Recent Advances in the Scaffold Engineering of Protein Binders.

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications.

A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation.

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.

Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine.

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.

Peptoid transporters: effects of cationic, amphipathic structure on their cellular uptake.

Two cationic, amphipathic peptoids (poly-N-substituted glycines) were developed as new molecular transporters, which have extensive cellullar uptake and utilize different internalization mechanisms from purely cationic polyguanidine comparators.

Anticalins small engineered binding proteins based on the lipocalin scaffold.

Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.

Engineering knottins as novel binding agents.

Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.

Isolation and identification of diadenosine 5',5'''-P1,P4-tetraphosphate binding proteins using magnetic bio-panning.

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.

Fmoc synthesis of peptide thioesters without post-chain-assembly manipulation.

An operationally simple method for the synthesis of peptide thioesters is developed using standard Fmoc solid-phase peptide synthesis procedures. The method relies on the use of a premade enamide-containing amino acid which, in the final TFA cleavage step, renders the desired thioester functionality through an irreversible intramolecular N-to-S acyl transfer.