RNAi: for functional analysis and target validation

The third annual conference on discovery on target, organised by the Cambridge Healthtech Institute was held on 19 - 20 October 2005, in Boston. More than 300 delegates from both academic and industrial institutes attended the meeting. The presentations provided insights into understanding the RNA interference technology as a useful tool to identify and validate new targets for therapeutic intervention. Discussions focused in the design of siRNA for effective gene silencing, RNAi screens to identify new targets, RNAi delivery and the in vivo validation of targets using this technology.     

RNAi: for functional analysis and target validation

The third annual conference on discovery on target, organised by the Cambridge Healthtech Institute was held on 19 – 20 October 2005, in Boston. More than 300 delegates from both academic and industrial institutes attended the meeting. The presentations provided insights into understanding the RNA interference technology as a useful tool to identify and validate new targets for therapeutic intervention. Discussions focused in the design of siRNA for effective gene silencing, RNAi screens to identify new targets, RNAi delivery and the in vivo validation of targets using this technology.     

Utilizing RNA interference to enhance cancer drug discovery

With the development of RNA interference (RNAi) libraries, systematic and cost-effective genome-wide loss-of-function screens can now be carried out with the aim of assessing the role of specific genes in neoplastic phenotypes, and the rapid identification of novel drug targets. Here, we discuss the existing applications of RNAi in cancer drug discovery and highlight areas in this process that may benefit from this technology in the future.     

Vector-based RNAi approaches for stable, inducible and genome-wide screens

RNA interference (RNAi) has revolutionized the study of biology and offers numerous applications in basic biology as well as in drug discovery research. Since the discovery of RNAi, several tools have been developed to enable loss-of-function studies in mammalian systems. The efficacy of RNAi is dependent on specific and versatile RNAi triggers that have evolved to enable transient, stable and in-vivo applications. Recently developed genome-wide short hairpin RNA (shRNA) and microRNA-adapted short hairpin RNA (shRNAmir) libraries incorporate advances in shRNA design and molecular 'barcodes' to enable more complex RNAi screens and the opportunity to progress to more complex genetics in whole animals.     

High-throughput analysis of an RNAi library identifies novel kinase targets in Trypanosoma brucei

New drugs are needed to treat human African trypanosomiasis because the currently approved treatments are toxic or limited in efficacy. One strategy for developing new drugs involves discovering novel genes whose products can be targeted for modulation by small‐molecule chemotherapeutic agents. The Trypanosoma brucei genome contains many genes with the potential to become such targets. Kinases represent one group of genes that regulate many important cell functions and can be modulated by small molecules, thus representing a promising group of enzymes to screen for potential therapeutic targets. RNAi screens could help identify the most promising kinase targets, but the lack of suitable assays represents a barrier for optimizing the use of this technology in T. brucei. Here, we describe an RNAi screen of a small RNAi library targeting 30 members of the T. brucei kinome utilizing a luciferase‐based assay. This screen both validated the luciferase‐based assay as a suitable method for conducting RNAi screens in T. brucei and also identified two kinases (CRK12 and ERK8) that are essential for normal proliferation by the parasite.     

Random mutagenesis in the mouse as a tool in drug discovery

The flood of raw information generated by large-scale data acquisition technologies in genomics, microarrays and proteomics is changing the early stages of the drug discovery process. Although many more potential drug targets are now available compared with the pre-genomics era, knowledge about the physiological context in which these targets act--information crucial to both discovery and development--is scarce. Random mutagenesis strategies in the mouse provide scalable approaches for both the gene-driven validation of candidate targets in vivo and the discovery of new physiological pathways by phenotype-driven screens.     

Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens

RNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.     

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.     

RNA interference and potential applications

RNA interference (RNAi) is the process of using specific sequences of double-stranded RNA (dsRNA) to knock down the expression level of sequence-homologous genes. Such ability of small interfering RNA (siRNA) in mammalian cells will undoubtedly revolutionize the study of functional genomics, the discovery of drug targets and even the treatment of human diseases. In this review we briefly describe the history of RNAi discovery, the RNAi mechanism and the general guideline for siRNA design as well as various methods for siRNA production and delivery. We also introduce the potential applications of siRNA, inducible siRNA and siRNA library in speeding up basic biomedical research and in acting as potential therapeutic agents for treatment of numerous human diseases.     

Antisense and RNAi: powerful tools in drug target discovery and validation

Drug target discovery and validation are complex processes that require significant resource investments and impose a substantial economic burden on the pharmaceutical industry. Technologies that accelerate or enhance the precision of target selection are, therefore, in high demand. Traditional antisense and RNA interference (RNAi) technologies are powerful tools with applications in multiple phases of drug target discovery and validation. These approaches elicit potent and highly selective cleavage of a target mRNA, permitting evaluation of the role of the corresponding protein based on a loss-of-function phenotype. Incorporation of these technologies into high-throughput screens, in vitro biological assays and in vivo disease models provides valuable insight into gene function. Efforts are also underway to develop these agents as drugs. This review presents recent studies involving antisense and RNAi, and discusses how these technologies are facilitating target selection at various stages of the drug development process.     

RNAi screening for therapeutic targets in human malignancies

The advent of RNA interference (RNAi) based library screening approaches has sparked a surge in loss-of-function genetic screens. Several recent screens have aimed to identify novel regulators of cancer-related phenotypes. These employ various tumor cell types to model malignant cell functions and use different RNAi effector library approaches to reveal a cache of novel tumor regulators. This review surveys recent RNAi screens conducted in transformed human cells.     

RNA interference as a novel and powerful tool in immunopharmacological research

RNA interference (RNAi), as an evolutionarily conserved mechanism for silencing gene expression, is realized through the actions of both small interference RNA (siRNA) and microRNA. Since its discovery, siRNA has been rapidly deployed not only for the elucidation of gene function, but also for identification of drug targets and as a powerful therapeutic approach for a variety of diseases. In this review, we briefly introduce the mechanisms of RNAi, methods of siRNA design and delivery, and summarized recent researches on the therapeutic potential of RNAi for immune diseases.     

Human protein-protein interaction networks and the value for drug discovery

Systematic genome-wide and pathway-specific protein-protein interaction screens have generated a putative, organizing framework of the spatial interconnectivity of a large number of human proteins, including numerous therapeutically relevant disease-associated proteins. The intrinsic value for drug discovery is that these physical protein-protein interaction networks may contribute to a mechanistic understanding of the pathophysiology of disease and can aid in the identification and prioritization of tractable targets and generate hypotheses on how to best drug non-tractable, disease-associated targets. Here, we review the 'therapeutic potential' of the 1st generation sub-genome-scale human interaction networks and disease-associated protein networks generated by yeast two-hybrid screens and affinity purification-mass spectrometry approaches.     

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.     

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.     

RNA interference for viral infections

The treatment of viral infections has relied on pre-emptive vaccination or use of a limited range of anti-viral drugs. However, the majority of viruses have no available drugs and treatment is merely supportive. RNA interference (RNAi) offers the ability to directly and rapidly treat virus infections via the targeting of viral genes. Indeed, clinical trials have already been undertaken with promising results. Here we review the current state of the RNAi field for the treatment of viral infections such as HIV, human papillomavirus and HCV. We also review novel strategies including the concept of targeting self-genes to limit viral infection and activating the immune system for improved outcomes. Finally we examine innovative approaches being pursued at the Australian Infectious Diseases Research Centre including the use of highthroughput siRNA screens to identify new antiviral targets.     

Expanding the repertoire of RNA interference screens for developing new anticancer drug targets

RNA interference (RNAi) screening for cancer drug target identification has been growing, in both the number of laboratories carrying out screens and in the scale of the screens themselves, from the first screens that were published a few years ago. This growth is directly related to the significant new insights into cancer cell biology that have been defined by relatively few studies. Recently, such screens have moved from general studies of cancer cell function (finding new mechanisms of malignancy and tumor suppression), to screens that explain the clinical problems, such as resistance to chemotherapeutics. In light of the progression observed in these published studies, it is now possible to consider how RNAi screening can be used to characterize other areas of cancer research that have been proposed to explain the development of clinical cancers. Examples include: oncogene addiction/oncogenic shock, cancer stem cells, lineage dependency and the epithelial-mesenchymal transition. RNAi screening can enable critical evaluations of both the roles of these concepts in tumor development and provide starting points for new therapeutic programs targeting emerging areas of cancer cell biology.     

The zebrafish: a powerful platform for in vivo, HTS drug discovery

The zebrafish (Danio rerio) is an emerging vertebrate model for drug discovery that permits whole animal drug screens with excellent throughput, combined with ease of use and low cost. This review will begin with a discussion on the background, suitability, and advantages of this vertebrate model system and then, citing specific examples, will describe the utility of zebrafish at specific stages in the drug development pipeline. We will end with a synopsis of recent drug screens based on morphological disruptions, genetic disease models, fluorescent markers, behavioral changes, and specific targets. The numerous advantages of this whole animal approach provide new promise for the discovery of safe, specific, and powerful new drugs.     

Expanding the repertoire of RNA interference screens for developing new anticancer drug targets

RNA interference (RNAi) screening for cancer drug target identification has been growing, in both the number of laboratories carrying out screens and in the scale of the screens themselves, from the first screens that were published a few years ago. This growth is directly related to the significant new insights into cancer cell biology that have been defined by relatively few studies. Recently, such screens have moved from general studies of cancer cell function (finding new mechanisms of malignancy and tumor suppression), to screens that explain the clinical problems, such as resistance to chemotherapeutics. In light of the progression observed in these published studies, it is now possible to consider how RNAi screening can be used to characterize other areas of cancer research that have been proposed to explain the development of clinical cancers. Examples include: oncogene addiction/oncogenic shock, cancer stem cells, lineage dependency and the epithelial–mesenchymal transition. RNAi screening can enable critical evaluations of both the roles of these concepts in tumor development and provide starting points for new therapeutic programs targeting emerging areas of cancer cell biology.