Aptamers as reagents for high-throughput screening

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.     

Reactive oligonucleotide derivatives as gene-targeted biologically active compounds and affinity probes

Development of efficient methods for synthesis of oligonucleotides and oligonucleotide analogs has opened up the possibility of designing a broad spectrum of affinity reagents for specific modification of nucleic acids and proteins. These affinity reagents are used for investigation of the topology of ribosomes and nucleic acid polymerases. Oligonucleotides and their analogs are already used for suppression of specific gene expression and for elucidation of the physiological role of their products. Oligonucleotide derivatives appear to offer considerable promise as potential gene-targeted drugs such as antivirals and specific inhibitors of oncogene expression.     

Styrene and ethylbenzene absorption in ionic liquids: comparing DFT affinity calculations with experimental data

Styrene is a widely used bulk chemical produced by dehydrogenation of ethylbenzene (EB). Purification of styrene to contain < 100 ppm EB is not cost-effective by conventional separation methods. One separation method is extractive distillation with an ionic liquid (IL) as a binding agent for one of the components, thereby lowering the vapour pressure of this component. In this study, using quantum density functional theory (DFT), we have simulated 22 IL anion–cation pairs, styrene and EB affinities to them, and ion-pair dimer affinities of the ILs. These are compared with experimental liquid–liquid equilibrium studies of M.T.G. Jongmans, B. Schuur, and A.B. de Haan, Ind. Eng. Chem. Res. 50 (2011), pp. 10800–10810. It is shown that experimental selectivity and distribution coefficients of styrene and EB in the ILs are related to computed gas phase anion–cation stabilisation energies and ion-pair–ion-pair dimer affinities. The inverse of molar volume is found to strongly correlate with the selectivity. The computational results also qualitatively correlate with molar volume, and consequently, it is possible to use DFT calculations as a qualitative prediction tool in screening of ILs for this separation process. This tool does not account for effects caused by long alkyl chains, as the length does not seem to affect dimer stabilisation energy beyond ethyl group.     

Alginate beads as an affinity material for alpha amylases

Calcium-alginate beads were found to bind a variety of enzymes in a nonspecific fashion. However, alpha amylases from porcine pancreas, Bacillus subtilis (BAN 240L) and wheat germ bound at a significant level and B. subtilis and wheat germ amylases could be eluted with 1M maltose. The wheat germ alpha amylase could be purified 45 fold with 70% recovery. The SDS - PAGE pattern showed significant purification by this single step strategy.     

The Screening of RNA Aptamers Specific for Carbonic Anhydrase I Using the Systematic Evolution of Ligands by an Exponential Enrichment Method (SELEX)

Carbonic anhydrases (CA) or carbonate dehydratases are a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate. CA I is the most abundant protein in the cytosol and has been reported to the partially associated with a number of fatal diseases. A newly established Systematic Evolution of Ligands by EXponential enrichment (SELEX) method referred to as Protein-SELEX was used to select RNA aptamers against the human erythrocyte CA I (CA I) protein. After five rounds of selection and counter selection the specific binding of the 6th cycle in vitro transcribed RNA library to CA I was detected by an Electrophoretic Mobility Shift Assay (EMSA). Three Specific sequences were identified as binding candidates after cloning and sequence analysis and one of the selected CA I specific RNA aptamers, CAapt1, was used to confirm specific binding and the Kd values were determined using an EMSA. The CAapt1 RNA aptamer showed no affinity towards any other protein and in comparison to the “0” cycle library, a significant enrichment was obtained. This methodology permitted us to successfully investigate the ssRNA aptamer CAapt1 for CA I protein.     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

Antibody-based tissue profiling as a tool for clinical proteomics

Here, we show a strategy for high-throughput antibody-based tissue profiling with the aim to create an atlas of protein expression patterns in normal human tissues and cancer tissues representing the 20 most prevalent cancer types. A set of standardized tissue microarrays (TMAs) was produced to allow for rapid screening of a multitude of different cells and tissues using immunohistochemistry. Eight TMA blocks were produced containing 48 different normal human tissues in triplicate and cancer tissue from 216 individually different tumors in duplicate. Sections from these blocks were immunohistochemically stained using five commercial and five in-house generated antibodies. Digital images for annotation of expression profiles were generated using a semiautomated approach. Five hundred seventy-six images and annotation data corresponding to a total of 30 Gbytes of data were collected for each antibody. The data presented here suggest that antibody-based profiling of protein expression in tissues can be used as a valuable tool in clinical proteomics.     

Methods for selection of aptamers to protein targets

Aptamers are synthetic single-stranded RNA or DNA molecules capable of specific binding to other target molecules. In this review, the main aptamer properties are considered and methods for selection of aptamers against various protein targets are described. Special attention is given to the methods for directed selection of aptamers, which allow one to obtain ligands with specified properties.     

Low affinity use-dependent NMDA receptor antagonists show promise for clinical development

Summary. The success of the low affinity use-dependent NMDA receptor antagonists to reach clinical trials can be readily attributed to their wider margins of safety and lack of neurotoxicity at higher doses. Several mechanistic differences distinguish the low affinity from the high affinity use-dependent antagonists: 1) Differential regional affinities for the various NMDA receptor subtypes; 2) The static receptor blockade due to the faster on/off rate receptor kinetics which limit, but do not totally prevent the amount of Ca⁺² entry into the cell during glutamate-induced depolarization; and 3) Rapid egress of the compounds from the ion channel during recovery resulting in less membrane trapping between transmission pulses. Advanced clinical trials are in progress for the following indications: epilepsy, stroke, head trauma, tardive dyskinesia, pain plus Parkinson's, Huntington's and Alzheimer's diseases. Received August 31, 1999 Accepted September 20, 1999     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable isotope-coded affinity tag reagents

Quantitative profiling of proteins, the direct effectors of nearly all biological functions, will undoubtedly complement technologies for the measurement of mRNA. Systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with isotope-coded affinity tag reagents and software tools for high-throughput analysis of LC-MS/MS data. We provide here the first such study achieving quantitative, global proteomic measurement of a time-course gene expression experiment in a model eukaryote, the budding yeast Saccharomyces cerevisiae, during the cell cycle. We sampled 48% of all predicted ORFs, and provide the data, including identifications, quantitations, and statistical measures of certainty, to the community in a sortable matrix. We do not detect significant concordance in the dynamics of the system over the time-course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures. Our proteomic dataset therefore provides a necessary and complementary measure of eukaryotic gene expression, establishes a rich database for the functional analysis of S. cerevisiae proteins, and will enable further development of technologies for global proteomic analysis of higher eukaryotes.     

Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents

ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Medium format enhancement: design of an immobilized heparin affinity media for industrial bioprocessing

Affinity chromatography is one of the most powerful and selective separation methods available. Recently, affinity methods are being incorporated into industrial processes with some frequency. One of the reasons for this is that affinity media robust enough for industrial bioprocessing are now available. By robust is meant that the media meet stringent requirements for cleanability, sanitization, physical and chemical stability, regulatory and technical support, batch-to-batch reproducibility and reliability of supply. This paper describes a medium format enhancement program to adapt a widely known group-specific affinity medium, Heparin Sepharose( CL6B, to the requirements for industrial bioprocessing. The new medium, Heparin Sepharose( 6 Fast Flow was designed for the recovery of antithrombin 3 (AT3) at industrial scale. The medium is based upon a highly cross-linked 6% agarose, which is produced in very large scale and is familiar to regulatory agencies. The ligand, heparin, is attached to the matrix by a reductive amination chemistry. The resulting linkage is stable in 0.1 N NaOH for 150 h, showing no decrease in AT3 binding affinity at that time. Heparin has a broad biological functionality and thus is useful chromatographically for the purification of a number of proteins which have an affinity for heparin. Heparin, as a complex sugar, is also a highly charged polyanion and thus has interesting ion-exchange properties. Because of its broad applicability to a number of purification problems, immobilized heparin is a useful case study in medium format enhancement. © 1997 John Wiley & Sons, Ltd.     

Weak affinity chromatography as a new approach for fragment screening in drug discovery

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–μM in dissociation constant (K_D)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.     

Chemical affinity matrix-based identification of prohibitin as a binding protein to anti-resorptive sulfonyl amidine compounds

In order to identify the binding proteins to anti-resorptive 5-chloro-1-(2,6-dimethylpiperidin-1-yl)-N-tosylpentan-1-imine (1), the chemical affinity matrix for the compound 1 (2b) was designed and synthesized. Using 2b-based chemical proteomics, prohibitin was identified as one of strong binding proteins for 2b.     

Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

用于MALDI-TOF MS分析的毛细管层析柱制备方法研究

研究了一种用于MALDI-TOF MS分析中样品预处理毛细管亲和层析柱的制备方法。首先采用硫酸和双氧水氧化法将羟基引入到石英毛细管内表面,进一步使用氨基丙基三乙氧基硅烷（APTES）对毛细管进行修饰以将氨基偶联到毛细管内表面;采用1,4-丁二醇二缩水甘油醚（BDDE）将魔芋葡甘聚糖（KGM）进行活化,将活化后的KGM与毛细管上的氨基进行了偶联,在此基础上将模型蛋白（胰蛋白酶）偶联到毛细管内KGM,成功制备出毛细管亲和层析柱,考察了KGM和环氧基含量对模型蛋白偶联量及其样品预处理效果的影响。结果表明,KGM分子量是影响毛细管层析柱上蛋白偶联量的关键因素,以胰蛋白酶抑制剂为目标物结合MALDI-TOF MS对亲和层析柱的分离效果进行了评价,证明了偶联胰蛋白酶的毛细管层析柱可有效实现胰蛋白酶抑制剂的分离和浓缩。基于表面处理和KGM衍生的毛细管亲和层析柱制备技术具有可行性,并可用于MALDI-TOF MS分析的样品预处理。