Aptamers as tools for target prioritization and lead identification

The increasing number of potential drug target candidates has driven the development of novel technologies designed to identify functionally important targets and enhance the subsequent lead discovery process. Highly specific synthetic nucleic acid ligands – also known as aptamers – offer a new exciting route in the drug discovery process by linking target validation directly with HTS. Recently, aptamers have proven to be valuable tools for modulating the function of endogenous cellular proteins in their natural environment. A set of technologies has been developed to use these sophisticated ligands for the validation of potential drug targets in disease models. Moreover, aptamers that are specific antagonists of protein function can act as substitute interaction partners in HTS assays to facilitate the identification of small-molecule lead compounds.     

Nucleic Acid Aptamers: New Methods for Selection,Stabilization, and Application in Biomedical Science

The adoption of oligonucleotide aptamer is well on the rise, serving an ever increasing demand for versatility in biomedical field. Through the SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamer that can bind to specific target with high affinity and specificity can be obtained. Aptamers are single-stranded nucleic acid molecules that can fold into complex threedimensional structures, forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target. Recently, aptamers have attracted much attention because they not only have all of the advantages of antibodies, but also have unique merits such as thermal stability, ease of synthesis, reversibility, and little immunogenicity. The advent of novel technologies is revolutionizing aptamer applications. Aptamers can be easily modified by various chemical reactions to introduce functional groups and/or nucleotide extensions. They can also be conjugated to therapeutic molecules such as drugs, drug containing carriers, toxins, or photosensitizers. Here, we discuss new SELEX strategies and stabilization methods as well as applications in drug delivery and molecular imaging.     

PDBLIG: classification of small molecular protein binding in the Protein Data Bank

It is known that proteins can adopt different folds while sharing similar features for recognition of similar substrates or ligands, for example, in the binding sites of enzyme cofactors such as ATP. On the other hand, proteins that have highly flexible binding sites or belong to large and diverse protein families can bind structurally dissimilar ligands, as, for example, in the case of the matrix metalloprotease family. We have developed a database, PDBLIG, that classifies protein domains and ligands. The information stored includes each protein's function, domain class(es), which ligand(s) it binds, and so on. The database can provide valuable knowledge for drug discovery, supporting the answering of questions such as whether the same drug molecule can bind different target protein families and whether these families are related functionally or structurally, which ligand classes (such as metabolites or organic molecules) bind to a particular protein family and whether the ligands are druglike, and which target families bind a wide variety of ligands and whether different ligands are associated with different subfamilies.     

RNA aptamers: from basic science towards therapy

The SELEX technique (systematic evolution of ligands by exponential enrichment) provides a powerful tool for the in vitro selection of nucleic acid ligands (aptamers) from combinatorial oligonucleotide libraries against a target molecule. In the beginning of the technique's use, RNA molecules were identified that bind to proteins that naturally interact with nucleic acids or to small organic molecules. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands (aptamers) for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do. The in vitro selection of oligonucleotides with enzymatic activity, denominated aptazymes, allows the direct transduction of molecular recognition to catalysis. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as membrane-bound receptors, and even entire cells. RNA aptamers have also been expressed in living cells. These aptamers, also called intramers, can be used to dissect intracellular signal transduction pathways. The utility of RNA aptamers for in vivo experiments, as well as for diagnostic and therapeutic purposes, is considerably enhanced by chemical modifications, such as substitutions of the 2'-OH groups of the ribose backbone in order to provide resistance against enzymatic degradation in biological fluids. In an alternative approach, Spiegelmers are identified through in vitro selection of an unmodified D-RNA molecule against a mirror-image (i.e. a D-peptide) of a selection target, followed by synthesis of the unnatural nuclease-resistant L-configuration of the RNA aptamer that recognizes the natural configuration of its selection target (i.e. a L-peptide). Recently, nuclease-resistant inhibitory RNA aptamers have been developed against a great variety of targets implicated in disease. Some results have already been obtained in animal models and in clinical trials.     

ATP-Responsive Drug Delivery Systems

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle–aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle–aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.     

Low efficient generation of ssDNA aptamer using chemical modified spacer primer

Aptamers are oligonucleotides (ssDNA or RNA) with an appropriate size of 100 bps that bind with high affinity and specificity to a wide range of target molecules, including virtually any class of protein, drugs or small organic/inorganic molecules. The in vitro selection process referred to as SELEX provides a powerful tool to identify specific aptamers with high affinity and even discriminate between closely related targets. Aptamers have various applications such as analytical tools, disease diagnosis and prediction, pharmaceutical research, drug development, therapy and even for environmental monitoring. Nowadays, with the development of SELEX methods, generation of aptamer becomes more efficient, less time consuming and even automatically. The whole SELEX process includes binding, separation, and nucleic acid amplification. As amplification of nucleotides is an important process in successive SELEX, we will compare several methods for generation of aptamer in this report.     

Characterisation of aptamers for therapeutic studies

Nucleic acid aptamers, like monoclonal antibodies, bind to their target molecule with high affinity and specificity. Owing to their molecular recognition properties that are based on their three-dimensional structure, aptamers have been used as powerful tools in various fields, such as detection, separation or purification of target molecules. In addition, their intrinsic characteristics make aptamers excellent candidates for therapeutic applications. Due to their high specificity, stability, low toxicity and apparent non-immunogenicity, they provide viable alternatives to antibody- and small molecule-based therapeutics. Unlike antibodies, they can be generated against toxic, unstable or difficult to purify proteins. Furthermore, they can be chemically derivatised to extend their bioavailability and lifetimes in biological fluids, and they even offer the possibility for fast and efficient regulation. Similar to other therapeutic nucleic acids, such as antisense oligonucleotides, ribozymes or siRNA, problems with cellular uptake and susceptibility to nucleolytic attack have to be overcome for their successful application. With the first aptamer-based drug having entered the market and a few in Phase II trials, the road has been paved for more to follow. In this review, the requirements and properties of therapeutic aptamers are discussed and the recent progress in aptamer research towards drug development is highlighted.     

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.     

指数富集的配体系统进化技术的发展及其在临床治疗中的应用

基于作为配基的寡核苷酸的三维立体结构,指数富集的配体系统进化（SELEX）技术能够筛选获得高效、特异结合靶分子的寡核苷酸适体。该配基由于其亲和力高、特异性强、免疫原性低、筛选周期短及适用范围广等特性,在临床诊断和疾病治疗中拥有多项优势,具有被研制成新型适体药物应用于临床的巨大潜力。本文从SELEX技术的发展、配基的修饰改造、临床治疗的优势、配基作为靶蛋白功能阻断剂和药物分子靶向载体进行的临床应用等几个方面,对寡核苷酸适体在临床疾病治疗中的应用进行综述。     

Aptamers: new arrows to target dendritic cells

Aptamers, as a novel class of molecular probes for diagnosis, imaging and targeting therapy, have attracted increasing attention in recent years. Aptamers are generated from libraries of single-stranded nucleic acids against different molecules via the “systematic evolution of ligands by exponential enrichment” (SELEX) method. SELEX is a repetitive process of a sequential selection procedure in which a DNA or RNA library pool is incubated separately with target and control molecules to select specific oligonucleotide aptamers with high affinities and specificities. Cell-SELEX is a modified version of the SELEX process in which whole living cells are used as targets for the aptamers. Dendritic cell (DC) targeting, as a new therapeutic approach, can improve the efficiency of immunotherapy in the treatment of allergies and cancers. DCs use various receptors to continuously induce adaptive immunity via capture and presentation of antigens to naïve T cells. DCs are considered as the best targets in modulating immune responses against cancer, autoimmunity, allergy and transplantation. Aptamers, as a new agent, can be applied in DC targeting. The purpose of this review is to present some general concepts of aptamer production and DC targeting by aptamer molecules.     

Development of nucleic acid transfection technology to the kidney

The kidney is one of the most important organs that play a crucial role in homeostasis and, therefore, congenital or acquired renal dysfunction causes refractory diseases, i.e., Alport's syndrome, Fabry's disease, diabetic nephropathy, IgA nephropathy, kidney cancer, transplant glomerulopathy. Nucleic acid transfection technology to the kidney is indispensable for the progress of biomedical research and the realization of gene therapy and nucleic acid drug for renal diseases. Control of renal nucleic acid transfection was difficult because of the structural complexity; however, the study of recombinant virus, synthetic carrier and physical force-mediated nucleic acid transfection to the kidney has advanced. Recombinant virus and synthetic carrier-mediated methods require long-term block of the blood or urinary flow for efficient transfection of nucleic acid because of the rich blood flow of the kidney. In contrast, physical force-mediated methods that transfect with nucleic acid via transient membrane permeability do not apprehend ischemia-reperfusion injury and, therefore, may be beneficial for nucleic acid transfection to the kidney. In this article, we collect the information of therapeutic gene, target molecule of the nucleic acid drug and target cells for renal diseases and structural property of the kidney from the point of view of nucleic acid transfection. Additively, current status of nucleic acid transfection technology to the kidney is reviewed.     

Nucleic acid aptamers against proteases

Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, protein-aptamer recognition, protease inhibition, and advantages of aptamers for pharmacological intervention with pathophysiological functions of proteases. Aptamers can be selected so that they bind their targets highly specifically and with affinities corresponding to KD values in the nM range. Aptamers can be selected so that they recognize their targets conformation-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers.     

New hydrogen-bond potentials for use in determining energetically favorable binding sites on molecules of known structure

An empirical energy function designed to calculate the interaction energy of a chemical probe group, such as a carbonyl oxygen or an amine nitrogen atom, with a target molecule has been developed. This function is used to determine the sites where ligands, such as drugs, may bind to a chosen target molecule which may be a protein, a nucleic acid, a polysaccharide, or a small organic molecule. The energy function is composed of a Lennard-Jones, an electrostatic and a hydrogen-bonding term. The latter is dependent on the length and orientation of the hydrogen bond and also on the chemical nature of the hydrogen-bonding atoms. These terms have been formulated by fitting to experimental observations of hydrogen bonds in crystal structures. In the calculations, thermal motion of the hydrogen-bonding hydrogen atoms and lone-pair electrons may be taken into account. For example, in a alcoholic hydroxyl group, the hydrogen may rotate around the C-O bond at the observed tetrahedral angle. In a histidine residue, a hydrogen atom may be bonded to either of the two imidazole nitrogens and movement of this hydrogen will cause a redistribution of charge which is dependent on the nature of the probe group and the surrounding environment. The shape of some of the energy functions is demonstrated on molecules of pharmacological interest.     

Nucleic Acid Aptamers Stabilize Proteins Against Different Types of Stress Conditions

It has been observed that the same osmolyte cannot provide protection to a protein exposed to more than one stress condition. We wanted to study the effect of nucleic acid aptamers on the stabilization of proteins against a variety of stress conditions. Adjuvanted tetanus toxoid was exposed to thermal, freeze–thawing, and agitation stress. The stability and antigenicity of the toxoid were measured. Using nucleic acid aptamers selected against tetanus toxoid, we show that these specific RNA sequences were able to stabilize alumina-adsorbed tetanus toxoid against thermal-, agitation-, and freeze–thawing-induced stress. Binding affinity of the aptamer–protein complex did not show any significant change at elevated temperature as compared with that at room temperature, indicating that the aptamer protected the protein by remaining bound to it under stress conditions and did not allow either the protein to unfold or to promote protein–protein interaction. Thus, we show that by changing the stabilization strategy from a solvent-centric to a protein-centric approach, the same molecule can be employed as a stabilizer against more than one stress condition and thus probably reduce the cost of the product during its formulation. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:100–106, 2014     

Technological Advances in High-Throughput Screening

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the possibility of candidate drug. SPR can measure the quantity of a complex formed between two molecules in real-time without the need for fluorescent or radioisotopic labels. SPR is capable of characterizing unmodified biopharmaceuticals, studying the interaction of drug candidates with macromolecular targets, and identifying binding partners during ligand fishing experiments. DNA microarrays can be used in HTS be used to further investigate the expression of biological targets associated with human disease, which then opens new and exciting opportunities for drug discovery. Without doubt, the addition of new technologies will further increase the application of HTS in drug screening and its related fields.     

Identification and characterization of peptide probes directed against PKCα conformations

Abstract: Phage display is a powerful technology that allows identification of high affinity peptides that bind specifically to a given molecular target. Using a highly complex peptide display library, we have identified separate classes of peptides that bind to protein kinase C alpha (PKCα) only under activation conditions. Furthermore, peptide binding was specific to PKCα and not to any of the other closely related PKC isoforms. The conformational and isoform specificity of the peptide binding was demonstrated using surface plasmon resonance as well as time‐resolved fluorescence assays. Kinase assays showed that these peptides were not direct substrates for PKC nor did they inhibit phosphorylation of PKC substrates. These peptides are most likely directed against protein–protein interaction sites on PKC. The data presented here offers another example of application of phage display technology to identify conformation‐dependent peptide probes against therapeutically important drug targets. These peptides are ideally suited to be used as surrogate ligands to identify compounds that bind specifically to PKCα, as well as conformational probes to detect activated forms of PKCα.     

Peptide aptamers as guides for small-molecule drug discovery

Peptide aptamers are combinatorial protein reagents that bind to target proteins with a high specificity and a strong affinity. By so doing, they can modulate the function of their cognate targets. Because peptide aptamers introduce perturbations that are similar to those caused by therapeutic molecules, their use identifies and/or validates therapeutic targets with a higher confidence level than is typically provided by methods that act upon protein expression levels. The unbiased combinatorial nature of peptide aptamers enables them to 'decorate' numerous polymorphic protein surfaces, whose biological relevance can be inferred through characterization of the peptide aptamers. Bioactive aptamers that bind druggable surfaces can be used in displacement screening assays to identify small-molecule hits to the surfaces. The peptide aptamer technology has a positive impact on drug discovery by addressing major causes of failure and by offering a seamless, cost-effective process from target validation to hit identification.     

RNA aptamers as potential pharmaceuticals against infections with African trypanosomes

Protozoal pathogens cause symptomatic as well as asymptomatic infections. They have a worldwide impact, which in part is reflected in the long-standing search for antiprotozoal chemotherapy. Unfortunately, effective treatments for the different diseases are by and large not available. This is especially true for African trypanosomiasis, also known as sleeping sickness. The disease is an increasing problem in many parts of sub-Saharan Africa, which is due to the lack of new therapeutics and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for "systematic evolution of ligands by exponential enrichment". The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions, and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognize ligands of different chemical origin. Aptamers typically bind their target with high affinity and high specificity and have successfully been converted into pharmaceutically active compounds. Here we summarize the recent examples of the SELEX technique within the context of identifying high-affinity RNA ligands against the surface of the protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness.     

Optical encoding of microbeads for gene screening: alternatives to microarrays

Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly in relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.