Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.     

Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors

RNA plays a pivotal role in the replication of all organisms, including viral and bacterial pathogens. The development of small molecules that selectively interfere with undesired RNA activity is a promising new direction for drug design. Currently, there are no anti-HIV treatments that target nucleic acids. This article presents the HIV-1 Rev response element (RRE) as an important focus for the development of antiviral agents that target RNA. The Rev binding site on the RRE is highly conserved, even between different groups of HIV-1 isolates. Compounds that inhibit HIV replication by binding to the RRE and displacing Rev are therefore expected to retain activity across groups of genetically diverse HIV infections. Systematic evaluations of both the RRE affinity and specificity of numerous small molecule inhibitors are essential for deciphering the parameters that govern effective RRE recognition. This article discusses fluorescence-based techniques that are useful for probing a small molecule's RRE affinity and its ability to inhibit Rev-RRE binding. Rev displacement experiments can be conducted by observing the fluorescence anisotropy of a fluorescein-labeled Rev peptide, or by quantifying its displacement from a solid-phase immobilized RRE. Experiments conducted in the presence of competing nucleic acids are useful for evaluating the RRE specificity of Rev-RRE inhibitors. The discovery and characterization of new RRE ligands are described. Eilatin is a polycyclic aromatic heterocycle that has at least one binding site on the RRE (apparent Kd is approximately 0.13 microM), but it does not displace Rev upon binding the RRE (IC50 > 3 microM). In contrast, ethidium bromide and two eilatin-containing metal complexes show better consistency between their RRE affinity and their ability to displace a fluorescent Rev peptide from the RRE. These results highlight the importance of conducting orthogonal binding assays that establish both the RNA affinity of a small molecule and its ability to inhibit the function of the RNA target. Some Rev-RRE inhibitors, including ethidium bromide, Lambda-[Ru(bpy)(2)eilatin]2+, and Delta-[Ru(bpy)(2)eilatin]2+ also inhibit HIV-1 gene expression in cell cultures (IC50 = 0.2-3 microM). These (and similar) results should facilitate the future discovery and implementation of anti-HIV drugs that are targeted to viral RNA sites. In addition, a deeper general understanding of RNA-small molecule recognition will assist in the effective targeting of other therapeutically important RNA sites.     

Expanding the DNA-encoded library toolbox: identifying small molecules targeting RNA

DNA-encoded library (DEL) technology is a powerful tool for small molecule identification in drug discovery, yet the reported DEL selection strategies were applied primarily on protein targets in either purified form or in cellular context. To expand the application of this technology, we employed DEL selection on an RNA target HIV-1 TAR (trans-acting responsive region), but found that the majority of signals were resulted from false positive DNA–RNA binding. We thus developed an optimized selection strategy utilizing RNA patches and competitive elution to minimize unwanted DNA binding, followed by k-mer analysis and motif search to differentiate false positive signal. This optimized strategy resulted in a very clean background in a DEL selection against Escherichia coli FMN Riboswitch, and the enriched compounds were determined with double digit nanomolar binding affinity, as well as similar potency in functional FMN competition assay. These results demonstrated the feasibility of small molecule identification against RNA targets using DEL selection. The developed experimental and computational strategy provided a promising opportunity for RNA ligand screening and expanded the application of DEL selection to a much wider context in drug discovery.     

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ～23.1 μM).     

RNAi and RNAa--the yin and yang of RNAome

RNA interference (RNAi) is a powerful technology with huge applications for functional genomics, target identification in drug discovery and elucidation of molecular signaling pathways. Current RNAi studies have demonstrated the clinical potential of small interfering RNAs (siRNAs) in metabolic diseases, cancer, AIDS, malaria, neurodegenerative disorders, dental diseases and other illnesses. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs could effectively activate a particular gene in a sequence specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed as RNA activation (RNAa). The paradoxical concept of Yin and Yang, which describe two primal opposing but complementary principles, can potentially be applied to elucidate the complex phenomenon of RNAa/RNAi in the RNAome. This warrants a proper understanding of the RNAi/RNAa molecular pathways in living organisms before any of the small dsRNAs can potentially be exploited for therapeutics in human beings.     

RNA as a small molecule druggable target

Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents.     

Fragment-based lead discovery: a chemical update

Fragment-based lead discovery constructs drug leads from small molecular fragments. In theory, this is a highly efficient method for drug discovery, and the technique has become enormously popular in the past few years. In this review, I describe how a variety of approaches in fragment-based lead discovery--including NMR, X-ray crystallography, mass spectrometry, functional screening, and in silico screening--have produced drug leads. Although the examples show that the technique can reliably generate potent molecules, there is still much work to be done to maintain the efficiency of molecules' binding affinities as fragments are linked, expanded, and otherwise improved.     

Discovery of a novel small molecule binding site of human survivin

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.     

The use of calorimetry in the biophysical characterization of small molecule alkaloids binding to RNA structures

BackgroundRNA has now emerged as a potential target for therapeutic intervention. RNA targeted drug design requires detailed thermodynamic characterization that provides new insights into the interactions and this together with structural data, may be used in rational drug design. The use of calorimetry to characterize small molecule–RNA interactions has emerged as a reliable and sensitive tool after the recent advancements in biocalorimetry.Scope of the reviewThis review summarizes the recent advancements in thermodynamic characterization of small molecules, particularly some natural alkaloids binding to various RNA structures. Thermodynamic characterization provides information that can supplement structural data leading to more effective drug development protocols.Major conclusionsThis review provides a concise report on the use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques in characterizing small molecules, mostly alkaloids–RNA interactions with particular reference to binding of tRNA, single stranded RNA, double stranded RNA, poly(A), triplex RNA.General significanceIt is now apparent that a combination of structural and thermodynamic data is essential for rational design of specific RNA targeted drugs. Recent advancements in biocalorimetry instrumentation have led to detailed understanding of the thermodynamics of small molecules binding to various RNA structures paving the path for the development of many new natural and synthetic molecules as specific binders to various RNA structures. RNA targeted drug design, that remained unexplored, will immensely benefit from the calorimetric studies leading to the development of effective drugs for many diseases. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Engineering regulatory RNAs

RNA has long been a favoured medium for in vitro evolution and engineering. Functional RNAs produced in vitro can bind small molecules (aptamers), possess catalytic activity (ribozymes) or do both (aptazymes). A plethora of recent work has shown similar strategies used naturally for gene regulation in bacteria. Interest in these natural systems has inspired an effort to engineer and evolve this activity in vivo. A recent paper by Isaacs et al. describes the engineering and in vivo activity of a small RNA that removes translation inhibition by binding the 5' untranslated region of its target mRNA and making the ribosome-binding site accessible.     

miRNA，siRNA，saRNA与基因表达调控

近年来的研究发现,生物体内存在着大量的非编码RNA（non．codingRNAs,ncRNA）,它们在染色质修饰、基因转录、RNA剪接和mRNA翻译等多种水平上参与了基因表达的调控。ncRNA中的小分子RNA如miRNA能够识别特定的目标mRNA,通过与mRNAs3’非翻译区结合,影响mRNA转录及蛋白质翻译;siRNA是RNA干扰的引发物,能够导致与dsRNA同源的mRNA降解,进而抑制相应基因表达;saRNA是目前最新发现的一种靶向目的基因启动子区的在转录水平激活目的基因表达的dsRNA。miRNA、siRNA和saRNA在生成机制、作用途径等方面关系密切,既区别又相互联系,小分子RNA的研究将是今后分子生物学的研究热点之一。     

Aptamers as reagents for high-throughput screening

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.     

A high-throughput screening assay for the identification of flavivirus NS5 capping enzyme GTP-binding inhibitors: implications for antiviral drug development

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5' end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE-guanosine triphosphate (GTP) and CE-RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE-GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE-GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.     

Antisense technologies. Improvement through novel chemical modifications.

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti-mRNA strategies can be distinguished. Firstly, the use of single stranded antisense-oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side-effects. Some of these problems have been solved in 'second generation' nucleotides with alkyl modifications at the 2' position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA-cleaving ribozymes and deoxyribozymes, and the use of 21-mer double-stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.     

Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery.     

High-throughput siRNA-based functional target validation

The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.     

A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions

Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.     

功能RNA分子的构建、表达及其在基因功能鉴定中的应用

人工构建的siRNAs、aptazymes、maxizymes以及intramers等功能RNA分子,可以在mRNA或蛋白质水平上调控基因的功能.功能RNA分子可在活体内或转基因模式动、植物中抑制目标基因的表达,使目标基因和蛋白质功能丧失,进而引起表型变异.胞内表达的活性RNAs可作为有效的研究工具应用于基因及其编码蛋白的功能鉴定,并在药物开发和人类疾病治疗上有潜在的应用前景.     

Inhibition of the U1A-RNA complex by an aminoacridine derivative

The RNA recognition motif (RRM) is one of the most common RNA binding domains. There have been few investigations of small molecule inhibitors of RRM-RNA complexes, although these inhibitors could be valuable tools for probing biological processes involving RRM-RNA complexes and would have the potential to be effective drugs. In this paper, the inhibition by small molecules of the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA has been investigated. An aminoacridine derivative has been found to promote dissociation of the U1A-stem loop 2 RNA complex with an IC(50) value of 1 microM. Fluorescence experiments indicate that two aminoacridine ligands bind to each RNA target site. RNase A footprinting suggests that one binding site may be near the base pair that closes the loop and the other may be in a more flexible region of the loop. The addition of the aminoacridine derivative to stem loop 2 RNA increases the susceptibility of other portions of the loop to digestion by RNase A, which implies that binding of the ligand changes the conformation or dynamics of the stem loop target site. Either direct binding to the RNA or indirect alteration of the structure or dynamics of the loop would be likely to inhibit binding of the U1A protein to this RNA.