RESUMEN
When used as inhibitors of gene expression inâ vivo, oligonucleotides require modification of their structures to boost their binding affinity for complementary target RNAs. To date, hundreds of modifications have been designed and tested but few have proven to be useful. Among those investigated are mono- and polyamino-groups. These are positively charged at physiological pH and have been appended to oligonucleotides in an effort to reduce electrostatic repulsion during hybridization to RNAs, but have generally shown relatively minor benefits to binding. We conjugated spermine to uracils in oligonucleotides via a triazole linker so that the polyamine fits in the major groove of a subsequently formed RNA-duplex. The modifications produced large increases in target-binding affinity of the oligonucleotides. Using surface plasmon resonance-based assays, we showed that the increases derived mainly from faster annealing (kon ). We propose that the spermine fragments play a similar role to that of natural polyamines during oligonucleotide-target interactions in cells, and may be advantageous for oligonucleotides that operate catalytic mechanisms.
Asunto(s)
Oligonucleótidos/química , Secuencia de Bases , Línea Celular , Dicroismo Circular , Terapia Genética , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/metabolismo , Poliaminas/química , ARN/metabolismo , Espermina/química , Electricidad Estática , Resonancia por Plasmón de Superficie , Triazoles/química , Uracilo/análogos & derivados , Uracilo/químicaRESUMEN
The protein Lin28 and microRNA let-7 play critical roles in mammalian development and human disease. Lin28 inhibits let-7 biogenesis through direct interaction with let-7 precursors (pre-let-7). Accumulating evidence in vitro and in vivo suggests this interaction plays a dominant role in embryonic stem cell self-renewal and tumorigenesis. Thus the Lin28-let-7 interaction might be an attractive drug target, if not for the well-known difficulties in targeting protein-RNA interactions with drugs. The identification and development of suitable probe molecules to further elucidate therapeutic potential, as well as mechanistic details of this pathway will be valuable. We report the development and application of a biophysical high-throughput screening assay for the identification of small molecule inhibitors of the Lin28-pre-let-7 interaction. A library of pharmacologically active small molecules was screened and several small molecule inhibitors were identified and biochemically validated. Of these four validated inhibitors, two compounds successfully restored processing of pre-let-7g in the presence of Lin28, validating the concept. Thus, we have identified examples of small molecule inhibitors of the interaction between Lin28 and pre-let-7. This study provides a proof of concept for small molecule inhibitors that antagonise the effects of Lin28 and enhance processing of let-7 miRNA.
Asunto(s)
MicroARNs/metabolismo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Bases , Evaluación Preclínica de Medicamentos , Polarización de Fluorescencia , MicroARNs/genética , Precursores del ARN/genéticaRESUMEN
Recent progress with techniques for monitoring RNA structure in cells such as 'DMS-Seq' and 'Structure-Seq' suggests that a new era of RNA structure-function exploration is on the horizon. This will also include systematic investigation of the factors required for the structural integrity of RNA. In this context, much evidence accumulated over 50 years suggests that polyamines play important roles as modulators of RNA structure. Here, we summarize and discuss recent literature relating to the roles of these small endogenous molecules in RNA function. We have included studies directed at understanding the binding interactions of polyamines with polynucleotides, tRNA, rRNA, mRNA and ribozymes using chemical, biochemical and spectroscopic tools. In brief, polyamines bind RNA in a sequence-selective fashion and induce changes in RNA structure in context-dependent manners. In some cases the functional consequences of these interactions have been observed in cells. Most notably, polyamine-mediated effects on RNA are frequently distinct from those of divalent cations (i.e. Mg2+) confirming their roles as independent molecular entities which help drive RNA-mediated processes.
Asunto(s)
Poliaminas/metabolismo , ARN/metabolismo , Poliaminas/química , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Oligonucleotide delivery in vivo is commonly seen as the principal hurdle to the successful development of oligonucleotide drugs. In an analysis of 26 oligonucleotide drugs recently evaluated in late-stage clinical trials we found that to date at least half have demonstrated suppression of the target mRNA and/or protein levels in the relevant cell types in man, including those present in liver, muscle, bone marrow, lung, blood and solid tumors. Overall, this strongly implies that the drugs are being delivered to the appropriate disease tissues. Strikingly we also found that the majority of the drug targets of the oligonucleotides lie outside of the drugable genome and represent new mechanisms of action not previously investigated in a clinical setting. Despite the high risk of failure of novel mechanisms of action in the clinic, a subset of the targets has been validated by the drugs. While not wishing to downplay the technical challenges of oligonucleotide delivery in vivo, here we demonstrate that target selection and validation are of equal importance for the success of this field.
Asunto(s)
Oligonucleótidos/uso terapéutico , ARN Mensajero/metabolismo , Ensayos Clínicos como Asunto , Regulación hacia Abajo , Humanos , Neoplasias/tratamiento farmacológico , Oligonucleótidos/administración & dosificación , Oligonucleótidos/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/uso terapéutico , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas/metabolismoRESUMEN
RNA is a major player in cellular function, and consequently can drive a number of disease pathologies. Over the past several years, small molecule-RNA targeting (smRNA targeting) has developed into a promising drug discovery approach. Numerous techniques, tools, and assays have been developed to support this field, and significant investments have been made by pharmaceutical and biotechnology companies. To date, the focus has been on identifying disease validated primary targets for smRNA drug development, yet RNA as a secondary (off) target for all small molecule drug programs largely has been unexplored. In this perspective, we discuss structure, target, and mechanism-driven safety aspects of smRNAs and highlight how these parameters can be evaluated in drug discovery programs to produce potentially safer drugs.
RESUMEN
Large compound libraries utilised for HTS often include metal contaminated compounds which can interfere with assay signal or target biology, and therefore appear as hits. Pursuit of these compounds can divert considerable time and resource away from more propitious hits, yet there is currently no established method of detecting metal impurities in a rapid and effective manner. Here we describe the development and application of a high-throughput method to identify metal contaminants using acoustic mist ionisation mass spectrometry (AMI-MS). Although metals species by themselves are not detectable by AMI-MS, we have identified two compounds that chelate metal ions and enable their detection. 6-(diethylamino)-1,3,5-triazine-2,4(1H,3H)-dithione (DMT) and 1-(3-{[4-(4-cyanophenyl)-1-piperidinyl]carbonyl}-4-methylphenyl)-3-ethylthiourea (TU) can form complexes with a range of metal ions. Using a collection of metal catalysts, we have developed two metal chelator assays that collectively allow for the detection of Ag, Au, Co, Cu, Fe, Pd, Pt and Zn. We employed these assays to profile the hit outputs of a Zn liable target, and a Pd liable target, and identified significant quantities of metal contaminated compounds in the HTS outputs. This work provides a method of rapidly identifying metal impurities in hit compounds and has become part of an established workflow in triaging HTS outputs at AstraZeneca, facilitating faster identification of robust lead series.
Asunto(s)
Bioensayo , Ensayos Analíticos de Alto Rendimiento , Bioensayo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de MasasRESUMEN
Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.
Asunto(s)
MicroARNs/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Ribonucleasa III/antagonistas & inhibidores , Ribonucleasa III/genética , Animales , Secuencia de Bases , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica/genética , Precursores del ARN/antagonistas & inhibidores , Precursores del ARN/química , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Ribonucleasa III/metabolismo , Relación Estructura-ActividadRESUMEN
RNA molecules have a variety of cellular functions that can drive disease pathologies. They are without a doubt one of the most intriguing yet controversial small-molecule drug targets. The ability to widely target RNA with small molecules could be revolutionary, once the right tools, assays, and targets are selected, thereby defining which biomolecules are targetable and what constitutes drug-like small molecules. Indeed, approaches developed over the past 5-10 years have changed the face of small molecule-RNA targeting by addressing historic concerns regarding affinity, selectivity, and structural dynamics. Presently, selective RNA-protein complex stabilizing drugs such as branaplam and risdiplam are in clinical trials for the modulation of SMN2 splicing, compounds identified from phenotypic screens with serendipitous outcomes. Fully developing RNA as a druggable target will require a target engagement-driven approach, and evolving chemical collections will be important for the industrial development of this class of target. In this review we discuss target-directed approaches that can be used to identify RNA-binding compounds and the chemical knowledge we have today of small-molecule RNA binders.
Asunto(s)
Terapia Molecular Dirigida , ARN/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Compuestos Azo/uso terapéutico , Diseño de Fármacos , Humanos , Complejos Multiproteicos/genética , Fármacos Neuromusculares/uso terapéutico , Pirimidinas/uso terapéutico , ARN/genética , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
G-quadruplexes are secondary structures formed in G-rich sequences in DNA and RNA. Considerable research over the past three decades has led to in-depth insight into these unusual structures in DNA. Since the more recent exploration into RNA G-quadruplexes, such structures have demonstrated their in cellulo existence, function and roles in pathology. In comparison to Watson-Crick-based secondary structures, most G-quadruplexes display highly redundant structural characteristics. However, numerous reports of G-quadruplex motifs/structures with unique features (e.g. bulges, long loops, vacancy) have recently surfaced, expanding the repertoire of G-quadruplex scaffolds. This review addresses G-quadruplex formation and structure, including recent reports of non-canonical G-quadruplex structures. Improved methods of detection will likely further expand this collection of novel structures and ultimately change the face of quadruplex-RNA targeting as a therapeutic strategy.
Asunto(s)
ADN/química , ARN/química , Animales , G-Cuádruplex , Humanos , Modelos MolecularesRESUMEN
The development of small molecule kinase drugs is a rapidly evolving field and represents one of the most important research areas within oncology. This innovation letter provides an overview and analysis of approved kinase drugs according to their WHO registration (INN) dates, primary biological targets, and selectivity and structural similarities, which are also depicted in an associated poster. It also discusses new trends in kinase drug discovery programs such as new kinase targets, novel mechanisms of action, and diverse indications.
RESUMEN
RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2'-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required anti conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs.
RESUMEN
BACKGROUND: Oligonucleotide drug development has revolutionised the drug discovery field. Within this field, 'small' or 'short' activating RNAs (saRNA) are a more recently discovered category of short double-stranded RNA with clinical potential. saRNAs promote transcription from target loci, a phenomenon widely observed in mammals known as RNA activation (RNAa). OBJECTIVE: The ability to target a particular gene is dependent on the sequence of the saRNA. Hence, the potential clinical application of saRNAs is to increase target gene expression in a sequence-specific manner. saRNA-based therapeutics present opportunities for expanding the "druggable genome" with particular areas of interest including transcription factor activation and cases of haploinsufficiency. RESULTS AND CONCLUSION: In this mini-review, we describe the pre-clinical development of the first saRNA drug to enter the clinic. This saRNA, referred to as MTL-CEBPA, induces increased expression of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα), a tumour suppressor and critical regulator of hepatocyte function. MTL-CEBPA is presently in Phase I clinical trials for hepatocellular carcinoma (HCC). The clinical development of MTL-CEBPA will demonstrate "proof of concept" that saRNAs can provide the basis for drugs which enhance target gene expression and consequently improve treatment outcome in patients.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , ARN Interferente Pequeño/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/administración & dosificación , Regulación de la Expresión Génica , Genes Supresores de Tumor/fisiología , Humanos , ARN Bicatenario/administración & dosificación , ARN Bicatenario/genética , ARN Interferente Pequeño/administración & dosificación , ARN Pequeño no Traducido/administración & dosificación , ARN Pequeño no Traducido/genética , Factores de Transcripción/genéticaRESUMEN
Chemical modification and/or the conjugation of small functional molecules to oligonucleotides have significantly improved their biological and biophysical properties, addressing issues such as poor cell penetration, stability to nucleases and low affinity for their targets. Here, the authors review the literature reporting on the biophysical, biochemical and biological properties of one particular class of modification - polyamine-oligonucleotide conjugates. Naturally derived and synthetic polyamines have been grafted onto a variety of oligonucleotide formats, including antisense oligonucleotides and siRNAs. In many cases this has had beneficial effects on their properties such as target hybridization, nuclease resistance, cellular uptake and activity. Polyamine-oligonucleotide conjugation, therefore, represents a promising direction for the further development of oligonucleotide-based therapeutics and tools.
Asunto(s)
Descubrimiento de Drogas , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacología , Oligonucleótidos/química , Oligonucleótidos/farmacología , Poliaminas/química , Poliaminas/farmacología , Animales , Descubrimiento de Drogas/métodos , Humanos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacologíaRESUMEN
The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and developmental timing and a candidate tumor suppressor. Here we show that LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 processing in Caenorhabditis elegans. We demonstrate that lin-28 is necessary and sufficient to block let-7 activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. Moreover, we have identified a poly(U) polymerase, PUP-2, which regulates the stability of LIN-28-blockaded let-7 pre-miRNA and contributes to LIN-28-dependent regulation of let-7 during development. We show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 pre-miRNA by PUP-2 in vitro. Our results demonstrate that LIN-28 and let-7 form an ancient regulatory switch, conserved from nematodes to humans, and provide insight into the mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 ortholog might regulate let-7 and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and cancer biology, we propose that such poly(U) polymerases are potential therapeutic targets.