Aptamers and aptazymes: accelerating small molecule drug discovery

Synthetic nucleic acid ligands, known as aptamers, are versatile tools that can greatly enhance the efficiency of modern drug development. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Thus, aptamer technology can be exploited to address the growing demand for multi-parallel analysis of proteomes, functional prioritization of potential drug targets and accelerated small molecule lead identification. The unique advantages of this technology are the rapid automated generation of sophisticated ligands against almost any target molecule and the convenient structural or chemical modification of the nucleic acid probes. Depending on the strategy, an RNA aptamer can be expressed transgenically to investigate and inactivate an endogenous protein in an animal model, or it can be designed to function as a highly sensitive nucleic acid biosensor. More recently, the technology has been extended to directly link functional target validation with HTS, accelerating the process of drug discovery.     

Chemical ligands, genomics and drug discovery

The sequencing of the human genome and numerous pathogen genomes has resulted in an explosion of potential drug targets. These targets represent both an unprecedented opportunity and a technological challenge for the pharmaceutical industry. A new strategy is required to initiate small-molecule drug discovery with sets of incompletely characterized, disease-associated proteins. One such strategy is the early application of combinatorial chemistry and other technologies to the discovery of bioactive small-molecule ligands that act on candidate drug targets. Therapeutically active ligands serve to concurrently validate a target and provide lead structures for downstream drug development, thereby accelerating the drug discovery process.     

Peptide ligands in antibacterial drug discovery: use as inhibitors in target validation and target-based screening

There is an urgent need to develop novel classes of antibiotics to counter the inexorable rise of resistant bacterial pathogens. Modern antibacterial drug discovery is focused on the identification and validation of novel protein targets that may have a suitable therapeutic index. In combination with assays for function, the advent of microbial genomics has been invaluable in identifying novel antibacterial drug targets. The major challenge in this field is the implementation of methods that validate protein targets leading to the discovery of new chemical entities. Ligand-directed drug discovery has the distinct advantage of having a concurrent analysis of both the importance of a target in the disease process and its amenability to functional modulation by small molecules. VITA is a process that enables a target-based paradigm by using peptide ligands for direct in vitro and in vivo validation of antibacterial targets and the implementation of high-throughput assays to identify novel inhibitory molecules. This process can establish sufficient levels of confidence indicating that the target is relevant to the disease process and inhibition of the target will lead to effective disease treatment.     

Novel trends in high-throughput screening

Lead discovery by high-throughput screening (HTS) has evolved into a mature scientific discipline in modern drug discovery since its beginning about 10–15 years ago. Owing to the strong efforts in automation and miniaturization, even relatively large compound collections of over one million compounds or more can be screened against a large number of biological targets in relatively short time and at relatively low cost compared to the efforts of just 5 or 10 years ago. This was only possible with the concomitant development of high-quality readout technologies for highly miniaturized screening. Whereas most of the conventional drug targets can be approached via current HTS-readout technologies, the challenge goes toward the hitherto non-tractable families of drug targets. Future trends will focus strongly toward these novel target classes such as ion channels, transporters, protein–protein interactions, among many others. It will be essential to make proper readout technologies and adequate chemical libraries available for these target classes. Chemical libraries derived from natural products, but also derived from combinatorial chemistry and automated synthesis will be a key prerequisite for success in the field, as long as enough diversity and drug-like properties are included in these chemical libraries [25]. The proper readout technologies for screening of large chemical libraries have seen strong advances in recent years [^{[2], [7] and [22••]}], nevertheless none of these technologies is void of artifacts, in particular artifacts derived from the inherent physical nature of chemical compounds in aqueous buffer [11^••]. We therefore propose that future lead discovery should pay more attention toward unambiguous identification of these compound related artifacts and toward efficient removal of these false-positive compounds from the HTS hit-lists. We strongly recommend the use of biophysical and enzymological studies in the HTS hit-list follow-up phase (‘hit validation’) in order to deliver information of the highest possible quality for subsequent hit-to-lead studies. Finally, the science and art of HTS has evolved in various phases from its beginning in the early 1990s toward today's state-of-the-art operation in lead discovery. During these 15 years, one can distinguish three phases (‘generations’) of HTS operations: during the first phase, HTS has been just the same as laboratory screening, albeit at much larger capacity; in the second phase (‘second generation HTS’), HTS has evolved toward more sophisticated assay development/adaptation, more toward dedicated tool production, but also more toward counter-screening and hit-list follow-up; in the current phase (‘third generation HTS’) we see much more flexibility with regards to the applied processes for lead discovery, a stronger focus on quality and validation of the obtained results and a better awareness for choosing a proper lead finding strategy in a target-by-target specific manner.Taken together, we can conclude that better flexibility and creativity, more quality and the use of project-related, tailor-made lead finding strategies in the discovery process will become the key drivers for the successful application of high-throughput screening in the Pharmaceutical, Biotech, and Academic drug discovery programs of the future.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

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.     

Computational Advances for the Development of Allosteric Modulators and Bitopic Ligands in G Protein-Coupled Receptors

Allosteric modulators of G protein-coupled receptors (GPCRs), which target at allosteric sites, have significant advantages against the corresponding orthosteric compounds including higher selectivity, improved chemical tractability or physicochemical properties, and reduced risk of receptor oversensitization. Bitopic ligands of GPCRs target both orthosteric and allosteric sites. Bitopic ligands can improve binding affinity, enhance subtype selectivity, stabilize receptors, and reduce side effects. Discovering allosteric modulators or bitopic ligands for GPCRs has become an emerging research area, in which the design of allosteric modulators is a key step in the detection of bitopic ligands. Radioligand binding and functional assays ([³⁵S]GTPγS and ERK1/2 phosphorylation) are used to test the effects for potential modulators or bitopic ligands. High-throughput screening (HTS) in combination with disulfide trapping and fragment-based screening are used to aid the discovery of the allosteric modulators or bitopic ligands of GPCRs. When used alone, these methods are costly and can often result in too many potential drug targets, including false positives. Alternatively, low-cost and efficient computational approaches are useful in drug discovery of novel allosteric modulators and bitopic ligands to help refine the number of targets and reduce the false-positive rates. This review summarizes the state-of-the-art computational methods for the discovery of modulators and bitopic ligands. The challenges and opportunities for future drug discovery are also discussed.Key words: allosteric modulators, bitopic ligands, computational approaches, drug discovery, drug target discovery, G protein-coupled receptors     

Structural bioinformatic approaches to the discovery of new antimycobacterial drugs

Integrated bioinformatic approaches to drug discovery exploit computational techniques to examine the flow of information from genome to structure to function. Informatics is being be used to accelerate and rationalize the process of antimycobacterial drug discovery and design, with the immediate goals to identify viable drug targets and produce a set of critically evaluated protein target models and corresponding set of probable lead compounds. Bioinformatic approaches are being successfully applied in the selection and prioritization of putative mycobacterial drug target genes; computational modelling and x-ray structure validation of protein targets with drug lead compounds; simulated docking and virtual screening of potential lead compounds; and lead validation and optimization using structure-activity and structure-function relationships. By identifying active sites, characterizing patterns of conserved residues and, where relevant, predicting catalytic residues, bioinformatics provides information to aid the design of selective and efficacious pharmacophores. In this review, we describe selected recent progress in antimycobacterial drug design, illustrating the strengths and limitations of current structural bioinformatic approaches as tools in the fight against tuberculosis.     

Affinity-based screening techniques for enhancing lead discovery

Contemporary, rational small-molecule lead discovery methods, comprising target identification, assay development, high-throughput screening (HTS), hit characterization and medicinal chemistry optimization, dominate early-stage drug discovery strategies in many pharmaceutical companies. There is a growing disparity between the increasing cost of funding these methods and the decreasing number of new drugs reaching the market. New strategies must be adopted to reverse this trend. The use of genomics- and proteomics-based target discovery efforts can aid the process by dramatically increasing the number of novel, more highly validated targets entering the discovery process, but HTS must meet this increased demand with faster, cheaper technologies. Although activity-based screening strategies are typically efficient, allowing one scientist to interrogate tens of thousands of compounds per day, affinity-based screening strategies can allow much greater efficiency in the overall process. Affinity-based methods can play a role in both facilitating the screening of a greater number of targets and in efficiently characterizing the primary hits discovered.     

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality. Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery. What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis. Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.     

HTS in the new millennium: the role of pharmacology and flexibility

Over the past decade, high throughput screening (HTS) has become the focal point for discovery programs within the pharmaceutical industry. The role of this discipline has been and remains the rapid and efficient identification of lead chemical matter within chemical libraries for therapeutics development. Recent advances in molecular and computational biology, i.e., genomic sequencing and bioinformatics, have resulted in the announcement of publication of the first draft of the human genome. While much work remains before a complete and accurate genomic map will be available, there can be no doubt that the number of potential therapeutic intervention points will increase dramatically, thereby increasing the workload of early discovery groups. One current drug discovery paradigm integrates genomics, protein biosciences and HTS in establishing what the authors refer to as the "gene-to-screen" process. Adoption of the "gene-to-screen" paradigm results in a dramatic increase in the efficiency of the process of converting a novel gene coding for a putative enzymatic or receptor function into a robust and pharmacologically relevant high throughput screen. This article details aspects of the identification of lead chemical matter from HTS. Topics discussed include portfolio composition (molecular targets amenable to small molecule drug discovery), screening file content, assay formats and plating densities, and the impact of instrumentation on the ability of HTS to identify lead chemical matter.     

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality.

Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery.

What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis.

Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.     

The role of structure based virtual screening in the early phase of drug discovery

Identification of a viable lead is a critical step in drug discovery. The qualities of the lead set the stage for subsequent efforts to ameliorate therapeutic efficacy through potency, selectivity, pharmacokinetics, toxicity and side effects. In a retrospective view of drug research the lead identification has been realised mainly by in vivo methodologies. However, limitations of in vivo models were found to be critical factors when analysing attrition rates that prompted research groups to introduce in vitro tests and rational approaches at the frontline of discovery programs. Virtual screening (VS) methods merge in vitro high-throughput (HTS) and rational approaches. The VS methods can be classified as ligand and structure based techniques. Structure based approaches depart from the structural information of the target to identify potential interactions between the ligands and the protein. The advantages and disadvantages and the applicability of the structure based virtual screening approaches constituted the main aim of my studies. The glycogen synthase kinase 3beta (GSK-3beta), the beta-secretase and the c-jun N-terminal kinase 3 (JNK-3) were selected as primary targets for virtual screening. The performance of virtual screens can only be validated in parallel with HTS, therefore a head to head comparative analysis was my next goal.     

In silico research in drug discovery

Target and lead discovery constitute the main components of today's early pharmaceutical research. The aim of target discovery is the identification and validation of suitable drug targets for therapeutic intervention, whereas lead discovery identifies novel chemical molecules that act on those targets. With the near completion of the human genome sequencing, bioinformatics has established itself as an essential tool in target discovery and the in silico analysis of gene expression and gene function are now an integral part of it, facilitating the selection of the most relevant targets for a disease under study. In lead discovery, advances in chemoinformatics have led to the design of compound libraries in silico that can be screened virtually. Moreover, computational methods are being developed to predict the drug-likeness of compounds. Thus, drug discovery is already on the road towards electronic R&D.     

Chemoproteomics-driven drug discovery: addressing high attrition rates

The advent of multiple high-throughput technologies has brought drug discovery round almost full circle, from pharmacological testing of compounds in vivo to engineered molecular target assays and back to integrated phenotypic screens in cells and organisms. In the past, primary screens to identify new pharmacological agents involved administering compounds to an animal and monitoring a pharmacologic endpoint. For example, antihypertensive agents were identified by dosing spontaneously hypertensive rats with compounds and observing whether their blood pressure dropped. In taking this phenomenological approach, scientists were focused on the final goal, in this example lowering of blood pressure, rather than developing an understanding of the target, or targets, the compounds were impacting. With the evolution of rational target-based approaches, scientists were able to study the direct interaction of compounds with their intended targets, expecting that this would lead to more-selective and safer therapeutics. With the industrialization of screening, referred to as HTS, hundreds of thousands of compounds were screened in robot-driven assays against targets of interest (with this goal in mind). However, an unintentional outcome of the migration from in vivo primary screens to highly target-specific HTS assays was a reduction in biological context caused by the separation of the target from other cellular proteins and processes that might impact its function. Recognition of the potential consequences of this over-simplification drove the modification of HTS processes and equipment to be compatible with cellular assays.     

Phage display for target-based antibacterial drug discovery

Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as ‘surrogate ligands’ in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.     

Microbial genomics - new targets,new drugs

Genomics has changed our view of the biological world in the past decade, providing both new information and new tools to characterise biological systems. Over 100 microbial genomes - including many of substantial clinical importance - have been fully or partially sequenced, pushing the search for novel antimicrobial compounds into the post-genomic era. Genomic information and associated new technologies have the potential to revolutionise the drug discovery process. Genomic methods have created a wealth of potential new antimicrobial targets; strategies are evolving to provide validation for these targets before chemical inhibitors are identified. The ability to obtain large amounts of purified target proteins and advances in X-ray crystallography have caused significant increases in available protein structures, which may foreshadow an increased effort in structure-based drug design. The post-genomics strategies used in antimicrobial drug discovery may have application for small molecule drug discovery in numerous therapeutic areas.     

Microbial genomics – new targets,new drugs

Genomics has changed our view of the biological world in the past decade, providing both new information and new tools to characterise biological systems. Over 100 microbial genomes – including many of substantial clinical importance – have been fully or partially sequenced, pushing the search for novel antimicrobial compounds into the post-genomic era. Genomic information and associated new technologies have the potential to revolutionise the drug discovery process. Genomic methods have created a wealth of potential new antimicrobial targets; strategies are evolving to provide validation for these targets before chemical inhibitors are identified. The ability to obtain large amounts of purified target proteins and advances in X-ray crystallography have caused significant increases in available protein structures, which may foreshadow an increased effort in structure-based drug design. The post-genomics strategies used in antimicrobial drug discovery may have application for small molecule drug discovery in numerous therapeutic areas.     

Application of proteomic technologies in the drug development process

Proteins are the principal targets of drug discovery. Most large pharmaceutical companies now have a proteomics-oriented biotech or academic partner or have started their own proteomics division. Common applications of proteomics in the drug industry include target identification and validation, identification of efficacy and toxicity biomarkers from readily accessible biological fluids, and investigations into mechanisms of drug action or toxicity. Target identification and validation involves identifying proteins whose expression levels or activities change in disease states. These proteins may serve as potential therapeutic targets or may be used to classify patients for clinical trials. Proteomics technologies may also help identify protein-protein interactions that influence either the disease state or the proposed therapy. Efficacy biomarkers are used to assess whether target modulation has occurred. They are used for the characterization of disease models and to assess the effects and mechanism of action of lead candidates in animal models. Toxicity (safety) biomarkers are used to screen compounds in pre-clinical studies for target organ toxicities as well as later on in development during clinical trials. Complementary approaches such as metabolomics and genomics can be used in conjunction with proteomics throughout the drug development process to create more of a unified, systems biology approach.     

Direct methods for modulating protein function

During the past few years, the drug discovery process has shifted from a chemistry- to a biology-driven paradigm. Genome sciences have made a significant contribution to this shift, leading to a plethora of potential drug targets that are mainly proteins. Genetic methods will continue to be used to characterize proteins, but more direct methods are needed to determine the suitability of these protein targets for pharmacological intervention. In addition to the use of antibodies and aptamers, technologies focusing on the direct modulation of protein activity, including chemical genetics, analog-sensitive enzyme alleles and chromophore-assisted laser inactivation, should significantly contribute to the field of post-genomic drug discovery and development.