce more cytocompatible surfaces if compared with the physical adsorption method. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3176-3188, 2017.

• MeSH
• Adsorption MeSH
• Biocompatible Materials chemistry   MeSH
• Cell Line MeSH
• Fibroblasts cytology   MeSH
• Glucosamine chemistry   MeSH
• Kinetics MeSH
• Microscopy, Atomic Force MeSH
• Mice MeSH
• Polyesters chemistry   MeSH
• Surface Properties MeSH
• Cell Proliferation MeSH
• Tissue Scaffolds chemistry   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

• MeSH
• Anti-Inflammatory Agents chemistry   pharmacology   MeSH
• Bacterial Proteins chemistry   MeSH
• K562 Cells MeSH
• Th17 Cells drug effects   metabolism   MeSH
• Immunologic Factors chemistry   pharmacology   MeSH
• Interleukin-23 chemistry   physiology   MeSH
• Jurkat Cells MeSH
• Binding, Competitive MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Peptide Fragments chemistry   pharmacology   MeSH
• Drug Evaluation, Preclinical MeSH
• Receptors, Interleukin antagonists & inhibitors   physiology   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The 7-chloro-4-(piperazin-1-yl)quinoline structure is an important scaffold in medicinal chemistry. It exhibited either alone or as hybrid with other active pharmacophores diverse pharmacological profiles such as: antimalarial, antiparasitic, anti-HIV, antidiabetic, anticancer, sirtuin Inhibitors, dopamine-3 ligands, acetylcholinesterase inhibitors, and serotonin antagonists. In the presented review, a comprehensive discussion of compounds having this structural core is surveyed and illustrated.

• MeSH
• Quinolines chemistry   pharmacology   MeSH
• Humans MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.

• MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Gene Library MeSH
• Interferon-gamma metabolism   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Serum Albumin metabolism   MeSH
• Streptococcus chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

• MeSH
• Albumins metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• HeLa Cells MeSH
• Immobilized Proteins metabolism   MeSH
• Lactococcus lactis metabolism   MeSH
• Humans MeSH
• Cell Surface Display Techniques MeSH
• Protein Subunits metabolism   MeSH
• Surface Plasmon Resonance MeSH
• Protein Domains MeSH
• Flow Cytometry MeSH
• Recombination, Genetic genetics   MeSH
• Recombinant Proteins metabolism   MeSH
• Ribosomes metabolism   MeSH
• Sequence Homology, Amino Acid MeSH
• Shiga Toxin 1 chemistry   metabolism   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biotin chemistry   MeSH
• Biotinylation MeSH
• Cell Line MeSH
• Dendrimers chemical synthesis   pharmacology   MeSH
• Epithelial Cells cytology   drug effects   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression MeSH
• Rats MeSH
• Humans MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Nanoparticles chemistry   ultrastructure   MeSH
• Polyamines chemistry   MeSH
• Polyethylene Glycols chemistry   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Serum Albumin chemistry   MeSH
• Streptavidin chemistry   MeSH
• Streptomyces genetics   metabolism   MeSH
• Drug Delivery Systems * MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

• Publication type
• Journal Article MeSH