Pyrazolo-[1,5-a]-1,3,5-triazine corticotropin-releasing factor (CRF) receptor ligands

The syntheses and rat CRF receptor binding affinities of 'retro-pyrazolotriazine' corticotropin-releasing factor (CRF) ligands 4 are reported. Some have high affinity for rat CRF receptors (K(i)< or =10 nM). The data provide additional support for the hypothesis that it is possible to interchange isosteric cores with similar electronic properties in the design of high-affinity CRF receptor ligands, provided the peripheral pharmacophore elements are maintained in the same three-dimensional array.     

Investigation of restriction enzyme cofactor requirements: a relationship between metal ion properties and sequence specificity

Restriction enzymes are important model systems for understanding the mechanistic contributions of metal ions to nuclease activity. These systems are unique in that they combine distinct functions which have been shown to depend on metal ions: high-affinity DNA binding, sequence-specific recognition of DNA, and Mg(II)-dependent phosphodiester cleavage. While Ca(II) and Mn(II) are commonly used to promote DNA binding and cleavage, respectively, the metal ion properties that are critical to the support of these functions are not clear. To address this question, we assessed the abilities of a series of metal ions to promote DNA binding, sequence specificity, and cleavage in the representative PvuII endonuclease. Among the metal ions tested [Ca(II), Sr(II), Ba(II), Eu(III), Tb(III), Cd(II), Mn(II), Co(II), and Zn(II)], only Mn(II) and Co(II) were similar enough to Mg(II) to support detectable cleavage activity. Interestingly, cofactor requirements for the support of DNA binding are much more permissive; the survey of DNA binding cofactors indicated that Cd(II) and the heavier and larger alkaline earth metal ions Sr(II) and Ba(II) were effective cofactors, stimulating DNA binding affinity 20-200-fold. Impressively, the trivalent lanthanides Tb(III) and Eu(III) promoted DNA binding as efficiently as Ca(II), corresponding to an increase in affinity over 1000-fold higher than that observed under metal-free conditions. The trend for DNA binding affinity supported by these ions suggests that ionic radius and charge are not critical to the promotion of DNA binding. To examine the role of metal ions in sequence discrimination, we determined specificity factors [K(a)(specific)/K(a)(nonspecific)] in the presence of Cd(II), Ba(II), and Tb(III). Most interestingly, all of these ions compromised sequence specificity to some degree compared to Ca(II), by either increased affinity for a noncognate sequence, decreased affinity for the cognate sequence, or both. These results suggest that while amino acid-base contacts are important for specificity, the properties of metal ion cofactors at the catalytic site are also critical for sequence discrimination. This insight is invaluable to our efforts to understand and subsequently design sequence-specific nucleases.     

POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties

MOTIVATION: Both modeling of antigen-processing pathway including major histocompatibility complex (MHC) binding and immunogenicity prediction of those MHC-binding peptides are essential to develop a computer-aided system of peptide-based vaccine design that is one goal of immunoinformatics. Numerous studies have dealt with modeling the immunogenic pathway but not the intractable problem of immunogenicity prediction due to complex effects of many intrinsic and extrinsic factors. Moderate affinity of the MHC-peptide complex is essential to induce immune responses, but the relationship between the affinity and peptide immunogenicity is too weak to use for predicting immunogenicity. This study focuses on mining informative physicochemical properties from known experimental immunogenicity data to understand immune responses and predict immunogenicity of MHC-binding peptides accurately. RESULTS: This study proposes a computational method to mine a feature set of informative physicochemical properties from MHC class I binding peptides to design a support vector machine (SVM) based system (named POPI) for the prediction of peptide immunogenicity. High performance of POPI arises mainly from an inheritable bi-objective genetic algorithm, which aims to automatically determine the best number m out of 531 physicochemical properties, identify these m properties and tune SVM parameters simultaneously. The dataset consisting of 428 human MHC class I binding peptides belonging to four classes of immunogenicity was established from MHCPEP, a database of MHC-binding peptides (Brusic et al., 1998). POPI, utilizing the m = 23 selected properties, performs well with the accuracy of 64.72% using leave-one-out cross-validation, compared with two sequence alignment-based prediction methods ALIGN (54.91%) and PSI-BLAST (53.23%). POPI is the first computational system for prediction of peptide immunogenicity based on physicochemical properties. AVAILABILITY: A web server for prediction of peptide immunogenicity (POPI) and the used dataset of MHC class I binding peptides (PEPMHCI) are available at http://iclab.life.nctu.edu.tw/POPI     

Generation and evaluation of putative neuroregenerative drugs. Part 1: virtual point mutations to the polyketide rapamycin

This paper explores the use of computational methods to direct engineered biosynthesis based on the desired properties of the target compounds. The immunosuppressive properties of rapamycin are a result of the formation of the complex FKBP12-rapamycin-FRAP. Neuroregenerative properties are exhibited by the complex or complexes of rapamycin with FKBP proteins. Our objective has been to design biosynthetically available analogues of rapamycin that bind tightly to FKBP12 but not to FRAP. This has been carried out by successive single ketide deletions from the effector domain of rapamycin. The approach described here has yielded modified rapamycin analogues (RP2 and RP3) as targets for biosynthesis by modified polyketide synthases. RP2 and RP3 have an identical binding affinity (linear interaction energy calculation) to FKBP12 as rapamycin but little or no affinity for binding to FRAP.     

Affinity purification of immunoglobulin M using a novel synthetic ligand

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05–0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85–95%, as determined by densitometric scanning of sodium dodecyl sulfate–polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.     

High affinity extremes in combinatorial libraries and repertoires

By generating a large diversity of molecules, the immune system selects antibodies that bind antigens. Sharing the same approach, combinatorial biotechnologies use a large library of compounds to screen for molecules of high affinity to a given target. Understanding the properties of the best binders in the pool aids the design of the library. In particular, how does the maximum affinity increase with the size of the library or repertoire? We consider two alternative models to examine the properties of extreme affinities. In the first model, affinities are distributed lognormally, while in the second, affinities are determined by the number of matches to a target sequence. The second model more explicitly models nucleic acids (DNA or RNA) and proteins such as antibodies. Using extreme value theory we show that the logarithm of the mean of the highest affinity in a combinatorial library grows linearly with the square root of the log of the library size. When there is an upper bound to affinity, this “absolute maximum” is also approached approximately linearly with root log library size, reaching the upper limit abruptly. The design of libraries may benefit from considering how this plateau is reached as the library size is increased.     

Affinity chromatography supports: a look at performance requirements.

Because of its high selectivity, affinity chromatography is a preferred tool in the downstream processing of high-value proteins and peptides of therapeutic interest. This review examines the affinity supports currently available, and investigates the performance characteristics and properties required of the support matrices for improved affinity-based supports for large-scale purification of biomolecules. Parameters for optimizing an affinity chromatographic process, and the advantages of affinity-based separation for scaled-up systems are highlighted.     

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.     

Engineered affinity proteins—Generation and applications

The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.     

Agonist binding to multiple muscarinic receptors

The binding of agonists to muscarinic cholinergic receptors is well described by a binding model of multiple affinity states (superhigh, high, and low) in most central and peripheral tissues. Although previous studies of the influences by divalent cations, guanine nucleotides, and sulfhydryl reagents support the concept that these regulators act through closely related sites to alter the relative proportions of muscarinic agonist affinity states, it has become apparent that muscarinic receptor subtypes (as defined with the nonclassical antagonist pirenzepine) are differentially affected by the regulators. For example, in tissues that have few high-affinity [3H]pirenzepine-binding sites (heart, ileum, cerebellum), magnesium ions promote the formation of a high agonist affinity state, whereas exposure of these tissues to the sulfhydryl reagent N-ethylmaleimide (NEM) or guanine nucleotides promotes the formation of a low agonist affinity state. Conversely, tissues rich in high-affinity [3H]pirenzepine-binding sites (cerebral cortex, corpus striatum, hippocampus) show little, if any, change in agonist binding site affinity when magnesium ions or guanine nucleotides are present. Furthermore, NEM enhances the muscarinic binding site affinity for agonists in these tissues. Taken together, these results support the concept of muscarinic receptor heterogeneity, as proposed from previous physiological studies, and indicate that the aforementioned regulators (guanine nucleotides, magnesium ions, NEM) differentially alter the agonist-binding properties of these muscarinic receptor subtypes.     

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological,biochemical and therapeutic properties

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor–ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.     

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.     

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.     

Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides

An affinity matrix was constructed by synthesis of a DNA oligonucleotide on a Teflon fiber support followed by deblocking and hybridization of the complementary strand. It was used to purify a sequence-specific binding protein at least 100-fold to near homogeneity. This matrix is easily fabricated on an automated DNA synthesizer, contains high levels of attached DNA, and has superior mechanical properties. It should be generally useful for affinity chromatography of DNA binding proteins.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.     

Detection and isolation of oligosaccharides with Lea and Leb blood group activities by affinity chromatography using monoclonal antibodies

Affinity columns prepared by immobilizing monoclonal antibodies that specifically recognize the Lea or the Leb blood group antigens can be used for analytical or preparative isolation of oligosaccharides with the corresponding reactivities. The number of immobilized functional antibody combining sites on a column and the dissociation constants for standard oligosaccharides are determined by frontal analysis. By employing a simple approximation [K.-I. Kasai et al. (1986) J. Chromatogr. 376, 33-47] these parameters can be used to rationally design columns with properties appropriate for zonal affinity chromatography. The affinity for binding of the Lea-active oligosaccharide lacto-N-fucopentaose II (LNF II) by the anti-Lea antibody CO-514 doubles for each 8 degrees C downward shift in temperature between 37 and 4 degrees C. By zonal chromatography, Lea- or Leb-active oligosaccharides are recovered from a complex mixture of milk oligosaccharides containing more than a 20-fold molar excess of structurally similar but antigenically distinct oligosaccharides. The capacity for preparative isolation of an oligosaccharide increases in a linear fashion with the amount of antibody loaded on the solid support. The monoclonal antibodies used in these studies are products of hybridomas derived from mice immunized with human colorectal carcinoma cell lines [M. Blaszczyk et al. (1984) Arch. Biochem. Biophys. 233, 161-168]. The experiments establish that affinity chromatography applied to mixtures of oligosaccharides released by enzymatic or chemical cleavage of glycoconjugates may simplify the task of isolating and characterizing biologically interesting target antigens of monoclonal antibodies.     

Engineering cooperative tecto-RNA complexes having programmable stoichiometries

High affinity and specificity RNA-RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop-receptor interaction motifs. These interactions can be fused using flexible four-way junction motifs to create divalent, self-assembling scaffolding units ('tecto-RNA') that have favorable properties for nanomedicine and other applications. We describe the design and directed assembly of tecto-RNA units ranging from closed, cooperatively assembling ring-shaped complexes of programmable stoichiometries (dimers, trimers and tetramers) to open multimeric structures. The novelty of this work is that tuning of the stoichiometries of self-assembled complexes is achieved by precise positioning of the interaction motifs in the monomer units rather than changing their binding specificities. Structure-probing and transmission electron microscopy studies as well as thermodynamic analysis support formation of closed cooperative complexes that are highly resistant to nuclease digestion. The present designs provide two helical arms per RNA monomer for further functionalization aims.     

"Glutaraldehyde-P", a stable, reactive aldehyde matrix for affinity chromatography

A high-performance affinity chromatography support based on silica has been developed for the immobilization of proteins containing primary amino groups. A hydrophilic polymer covalently bound to the silica surface minimizes nonspecific protein binding to the support while preserving high binding capacity. The Schiff base reaction involved in the coupling of a ligand to the affinity medium is rapid, allows the use of mild conditions during the coupling process, and results in a very stable linkage. Reaction parameters were studied for protein coupling to the affinity support to determine optimum binding conditions and dynamic capacity as a function of protein size. The stability of the ligand-matrix bond was determined. The performance and reproducibility of the affinity support are demonstrated by its use in the analysis of nitrophenyl sugar derivatives, purification of glycoproteins, and isolation of anti-bovine immunoglobulin G developed in rabbit.     

Modeling of monolith-supported affinity chromatography

Ceramic monoliths have been used successfully as active support for affinity chromatography (1). A mathematical model was developed to simulate the adsorption-elution experimental behavior of asparaginase in an agarose-coated monolith support. The computer-based model allows precise determination of experimental parameters. Because of the simple geometry of ceramic monoliths used as support, the mathematical model can be used to design adsorption/elution cycles for the large throughput separation of biomolecules.