RNA interference: an emerging generation of biologicals

RNA interference (RNAi) is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. RNAi has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. RNAi was first described in animal cells by Fire and colleagues in the nematode, Caenorhabditis elegans. Knowledge of RNAi mechanism in mammalian cell in 2001 brought a storm in the field of drug discovery. During the past few years scientists all over the world are focusing on exploiting the therapeutic potential of RNAi for identifying a new class of therapeutics. The applications of RNAi in medicine are unlimited because all cells possess RNAi machinery and hence all genes can be potential targets for therapy. RNAi can be developed as an endogenous host defense mechanism against many infections and diseases. Several studies have demonstrated therapeutic benefits of small interfering RNAs and micro RNAs in animal models. This has led to the rapid advancement of the technique from research discovery to clinical trials.     

Unveiling the druggable RNA targets and small molecule therapeutics

The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets.     

AlphaFold2 protein structure prediction: Implications for drug discovery

The drug discovery process involves designing compounds to selectively interact with their targets. The majority of therapeutic targets for low molecular weight (small molecule) drugs are proteins. The outstanding accuracy with which recent artificial intelligence methods compile the three-dimensional structure of proteins has made protein targets more accessible to the drug design process. Here, we present our perspective of the significance of accurate protein structure prediction on various stages of the small molecule drug discovery life cycle focusing on current capabilities and assessing how further evolution of such predictive procedures can have a more decisive impact in the discovery of new medicines.     

Ribozymes: A modern tool in medicine

Since the discovery of ribozymes and self-splicing introns, it has been estimated that this biological property of RNA combined with other recombinant DNA technologies would become a tool to combat viral diseases and control oncogenes. These goals seem like a distinct possibility now. However, there is still a lot to be learned about the mobility of RNA inside the cells and the cellular factors that can impede ribozyme action in order to capitalize fully on the targeted RNA inactivation property of ribozymes. The most effective approach to maximize ribozyme function in a complex intracellular environment is to understand as much as possible about the intracellular fate of the RNA that is being targeted. As new techniques in cell biology become available, such understanding will be less problematic. Fundamental studies of ribozyme structure and mechanism of catalysis are flourishing both at the academic and industrial level and it can be expected that many new developments will continue to take place in these areas in the near future. Here, we review the design, stability and therapeutic application of these technologies illustrating relevant gene targets and applications in molecular medicine. Relevant problems in implementation of the technology, group I and II introns and the differences in applications, ribozyme structure and the application of this technology to virus attack and oncogene downregulation are discussed. Also some of the latest RNA-based technologies such as siRNA, RNA/DNA duplexes and RNA decoys have been introduced.     

Smart chemistry tackling complex biology

The pathway of drug discovery from gene and therapeutic targets to breakthrough medicines provides an extraordinary adventure that often includes high risks, creative insights, relentless perserverance, goal-oriented focus, interdisciplinary teamwork and a bit of good luck. From synthetic small molecules to natural products, peptides, peptidomimetics, proteins (including antibodies) and nucleic acids (RNA or DNA), there exists a plethora of molecular diversity that has resulted in the development of potent, selective, effective and safe-acting drugs to treat numerous diseases. Yet, despite the numerous success cases in this drug discovery adventure, do we really know what we're doing? Granted, powerful enabling technologies have been established to identify, validate and decipher therapeutic targets, and we have the human genome blueprint and the ability to unravel the human proteome systematically into discrete functional components of complex biological processes. Yet, as exquisite and well-defined as such biological processes may become, do we really know how to chemically design breakthrough medicines? Is it a game of probability (i.e. how many attempts it may take to hit the proverbial ‘bullseye’)? Perhaps this has been case. However, there is such a thing as ‘smart chemistry’, and it has a genuine beauty of its own.     

Bacillus brevis,a Host Bacterium for Efficient Extracellular Production of Useful Proteins

Abstract

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with “undruggable” therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture     

Minireview: The roles of small RNA pathways in reproductive medicine

The discovery of small noncoding RNA, including P-element-induced wimpy testis-interacting RNA, small interfering RNA, and microRNA, has energized research in reproductive medicine. In the two decades since the identification of small RNA, first in Caenorhabditis elegans and then in other animals, scientists in many disciplines have made significant progress in elucidating their biology. A powerful battery of tools, including knockout mice and small RNA mimics and antagonists, has facilitated investigation into the functional roles and therapeutic potential of these small RNA pathways. Current data indicate that small RNA play significant roles in normal development and physiology and pathological conditions of the reproductive tracts of females and males. Biologically plausible mRNA targets for these microRNA are aggressively being discovered. The next phase of research will focus on elucidating the clinical utility of small RNA-selective agonists and antagonists.     

Frontier of therapeutic antibody discovery:The challenges and how to face them

Therapeutic monoclonal antibodies have become an important class of modern medicines.The established technologies for therapeutic antibody discovery such as humanization of mouse antibodies,phage display of human antibody libraries and transgenic animals harboring human IgG genes have been practiced successfully so far,and many incremental improvements are being made constantly.These methodologies are responsible for currently marketed therapeutic antibodies and for the biopharma industry pipeline which are concentrated on only a few dozen targets.A key challenge for wider application of biotherapeutic approaches is the paucity of truly validated targets for biotherapeutic intervention.The efforts to expand the target space include taking the pathway approach to study the disease correlation.Since many new targets are multi-spanning and multimeric membrane proteins there is a need to develop more effective methods to generate antibodies against these difficult targets.The pharmaceutical properties of therapeutic antibodies are an active area for study concentrating on biophysical characteristics such as thermal stability and aggregation propensity.The immunogenicity of biotherapeutics in humans is a very complex issue and there are no truly predictive animal models to rely on.The in silico and T-cell response approaches identify the potential for immunogenicity;however,one needs contingency plans for emergence of antiproduct antibody response for clinical trials.     

A murine leukemia virus with Cre-LoxP excisible coding sequences allowing superinfection, transgene delivery, and generation of host genomic deletions

During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets.     

Diverse Ways to Control p27Kip1 Function: miRNAs Come into Play

microRNAs (miRNAs) are small RNA molecules controlling messenger RNA (mRNA) and protein abundance. Since their discovery, research has been aimed at identifying the functions and target genes of miRNAs. A number of computer algorithms have been developed capable of predicting putative targets for any given miRNA. However, they might predict many false-positive targets and on top of that some true targets could be missed. This reflects the incomplete knowledge we still have concerning the rules governing true and effective miRNA-mRNA interactions. To experimentally identify miRNA-target genes, we have recently developed a genetic approach and employed it on p27Kip1, a hapo-insufficient tumor suppressor and cell cycle inhibitor. Here we review the difficulties interpreting the data from available computer algorithms, and critically address the pros and cons of our genetic screening method. Additionally, we focus on the different ways in which p27 is managed, argue how miRNAs could be involved in the regulation of p27 in both normal and malignant conditions, and discuss possible use of this knowledge for cancer therapy.     

A Multidimensional Strategy to Detect Polypharmacological Targets in the Absence of Structural and Sequence Homology

Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.     

Specific RNA-Binding Antibodies with a Four-Amino-Acid Code

Numerous large non-coding RNAs are rapidly being discovered, and many of them have been shown to play vital roles in gene expression, gene regulation, and human diseases. Given their often structured nature, specific recognition with an antibody fragment becomes feasible and may help define the structure and function of these non-coding RNAs. As demonstrated for protein antigens, specific antibodies may aid in RNA crystal structure elucidation or the development of diagnostic tools and therapeutic drugs targeting disease-causing RNAs. Recent success and limitation of RNA antibody development has made it imperative to generate an effective antibody library specifically targeting RNA molecules. Adopting the reduced chemical diversity design and further restricting the interface diversity to tyrosines, serines, glycines, and arginines only, we have constructed a RNA-targeting Fab library. Phage display selection and downstream characterization showed that this library yielded high-affinity Fabs for all three RNA targets tested. Using a quantitative specificity assay, we found that these Fabs are highly specific, possibly due to the alternate codon design we used to avoid consecutive arginines in the Fab interface. In addition, the effectiveness of the minimal Fab library may challenge our view of the protein–RNA binding interface and provide a unique solution for future design of RNA-binding proteins.     

Yeast "N"-hybrid systems for protein-protein and drug-protein interaction discovery

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets.     

Ribozymes: recent advances in the development of RNA tools

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.     

Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.