Screening methods for influenza antiviral drug discovery

Introduction: Influenza antiviral high-throughput screens have been extensive, and yet no approved influenza antivirals have been identified through high-throughput screening. This underscores the idea that development of successful screens should focus on the exploitation of the underrepresented viral targets and novel, therapeutic host targets.

Areas covered: The authors review conventional screening applications and emerging technologies with the potential to enhance influenza antiviral discovery. Real-world examples from the authors' work in biocontained environments are also provided. Future innovations are discussed, including the use of targeted libraries, multiplexed assays, proximity-based endpoint methods, non-laboratory-adapted virus strains, and primary cells, for immediate physiological relevance and translational applications.

Expert opinion: The lack of successful anti-influenza drug discovery using high-throughput screening should not deter future efforts. Increased understanding of the functions of viral targets and host–pathogen interactions has broadened the target reservoir. Future screening efforts should focus on identifying new drugs against unexploited viral and host targets using currently developed assays, and on the development of novel, innovative assays to discover new drugs with novel mechanisms. Innovative screens must be designed to identify compounds that specifically inhibit protein–protein or protein–RNA interactions or other virus/host factor interactions that are crucial for viral replication. Finally, the use of recent viral isolates, increased biocontainment (for highly-pathogenic strains), primary cell lines, and targeted compound libraries must converge in efficient high-throughput primary screens to generate high-content, physiologically-relevant data on compounds with robust antiviral activity.     

Recent advances in pharmacophore modeling and its application to anti-influenza drug discovery

Introduction: The emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus and the recent global circulation of H1N1 swine-origin influenza virus in 2009 have highlighted the need for new anti-influenza therapies. This has been made all the more important with the emergence of antiviral-resistant strains. Recent progress in achieving three-dimensional (3D) crystal structures of influenza viral proteins and efficient tools available for pharmacophore-based virtual screening are aiding us in the discovery and design of new antiviral compounds.

Areas covered: This review discusses pharmacophore modeling as a potential cost-effective and time-saving technology for new drug discovery as an alternative to high-throughput screening. Based on this technical platform, the authors discuss current progress and future prospects for developing novel influenza antivirals against pre-existing or emerging novel targets.

Expert opinion: Although it might be at an infant stage of development, the availability of the 3D crystal structures of influenza viral proteins is expected to accelerate the application of structure-based drug design (SBDD) and pharmacophore modeling. Furthermore, the neuraminidase inhibitor, one of the most successful examples of a SBDD, still receives great attention because of its superb antiviral activities and the resistance of influenza strains to oseltamivir. However, despite much success, pharmacophore-based virtual screening exhibits limited predictive power in hit identification. Further improvements in pharmacophore detection algorithms, proper combinations of in silico methods as well as judicious choosing of compounds are expected to improve the hit rate. With the help of these technologies, the discovery of anti-influenza agents will be accelerated.     

Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors

The identification of novel HIV-1 inhibitors is facilitated by screening campaigns that combine the right screening strategy with a large diverse collection of drug-like compounds. Cell-based screening approaches offer some advantages in the quest for novel inhibitors because they can include multiple targets in a single screen and in some cases reveal targets and/or structures not captured in biochemical assays. However, follow-up activities for cell-based screens are often more complicated and resource intensive when compared to biochemical screens. Alternatively, biochemical screens usually offer the advantage of focusing on a single target with a well-defined set of follow-up assays. In this review we cover multiple cell-based and biochemical assay formats, many of which were designed to identify inhibitors that act through new mechanisms. Some of the assays discussed have been utilized in antiviral screens while others might be formatted for HTS or utilized as secondary assays in a screening campaign. As drug discovery efforts in the pharmaceutical industry shift away from traditional strategies, new approaches such as those presented here are likely to play a significant role in the identification of next generation HIV-1 inhibitors.     

A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals

The spread of highly pathogenic avian influenza across geographical and species barriers underscores the increasing need for novel antivirals to compliment vaccination and existing antiviral therapies. Identification of new antiviral lead compounds depends on robust primary assays for high-throughput screening (HTS) of large compound libraries. We have developed a cell-based screen for potential influenza antivirals that measures the cytopathic effect (CPE) induced by influenza virus (A/Udorn/72, H3N2) infection in Madin Darby canine kidney (MDCK) cells using the luminescent-based CellTiter Glo system. This 72 h assay is validated for HTS in 384-well plates and performs more consistently and reliably than methods using neutral red, with Z values>0.8, signal-to-background>30 and signal-to-noise>10. In a blinded pilot screen (n=10,781) at 10 microM concentration, four compounds (with previously demonstrated efficacy against influenza) inhibited viral-induced CPE by >50%, with EC50/CC50 values comparable to those determined by other cell-based assays, thereby validating this assay accuracy and ability to simultaneously evaluate compound cellular availability and/or toxicity. This assay is translatable for screening against other influenza strains, such as avian flu, and may facilitate identification of antivirals for other viruses that induce CPE, such as West Nile or Dengue.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Novel targets for antibiotics

The rapid and extensive emergence of antibiotic resistant bacteria has resulted in a clear cut need to discover new antibiotics. Because of the many years of extensive screening, it is likely that most of the easy discoveries have been made and, therefore, new targets for antibiotics and new screening strategies for their discovery need to be developed. The approaches described in this overview are divided into several categories that are associated with different probabilities for a successful discovery. Approaches that are more likely to be successful include a continuation of classical discovery tactics including the chemical modification of extant structures, the use of new screens for classical targets (for example, the use of the enzyme DNA gyrase to discover new 4-fluoroquinolones), and the development of novel methods of drug delivery. These approaches, however, are likely to yield small incremental advances. More novel approaches should yield radically new chemical structures, however, the likelihood for a successful discovery will be lower than the classical approaches. The novel approaches include rational drug design, the discovery of new essential targets for antibiotics and using them for the purpose of drug screening, and the intervention in pathways necessary for pathogenesis. A middle of the road approach is to discover new agents that interfere with mechanisms of antibiotic resistance. Implicit in this overview is the need to develop new methods that result in real technologic advances. This may require a complete re-thinking of how antibiotics are discovered including the restricted use of live microbe killing assays as a primary screening tool.     

Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.     

Discovery of modulators of protein-protein interactions: current approaches and limitations

Protein-protein interactions are involved in most of the essential processes that occur in living organisms from cell motility to DNA replication, which makes them interesting targets for drug discovery. However, due to the lack of deep pockets, and the large contact surfaces involved in these interactions, they are considered challenging targets and have been often times dismissed as "undruggable". Nonetheless, significant efforts in pharmaceutical and academic laboratories have been devoted to finding ways to exploit protein-protein interactions as drug targets. This article provides an overview of the principles underlying the main general strategies for discovering small-molecule modulators of protein-protein interactions, namely: high-throughput screening, fragment-based drug discovery, peptide-based drug discovery, protein secondary structure mimetics, and computer-aided drug discovery. In addition, examples of successful discovery of modulators of protein-protein interactions are discussed for each of those strategies.     

Application of NMR screening in drug discovery

The application of NMR screening in drug discovery has recently attained heightened importance throughout the pharmaceutical industry. NMR screening can be applied at various points in a drug discovery program, ranging from very early in the program, when new targets can be screened long before an HTS enzymatic assay is developed, to later in the program, as in the case where no useful hits have been detected by HTS using biological assays. The binders determined in primary NMR screens are used to guide secondary screens, which can be either completely NMR driven or use NMR in combination with other biophysical techniques. In this review we briefly discuss the methods and techniques used in NMR screening. Then, we describe in detail the NMR screening strategies and their applications to specific targets, including successful examples from actual drug design programs at our own and other pharmaceutical companies.     

Comprehensive essential gene identification as a platform for novel anti-infective drug discovery

In large part, antimicrobial drug discovery is driven by the breadth and quality of both potential drug targets and available chemical libraries to screen. Traditionally, targets have been few in number and have been limited to those with known function, from which biochemical assays could be implemented into drug screens. Iterations of this same basic approach, applied to a few biochemically-defined targets have identified a limited set of novel antibiotics and even fewer antifungal agents. Indeed, in the last 50 years less than 30 antimicrobial targets have been exploited commercially. Within infectious disease, the industry was driven largely by chemistry-based approaches, simply making new analogs to existing drugs to overcome the growing problem of drug resistance. Elitra Pharmaceutical s approach has been to enable true functional genomics on a genome-wide scale. Elitra s vision has been to identify all of the essential genes directly in the key pathogenic organisms. Having moved rapidly towards the completion of this goal, we are now faced with the enviable challenge of prioritizing enormous target sets and developing novel sensitive screens for those best suited as definitive drug targets. These highly sensitive, cell-based screening paradigms enable re-screening of even well screened chemical libraries to reveal new chemical entities displaying novel modes of action against new targets. In parallel, we have also begun to shift the paradigm from screening targets singly, towards genome-wide approaches to drug screening.     

High-throughput screening and small animal models,where are we?

Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.     

Novel antiviral drug discovery strategies to tackle drug-resistant mutants of influenza virus strains

Introduction: The emergence of drug-resistant influenza virus strains highlights the need for new antiviral therapeutics to combat future pandemic outbreaks as well as continuing seasonal cycles of influenza.

Areas covered: This review summarizes the mechanisms of current FDA-approved anti-influenza drugs and patterns of resistance to those drugs. It also discusses potential novel targets for broad-spectrum antiviral drugs and recent progress in novel drug design to overcome drug resistance in influenza.

Expert opinion: Using the available structural information about drug-binding pockets, research is currently underway to identify molecular interactions that can be exploited to generate new antiviral drugs. Despite continued efforts, antivirals targeting viral surface proteins like HA, NA, and M2, are all susceptible to developing resistance. Structural information on the internal viral polymerase complex (PB1, PB2, and PA) provides a new avenue for influenza drug discovery. Host factors, either at the initial step of viral infection or at the later step of nuclear trafficking of viral RNP complex, are being actively pursued to generate novel drugs with new modes of action, without resulting in drug resistance.     

Advantages of voltage-gated ion channels as drug targets

Many human diseases result from over- or underactivity in one or more critical physiologic systems. One of the foremost challenges in modern drug discovery is the identification and selection of cellular proteins that can be specifically targeted with therapeutic agents in order to normalize aberrant processes/systems. Suitable drug targets must be validated in the human disease state and ideally, the targeted protein will fulfill similar physiologic and pathologic functions in humans and at least one animal species so that in vivo efficacy and toxicology assays with some predictive clinical relevance may be developed. Nowadays, drug targets must also be amenable to high-throughput screening so that novel molecules, which are capable of modifying cellular protein function, can be identified in large libraries of compounds. Voltage-gated ion channels satisfy many of these requirements and, as a class, are viewed as promising drug targets. Nevertheless, despite their relevance to human disease, voltage-gated ion channels remain considerably underexploited. Therein lie some of the opportunities and advantages associated with voltage-gated ion channels as drug targets.     

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

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The use of Xenopus oocytes in drug screening

Introduction: The physiological roles of ion channels are receiving increased interest in both basic research and drug discovery, and a demand for pharmacological approaches that can characterize or screen ion channels and their ligands with higher throughput has emerged. Traditionally, screening of compound libraries at ion channel targets has been performed using assays such as binding assays, fluorescence-based assays and flux assays that allow high-throughput, but sacrifice high data quality. The use of these assays with ion channel targets can also be problematic, emphasizing the usefulness of automated Xenopus oocyte electrophysiological assays in drug screening. Areas covered: This review summarizes the use of Xenopus oocytes in drug screening, presents the advantages and disadvantages of the use of Xenopus oocytes as expression system, and addresses the options available for automated two-electrode voltage-clamp recordings from Xenopus oocytes. Expert opinion: Automated and manual Xenopus oocyte two-electrode voltage-clamp recordings are useful and important techniques in drug screening. Although they are not compatible with high-throughput experimentation, these techniques are excellent in combination or as alternatives to fluorescence-based assays for hit validation, screening of focused compound libraries and safety screening on ion channels with their high flexibility for the choice of molecular targets, quality of data and reproducibility.     

Type II transmembrane serine proteases as potential target for anti-influenza drug discovery

Introduction: The outbreak of an influenza pandemic as well as the continued circulation of seasonal influenza highlights the need for effective antiviral therapies. The emergence of drug-resistant strains further necessitates the development of novel antivirals that target the host factors crucial for viral replication.

Area covered: This review summarizes the current understanding of the structural and functional properties of type II transmembrane serine proteases (TTSPs) as a proteolytic activator of influenza virus infection and discusses their potential as antiviral targets. It also explores the experimental evidence accumulated for inhibitors of TTSPs as novel, broad-spectrum antivirals against various influenza virus subtypes. The review also provides an overview of the properties of small molecules, proteins, and peptides that efficiently inhibit the proteolytic activation of the influenza virus.

Expert opinion: TTSPs activate a wide range of influenza virus subtypes including avian influenza viruses, both in vitro and in vivo, via proteolytic cleavage of influenza hemagglutinin (HA) into infection-competent fusogenic conformation. Other viruses such as SARS-, MERS-coronaviruses and human metapneumoviruses may use the same host cell proteases for activation, implying that TTSP inhibition might be a novel strategy for developing broad-spectrum antiviral agents for respiratory viral infections.     

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.     

Protein–protein interaction modulator drug discovery: past efforts and future opportunities using a rich source of low- and high-throughput screening assays

Introduction: Historically, small-molecule drug discovery projects have largely focused on the G-protein-coupled receptor, ion-channel and enzyme target classes. More recently, there have been successes demonstrating that protein–protein interactions (PPIs) can be targeted by small-molecules and that this strategy has the potential to provide appropriate specificity and selectivity. However, a disadvantage is that compounds that modulate PPIs are often associated with relatively weak affinities as the targeted interaction surfaces are often relatively large. Moreover, from a small-molecule screening perspective, a large proportion of the initial screening Hits are often false positives and these need to be identified and excluded in order to focus on genuine modulators of the PPI being investigated.

Areas covered: The authors review previous efforts on PPI modulator drug discovery. Furthermore, they review assays that can be employed in small-molecule screening and/or Hit validation. The PPI assays are categorized as: i) low-throughput target-based biochemical assays, which are primarily employed for Hit validation at the post-screening stage; ii) high-throughput target-based biochemical assays that are suitable for screening campaigns; and iii) cell-based assays, which are suitable for high-throughput screening campaigns and/or Hit validation.

Expert opinion: Modulating the interaction of PPIs offers the potential to develop novel drugs to treat a wide range of diseases. New assay technologies are continually being developed and it is anticipated that these will be able to be directly used for small-molecule screening campaigns in the future.