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Low-Molecular Weight Small Molecules Can Potently Bind RNA and Affect Oncogenic Pathways in Cells
Abstract
RNA is challenging to target with bioactive small molecules, particularly those of low molecular weight that bind with sufficient affinity and specificity. In this report, we developed a platform to address this challenge, affording a novel bioactive interaction. An RNA-focused small-molecule fragment collection (n = 2500) was constructed by analyzing features in all publicly reported compounds that bind RNA, the largest collection of RNA-focused fragments to date. The RNA-binding landscape for each fragment was studied by using a library-versus-library selection with an RNA library displaying a discrete structural element, probing over 12.8 million interactions, the greatest number of interactions between fragments and biomolecules probed experimentally. Mining of this dataset across the human transcriptome defined a drug-like fragment that potently and specifically targeted the microRNA-372 hairpin precursor, inhibiting its processing into the mature, functional microRNA and alleviating invasive and proliferative oncogenic phenotypes in gastric cancer cells. Importantly, this fragment has favorable properties, including an affinity for the RNA target of 300 ± 130 nM, a molecular weight of 273 Da, and quantitative estimate of drug-likeness (QED) score of 0.8. (For comparison, the mean QED of oral medicines is 0.6 ± 0.2). Thus, these studies demonstrate that a low-molecular weight, fragment-like compound can specifically and potently modulate RNA targets.
... That is, structural information about the fragments in complex with RNA informs optimization to improve affinity and selectivity of individual fragments and also informs how to link two or more fragments together. 42 Indeed, novel strategies to identify fragments that bind RNAs and the optimization thereof have been developed by various laboratories, employing NMR spectroscopy, 43-45 mass spectrometry, 45,46 dynamic combinatorial chemistry (DCC), 47 equilibrium dialysis, 48 labeled ligand displacement methods, chemical cross-linking and isolation by pull-down (Chem CLIP), 49, 50 selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-Map), 51, 52 twodimensional combinatorial screening (2DCS), 52 and in silico methods. 42 Here, we describe the above-mentioned biophysical strategies employed to identify fragment binders, with one detailed example for each methodology. ...
... As proof of principle that it is indeed possible to define the molecular finger prints of fragments and apply this information to target RNA selectively, a 2,500-member, RNA-focused fragment library (250 Da average molecular weight) was studied for binding to 33 and 32 internal loop library (ILL) via 2DCS. 52 Three fragments selectively bound RNA structures that comprise the two RNA libraries, the affinity landscapes of which were defined. Interestingly, fragment 34 was predicted to bind the Dicer site of the miR-372 precursor (pre-miR-372) with highest affinity based on HiT-StARTS statistical analysis ( Figure 10C). ...
... The fragment also de-repressed a downstream target of miR-372, large tumor suppressor kinase 2 (LATS2) at the mRNA and protein levels while also reducing cell proliferation and invasion phenotypes associated with pre-miR-372-associated gastric cancer. 52 This study demonstrated that drug-like low molecular weight compounds can in fact be studied using 2DCS and that the method can detect high affinity binding interactions between fragments and RNA. Importantly, the fragment hit, 34, was bioactive in cells without any optimization. ...
Although fragment-based drug discovery (FBDD) has been successfully implemented and well-explored for protein targets, its feasibility for RNA targets is emerging. Despite the challenges associate with the selective targeting of RNA, efforts to integrate known methods of RNA binder discovery with fragment-based approaches has been fruitful, as a few bioactive ligands have been identified. Here, we review various fragment-based approaches implemented for RNA targets and provide insight into experimental design and outcomes, to guide future work in the area. Indeed, investigations surrounding the molecular recognition of RNA by fragments address rather important questions such as the limits of molecular weight that confers selective binding and the physicochemical properties favorable for RNA binding and bioactivity.
... Alternatively, a single fragment found to bind an RNA pocket can be synthetically extended into adjacent pocket space by adding functional groups or other fragment moieties (Fig. 1b). To date, there have been many examples of initial fragment hits that engage RNA [12], and two very recent studies show that it is possible to obtain high-affinity fragmentlike hits for RNA [24,25]. Going forward, FBLD e with a focus on fragment elaboration e is well suited to fill current knowledge gaps in small molecule-RNA interactions, as FBLD efficiently and simultaneously interrogates both small molecule chemical space and RNA structural space. ...
... For compounds that act by structural stabilization, ligand binding induces a change in the relative populations of RNA conformational states. This mechanism has been exploited to stabilize specific states for viral frameshift and IRES elements (compounds 17,19,25) [20,29], to alter splicing (compounds 23, 24) [28], and to stabilize the bound states of riboswitches (compounds 9, 26) [11, 21,30]. Ultimately, fragment-based ligands should be capable of modulating RNA-mediated www.sciencedirect.com ...
A subset of functional regions within large RNAs fold into complex structures able to bind small-molecule ligands with high affinity and specificity. Fragment-based ligand discovery (FBLD) offers notable opportunities for discovery and design of potent small molecules that bind pockets in RNA. Here we share an integrated analysis of recent innovations in FBLD, emphasizing opportunities resulting from fragment elaboration via both linking and growing. Analysis of elaborated fragments emphasizes that high-quality interactions form with complex tertiary structures in RNA. FBLD-inspired small molecules have been shown to modulate RNA functions by competitively inhibiting protein binding and by selectively stabilizing dynamic RNA states. FBLD is creating a foundation to interrogate the relatively unknown structural space for RNA ligands and for discovery of RNA-targeted therapeutics.
... Notably, Hanabusa, [9][10][11][12] Feringa, [13][14][15][16][17][18] and Tomioka [19][20][21] and other research groups [22][23][24][25][26][27][28][29][30][31][32][33] successfully constructed gelators for organic liquids. However, a more accurate and deeper understanding of these interactions is necessary to design more efficient gelators and apply these materials. ...
In this study, a low‐molecular‐weight organogelator derived from (l)‐amino acids was designed and synthesized. Gelation assays using (l)‐amino acid derivatives were performed to confirm the gelation ability, which was found to be high in several compounds. The (l)‐alanine derivatives were determined to be excellent gelators, forming good gels even when smaller amounts were added. These results led to a library of amino acid‐derived organogelators. In addition, the thermal properties of the (l)‐alanine derivatives with high gelation performance were measured. Differential scanning calorimetry measurements revealed that the thermal stability of the gels could be controlled by changing the gelator concentration. The surface states of the obtained gels were observed by field‐emission scanning electron microscopy and atomic force microscopy measurements, which confirmed the structure of the self‐molecular aggregates. Self‐molecular aggregates were observed to be helical or sheet‐like, and the gels were constructed by forming aggregates by self‐molecular recognition.
Stacking interactions contribute significantly to the interaction of small molecules with RNA, and harnessing the power of these interactions will likely prove important in the development of RNA-targeting inhibitors. To this end, we present a comprehensive computational analysis of stacking interactions between a set of 54 druglike heterocycles and the natural nucleobases. We first show that heterocycle choice can tune the strength of stacking interactions with nucleobases over a large range and that heterocycles favor stacked geometries that cluster around a discrete set of stacking loci characteristic of each nucleobase. Symmetry-adapted perturbation theory results indicate that the strengths of these interactions are modulated primarily by electrostatic and dispersion effects. Based on this, we present a multivariate predictive model of the maximum strength of stacking interactions between a given heterocycle and nucleobase that depends on molecular descriptors derived from the electrostatic potential. These descriptors can be readily computed using density functional theory or predicted directly from atom connectivity (e.g., SMILES). This model is used to predict the maximum possible stacking interactions of a set of 1854 druglike heterocycles with the natural nucleobases. Finally, we show that trivial modifications of standard (fixed-charge) molecular mechanics force fields reduce errors in predicted stacking interaction energies from around 2 kcal/mol to below 1 kcal/mol, providing a pragmatic means of predicting more reliable stacking interaction energies using existing computational workflows. We also analyze the stacking interactions between ribocil and a bacterial riboswitch, showing that two of the three aromatic heterocyclic components engage in near-optimal stacking interactions with binding site nucleobases.
Hits identified from screening diverse compound libraries against RNA targets can be used to inform design of RNA-focused libraries via computational techniques to calculate chemical similarity and physicochemical properties.
Halogenation is one of the most important transformations in organic synthesis. Halogenated compounds are employed in many reactions to prepare useful molecules. Many methods have been developed to introduce halogens into different compounds. Visible light‐mediated reactions are the efficient, low‐toxic, and mild‐condition methods applied for various organic chemistry transformations. Remarkably, there has been an increasing development in the application of visible light‐induced halogenation in recent years. Herein, we present a comprehensive summary of halogenation reactions including chlorination, bromination, and iodination under visible light irradiation since 2020.
An ideal DNA‐encoded library (DEL) selection requires the library to consist of diverse core skeletons and cover chemical space as much as possible. However, the lack of efficient on‐DNA synthetic approaches toward core skeletons has greatly restricted the diversity of DEL. To mitigate this issue, this work disclosed a “Mask & Release” strategy to streamline the challenging on‐DNA core skeleton synthesis. N ‐phenoxyacetamide is used as a masked phenol and versatile directing group to mediate diversified DNA‐compatible C‐H functionalization, introducing the 1st‐dimensional diversity at a defined site, and simultaneously releasing the phenol functionality, which can facilitate the introduction of the 2nd diversity. This work not only provides a set of efficient syntheses toward DNA‐conjugated drug‐like core skeletons such as ortho ‐alkenyl/sulfiliminyl/cyclopropyl phenol, benzofuran, dihydrobenzofuran but also provides a paradigm for on‐DNA core skeleton synthetic method development.
Previously, we have identified a novel human metastasis-inducing lncRNA (named SKAI1BC), that suppresses the KAI1/CD82 metastasis-suppressing gene and is upregulated in triple negative breast cancer and melanoma derived cell lines. Modeling of the SKAI1BC lncRNA secondary structure and its potential interaction with Inforna compounds, led us to identify several compounds that might bind the SKAI1BC lncRNA. We found that these compounds inhibit metastasis invasion and cell migration in culture, in all eight types of solid human cancers tested: several of which are the most lethal and/or frequent human malignancies. Moreover, in most cases, the mechanism of action of several of our compounds involves enhancement of KAI1/CD82 RNA level depending on the specific compound and the human tumor type. With the epigenetic inactivation of KAI1/CD82 in at least ten additional solid human cancers, this implies a very good chance to broaden the spectrum of human cancers affected by our compounds. This is the first time that modeling of a large lncRNA (> 700 bp) secondary structure followed by its potential interaction with Inforna like compounds database has led to the identification of potential biologically active small molecule drugs.
Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.
RNA-binding proteins (RBPs) play vital roles in organisms through binding with RNAs to regulate their functions. Small molecules affecting the function of RBPs have been developed, providing new avenues for drug discovery. Herein, we describe the perspectives on developing small molecule regulators of RBPs. The following types of small molecule modulators are of great interest in drug discovery: small molecules binding to RBPs to affect interactions with RNA molecules, bifunctional molecules binding to RNA or RBP to influence their interactions, and other types of molecules that affect the stability of RNA or RBPs. Moreover, we emphasize that the bifunctional molecules may play important roles in small molecule development to overcome the challenges encountered in the process of drug discovery.
The DNA-encoded chemical library (DEL) is a powerful hit selection technique in either basic science or innovative drug discovery. With the aim to circumvent the issue concerning DNA barcode damage in a conventional on-DNA copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), we have successfully developed the first DNA-compatible enolate-azide [3 + 2] cycloaddition reaction. The merits of this DEL chemistry include metal-free reaction and high DNA fidelity, high conversions and easy operation, broad substrate scope, and ready access to the highly substituted 1,4,5-trisubstituted triazoles. Thus, it will not only further enrich the DEL chemistry toolbox but also will have great potential in practical DEL synthesis.
Although fragment-based drug discovery (FBDD) has been successfully implemented and well-explored for protein targets, its feasibility for RNA targets is emerging. Despite the challenges associated with the selective targeting of RNA, efforts to integrate known methods of RNA binder discovery with fragment-based approaches have been fruitful, as a few bioactive ligands have been identified. Here, we review various fragment-based approaches implemented for RNA targets and provide insights into experimental design and outcomes to guide future work in the area. Indeed, investigations surrounding the molecular recognition of RNA by fragments address rather important questions such as the limits of molecular weight that confer selective binding and the physicochemical properties favorable for RNA binding and bioactivity.
Significance
RNA molecules encode proteins and play numerous regulatory roles in cells. Targeting RNA with small molecules, as is routine with proteins, would create broad opportunities for modulating biology and creating new drugs. However, this opportunity has been difficult to realize because creating novel small molecules that bind RNA, especially using modest resources, is challenging. This study integrates two widely used technologies, SHAPE chemical probing of RNA and fragment-based ligand discovery, to craft an innovative strategy for creating small molecules that bind to and modulate the activity of a structured RNA. The anticipated impact is high because the methods are simple, can be implemented in diverse research and discovery contexts, and lead to realistic druglike molecules.
Significance
Drug discovery generally investigates one target at a time, in sharp contrast to living organisms, which mold ligands and targets by evolution of highly complex molecular interaction networks. We recapitulate this modality of discovery by encoding drug structures in DNA, allowing the entire DNA-encoded library to interact with thousands of RNA fold targets, and then decoding both drug and target by sequencing. This information serves as a filter to identify human RNAs aberrantly produced in cancer that are also binding partners of the discovered ligand, leading to a precision medicine candidate that selectively ablates an oncogenic noncoding RNA, reversing a disease-associated phenotype in cells.
SARS‐CoV‐2 contains a positive single‐stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS‐CoV and SARS‐CoV‐2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex‐vivo structural probing experiments. These elements contain non‐base‐paired regions that potentially harbor ligand‐binding pockets. Here, we performed an NMR‐based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different ¹H‐based 1D NMR binding assays. The screening identified common as well as RNA‐element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS‐CoV‐2.
Rapid development within the fields of both fragment‐based drug discovery (FBDD) and medicinal targeting of RNA provides possibilities for combining technologies and methods in novel ways. This review provides an overview of fragment‐based screening (FBS) against RNA targets, including a discussion of the most recently used screening and hit validation methods such as NMR spectroscopy, X‐ray crystallography, and virtual screening methods. A discussion of fragment library design based on research from small‐molecule RNA binders provides an overview on both the currently limited guidelines within RNA‐targeting fragment library design, and future possibilities. Finally, future perspectives are provided on screening and hit validation methods not yet used in combination with both fragment screening and RNA targets.
This study aims to highlight the relationships between the structure of smell compounds and their odors. For this purpose, heterogeneous data sources were screened, and 6038 odorant compounds and their known associated odors (162 odor notes) were compiled, each individual molecule being represented with a set of 1024 structural fingerprint. Several dimensional reduction techniques (PCA, MDS, t-SNE and UMAP) with two clustering methods (k-means and agglomerative hierarchical clustering AHC) were assessed based on the calculated fingerprints. The combination of UMAP with k-means and AHC methods allowed to obtain a good representativeness of odors by clusters, as well as the best visualization of the proximity of odorants on the basis of their molecular structures. The presence or absence of molecular substructures has been calculated on odorant in order to link chemical groups to odors. The results of this analysis bring out some associations for both the odor notes and the chemical structures of the molecules such as “woody” and “spicy” notes with allylic and bicyclic structures, “balsamic” notes with unsaturated rings, both “sulfurous” and “citrus” with aldehydes, alcohols, carboxylic acids, amines and sulfur compounds, and “oily”, “fatty” and “fruity” characterized by esters and with long carbon chains. Overall, the use of UMAP associated to clustering is a promising method to suggest hypotheses on the odorant structure-odor relationships.
Significance
The development of selective bioactive compounds that target RNA structures is difficult. Here we report an approach to identify low molecular weight fragments that bind to a cancer-causing RNA. By determining where the fragments bind within an RNA target, they can be assembled to provide a high-affinity and potent inhibitor. This approach could be a general way to develop bioactive small molecules that target RNA, which previously was considered “undruggable.” It was applied to provide a precision lead medicine against an RNA that contributes to cancer metastasis.
RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed.
Significance Statement
With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.
Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif–small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between ‘undruggable’ biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.
We report here the nuclear magnetic resonance ¹⁹F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter‐screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow‐up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low‐micromolar binding affinity without losing binding specificity between two different terminator structures.
M. tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis, which kills more than 1.5 million people worldwide every year. Resistant strains to available antibiotics pose a significant healthcare problem. The enormous complexity of the ribosome poses a barrier for drug discovery. We have overcome this in a tractable way by using an RNA segment that represents the peptidyl transferase center as a target. By using a novel combination of NMR transverse relaxation times (T2) and computational chemistry approaches we have obtained improved inhibitors of Mtb ribosomal PTC. Two phenylthiazole derivatives were predicted by machine learning models as effective inhibitors and this was confirmed by their IC50 values, which were significantly improved over standard antibiotic drugs.
Riboswitches are naturally occurring RNA aptamers that regulate gene expression by binding to specific small molecules. Riboswitches control the expression of essential bacterial genes and are important models for RNA-small molecule recognition. Here, we report the discovery of a class of synthetic small molecules that bind to PreQ1 riboswitch aptamers. These molecules bind specifically and reversibly to the aptamers with high affinity and induce a conformational change. Furthermore, the ligands modulate riboswitch activity through transcriptional termination despite no obvious chemical similarity to the cognate ligand. X-ray crystallographic studies reveal that the ligands share a binding site with the cognate ligand but make different contacts. Finally, alteration of the chemical structure of the ligand causes changes in the mode of RNA binding and affects regulatory function. Thus, target- and structure-based approaches can be used to identify and understand the mechanism of synthetic ligands that bind to and regulate complex, folded RNAs.
Long non‐coding RNAs have recently become a key regulatory factor for cancers, whereas FER1L4, a newly discovered long non‐coding RNA, has been mostly studied in gastric carcinoma and colon cancer cases. The functions and molecular mechanism of FER1L4 have been rarely reported in glioma malignant phenotypes. In this study, it was found that the expression of LncRNA FER1L4 is upregulated in high‐grade gliomas than in low‐grade cases and that a high expression of LncRNA FER1L4 predicts poor prognosis of gliomas. Meanwhile, in vitro study suggests that expression of FER1L4 with SiRNA knockdown obviously suppresses cell cycle and proliferation. It is further demonstrated by experiments that the FER1L4 knockdown suppresses growth of in vivo glioma. Besides, it is found in our study that LncRNA FER1L4 expression is positively correlated with E2F1 mRNA expression. After knockdown of FER1L4 expression, E2F1 expression is significantly down‐regulated, whereas the expression of miR‐372 is significantly up‐regulated; the up‐regulation of miR‐372 leads to significant down‐regulation of FER1L4 and E2F1 expression. In addition, it is also found that FER1L4 can be used as competitive endogenous RNA to interact or bind with miR‐371 and thereby up‐regulate E2F1, thus promoting the cycle and proliferation of glioma cells. It may be one of the molecular mechanisms in which FER1L4 plays its oncogene‐like role in gliomas.
As a core kinase in the Hippo pathway, large tumor suppressor kinase 2 (LATS2) regulates cell proliferation, migration and invasion through numerous signaling pathways. However, its functions on cell proliferation, migration and invasion in glioma have yet to be elucidated. The present study revealed that LATS2 was downregulated in glioma tissues and cells, as determined by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. In addition, Cell Counting Kit-8, scratch wound healing and Transwell assays revealed that overexpression of LATS2 in U-372 MG cells inhibited cell proliferation, migration and invasion. Furthermore, western blot analysis indicated that the expression levels of phosphorylated (p)-yes-associated protein and p-tafazzin were increased in cells with LATS2 overexpression. These results indicated that LATS2 is a potential tumor suppressor, and downregulation of LATS2 in glioma may contribute to cancer progression.
We previously reported that LATS2, the upstream serine/threonine kinase of Yes-associated protein (YAP), is downregulated in gliomas and exhibits negative correlation with the prognosis of glioma patients. In this work, we aimed to explore the role and mechanism of large tumor suppressor kinase (LATS2) in the progression of malignant gliomas. We found that over-expression of LATS2 inhibited glioma cell proliferation and migration/invasion, while LATS2 downregulation promoted them. Mechanistically, LATS2 promoted the phosphorylation of YAP without affecting YAP mRNA expression. Inconsistent with some previous reports, both YAPWT and YAP5SA did not affect the level of LATS2, suggesting that LATS2 did not form a feedback loop with YAP in gliomas. Furthermore, mammalian Ste20-like kinases (MST1) over-expression did not affect the phosphorylation of YAP, suggesting that MST1 may not be the essential upstream serine/threonine kinase of Hippo/YAP pathway in gliomas. Together, LATS2 inhibits glioma cell proliferation and migration/invasion by inactivating YAP.
MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.
MicroRNAs (miRs) have previously been demonstrated to be important in the tumorigenesis and progression of breast cancer. miR-372 was previously revealed to be involved in various types of human cancer, however its function in breast cancer remains largely unknown. The present study demonstrated that miR-372 is frequently overexpressed in breast cancer cell lines and tissues. The downregulation of miR-372 markedly inhibited cell proliferation, arrested the cell cycle in the G1/S phase, and increased the apoptosis of breast cancer cells. Consistently, an in vivo xenograft study also demonstrated the suppressive effects of miR-372 knockdown on tumor growth. Further studies revealed that miR-372 modulated the expression of large tumor suppressor kinase 2 (LATS2) by directly targeting its 3-untranslated region in breast cancer cells. Furthermore, silencing of LATS2 was able to rescue the effect of the miR-372 inhibitor. Overall, the results suggest that miR-372 functions as an oncogenic miRNA in breast cancer by targeting LATS2.
Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.
RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif small molecule interactions identified via selection. Named High Throughput Structure Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nudeic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.
Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.
Hypoxia induces a complex circuit of gene expression that drives tumor progression and increases drug resistance. Defining these changes allows for an understanding of how hypoxia alters tumor biology and informs design of lead therapeutics. We probed the role of microRNA-544 (miR-544), which silences mammalian target of rapamycin (mTOR), in a hypoxic breast cancer model by using a small molecule (1) that selectively impedes the microRNA's biogenesis. Application of 1 to hypoxic tumor cells selectively inhibited production of the mature microRNA, sensitized cells to 5-fluorouracil, and de-repressed mRNAs affected by miR-544 in cellulo and in vivo. Thus, small molecule inhibition of miR-544 reverses a tumor cell's physiological response to hypoxia. Importantly, 1 sensitized tumor cells to hypoxia-associated apoptosis at a 25-fold lower concentration than a 2'-O-methyl RNA antagomir and was as selective. Further, the effect of 1 was suppressed by treatment of cell with rapamycin, a well-known inhibitor of the mTOR signaling pathway. Thus, RNA-directed chemical probes, which could also serve as lead therapeutics, enable interrogation of complex cellular networks in cells and animals.
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.
Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.
Through screening by NMR spectroscopy, we discovered a novel scaffold (DPQ: 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) that binds specifically to the influenza A virus RNA promoter. The solution structure of the RNA-DPQ complex reported here demonstrates that the internal loop is the binding site of DPQ. The scaffold exhibits antiviral activity against influenza viruses.
Riboswitches are regions of mRNA that bind selectively certain metabolites, thereby regulating gene expression. We have developed a method, which uses a combination of biophysical techniques (equilibrium dialysis, waterLOGSY and T2 relaxation-edited NMR spectroscopy and isothermal titration calorimetry) that allows screening and identification of novel ligands for riboswitches. By this method a library of 1300 fragments was screened on the E. coli thiamine pyrophosphate (TPP) riboswitchthiM, resulting in the identification of 17 hits. The compounds showed KD values from 22 to 670 μM, with four compounds having KD values <100 μM. The fragments are structurally diverse and their ligand efficiency indicates good prospects for structure-guided elaboration. In addition, a riboswitch functional assay based on in vitro transcriptiontranslation (IVTT) of the reporter geneluciferase was developed. This assay system is an alternative to in vivo assays and constitutes a valuable tool to determine the effect of small molecules on riboswitch-regulated gene expression.
The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. The human genome sequence provides the underlying code for human biology. Despite intensive study, especially in identifying protein-coding genes, our understanding of the genome is far from complete, particularly with regard to non-coding RNAs, alternatively spliced transcripts and regulatory sequences. Systematic analyses of transcripts and regulatory information are essential for the identification of genes and regulatory regions, and are an important resource for the study of human biology and disease. Such analyses can also provide comprehensive views of the organization and variability of genes and regulatory information across cellular contexts, species and individuals.
Drug-likeness is a key consideration when selecting compounds during the early stages of drug discovery. However, evaluation of drug-likeness in absolute terms does not reflect adequately the whole spectrum of compound quality. More worryingly, widely used rules may inadvertently foster undesirable molecular property inflation as they permit the encroachment of rule-compliant compounds towards their boundaries. We propose a measure of drug-likeness based on the concept of desirability called the quantitative estimate of drug-likeness (QED). The empirical rationale of QED reflects the underlying distribution of molecular properties. QED is intuitive, transparent, straightforward to implement in many practical settings and allows compounds to be ranked by their relative merit. We extended the utility of QED by applying it to the problem of molecular target druggability assessment by prioritizing a large set of published bioactive compounds. The measure may also capture the abstract notion of aesthetics in medicinal chemistry.
The increasing number of RNA crystal structures enables a structure-based approach to the discovery of new RNA-binding ligands. To develop the poorly explored area of RNA-ligand docking, we have conducted a virtual screening exercise for a purine riboswitch to probe the strengths and weaknesses of RNA-ligand docking. Using a standard protein-ligand docking program with only minor modifications, four new ligands with binding affinities in the micromolar range were identified, including two compounds based on molecular scaffolds not resembling known ligands. RNA-ligand docking performed comparably to protein-ligand docking indicating that this approach is a promising option to explore the wealth of RNA structures for structure-based ligand design.
Herein, we show that a naturally occurring RNA G-quadruplex element within the 5' UTR of the human NRAS proto-oncogene is a target for a small molecule that inhibits translation in vitro. The present study provides a first demonstration that natural 5' UTR mRNA G-quadruplexes have potential as molecular targets for small molecules that modulate translation.
RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing — by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade. The potential of therapeutically targeting RNA structures with small molecules is being increasingly recognized. Here, Disney and colleagues review strategies to identify, validate and optimize small-molecule RNA binders. Examples of existing RNA-targeted small molecules, as well as challenges and future directions in the field, are discussed.
The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.
Macrocyclic peptides are a promising class of compounds that can often engage challenging therapeutic targets. Display technologies, such as mRNA display, allow for the efficient discovery of macrocyclic peptides. This article reviews the current approaches for generating macrocyclic peptide libraries using mRNA display and highlights some recent examples of ribosomal incorporation of nonproteinogenic amino acids into macrocyclic peptides.
The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makesthem 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 tothe 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.
The biology of healthy and diseased cells is often mediated by RNA structures, desirable targets for small molecule chemical probes and lead medicines. Although structured regions are found throughout the transcriptome, some even with demonstrated functionality, human RNAs are considered recalcitrant to small molecule targeting. However, targeting structured regions with small molecules provides an important alternative to oligonucleotides that target sequence. In this Perspective, we describe challenges and progress in developing small molecules interacting with RNA (SMIRNAs) to capture their significant opportunities at the intersection of chemistry, biology, and medicine. Key to establishing a new paradigm in chemical biology and medicine is the development of methods to obtain, preferably by design, bioactive compounds that modulate RNA targets and the companion methods that validate their direct effects in cells and pre-clinical models. While difficult, demonstration of direct target engagement in the complex cellular milieu, along with methods to establish modes of action, are required to push this field forward We also describe frameworks for accelerated advancements in this burgeoning area, their implications, key new technologies for development of SMIRNAs, and milestones that have led to broader acceptance of RNA as a small molecule druggable target.
Potential RNA drug targets for small molecules are found throughout the human transcriptome, yet small molecules known to elicit a pharmacological response by directly targeting RNA are limited to antibacterials. Herein, we describe AbsorbArray, a small molecule microarray-based approach that allows for unmodified compounds, including FDA-approved drugs, to be probed for binding to RNA motif libraries in a massively parallel format. Several drug classes bind RNA including kinase and topoisomerase inhibitors. The latter avidly bound the motif found in the Dicer site of oncogenic microRNA (miR)-21 and inhibited its processing both in vitro and in cells. The most potent compound de-repressed a downstream protein target and inhibited a miR-21-mediated invasive phenotype. The compound's activity was ablated upon overexpression of pre-miR-21. Target validation via chemical crosslinking and isolation by pull-down showed direct engagement of pre-miR-21 by the small molecule in cells, demonstrating that RNAs should indeed be considered druggable.
Many RNAs cause disease; however, RNA is rarely exploited as a small-molecule drug target. Our programmatic focus is to define privileged RNA motif small-molecule interactions to enable the rational design of compounds that modulate RNA biology starting from only sequence. We completed a massive, library-versus-library screen that probed over 50 million binding events between RNA motifs and small molecules. The resulting data provide a rich encyclopedia of small-molecule RNA recognition patterns, defining chemotypes and RNA motifs that confer selective, avid binding. The resulting interaction maps were mined against the entire viral genome of hepatitis C virus (HCV). A small molecule was identified that avidly bound RNA motifs present in the HCV 30 UTR and inhibited viral replication while having no effect on host cells. Collectively, this study represents the first whole-genome pattern recognition between small molecules and RNA folds.
Structural studies of the 3'‐end of the oncogenic long non‐coding RNA Metastasis‐associated Lung Adenocarcinoma Transcript 1 (MALAT1) revealed a unique triple helix structure. This structure enables accumulation of the transcript, and high levels of MALAT1 are found in several cancers. Here, we synthesize a small molecule library based on an RNA‐binding scaffold, diphenylfuran (DPF), screen it against a variety of nucleic acid constructs, and demonstrate for the first time that the MALAT1 triple helix can be selectively targeted with small molecules. Computational analysis revealed a trend between subunit positioning and composition on DPF shape and intramolecular interactions, which in turn generally correlated with selectivity and binding strengths. This work thus provides design strategies toward chemical probe development for the MALAT1 triple helix and suggests that comprehensive analyses of RNA‐focused libraries can generate insights into selective RNA recognition.
RNA molecules are essential for cellular information transfer and gene regulation, and RNAs have been implicated in many human diseases. Messenger and non-coding RNAs contain highly structured elements, and evidence suggests that many of these structures are important for function. Targeting these RNAs with small molecules offers opportunities to therapeutically modulate numerous cellular processes, including those linked to 'undruggable' protein targets. Despite this promise, there is currently only a single class of human-designed small molecules that target RNA used clinically — the linezolid antibiotics. However, a growing number of small-molecule RNA ligands are being identified, leading to burgeoning interest in the field. Here, we discuss principles for discovering small-molecule drugs that target RNA and argue that the overarching challenge is to identify appropriate target structures — namely, in disease-causing RNAs that have high information content and, consequently, appropriate ligand-binding pockets. If focus is placed on such druggable binding sites in RNA, extensive knowledge of the typical physicochemical properties of drug-like small molecules could then enable small-molecule drug discovery for RNA targets to become (only) roughly as difficult as for protein targets.
A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.
Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC50 values ranging from 40 to 150 μM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.
MicroRNAs are known to be involved in carcinogenesis and tumor progression in glioma. Recently, microRNA-372 (miR-372) has been proved to play a substantial role in several human cancers, but its functions in glioma remain unclear. In this study, we confirmed that miR-372 was commonly upregulated in glioma cell lines and tissues. Downregulation of miR-372 markedly inhibited cell proliferation and invasion and induced G1/S arrest and apoptosis. Consistently, the xenograft mouse model also unveiled the suppressive effects of miR-372 knockdown on tumor growth. Further studies revealed that miR-372 modulated the expression of PHLPP2 by directly targeting its 3′-untranslated region (3′-UTR) and that miR-372 expression was inversely correlated with PHLPP2 expression in glioma samples. Silencing of PHLPP2 could rescue the inhibitory effect of miR-372 inhibitor. Moreover, miR-372 knockdown suppressed the phosphorylation levels of the major components of PI3K/Akt pathway including Akt, mTOR and P70S6K. Taken together, our results suggest that miR-372 functions as an oncogenic miRNA through targeting PHLPP2 in glioma. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Previous studies using fragment-based approaches identified small molecule "fragments" that bind this gene-regulatory mRNA domain. Crystallographic studies now show that, despite having micromolar Kds, four different fragments bind the TPP riboswitch site-specifically, occupying the pocket that recognizes the aminopyrimidine of TPP. Unexpectedly, the unoccupied site that would recognize the pyrophosphate of TPP rearranges into a structure distinct from that of the cognate complex. This idiosyncratic fragment-induced conformation, also characterized by small-angle X-ray scattering and chemical probing, represents a possible mechanism for adventitious ligand discrimination by the riboswitch, and suggests that off-pathway conformations of RNAs can be targeted for drug development. Our structures, together with previous screening studies, demonstrate the feasibility of fragment-based drug discovery against RNA targets.
MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and revealed to be oncogenic and to play a pivotal role in initiation and progression of these pathologies. It is now clear that the inhibition of oncogenic miRNAs, defined as blocking their biosynthesis or their function, could find an application in the therapy of different types of cancer in which these miRNAs are implicated. Here we report the design, the synthesis and the biological evaluation of new small-molecule RNA ligands targeting the production of oncogenic microRNAs. In this work we focused our attention on miR-372 and miR-373 that are implicated in the tumorigenesis of different types of cancer such as gastric cancer. These two oncogenic miRNAs are overexpressed in gastric cancer cells starting from their precursors pre-miR-372 and pre-miR-373: two stem-loop structured RNAs which lead to mature miRNAs after cleavage by the enzyme Dicer. The small molecules described herein consist of the conjugation of two RNA binding motives, i.e. the aminoglycoside neomycin and different natural and artificial nucleobases, in order to obtain RNA ligands with increased affinity and selectivity compared to parent compounds. After the synthesis of this new series of RNA ligands, we demonstrated that they are able to inhibit the production of the oncogenic miRNA-372 and -372 by binding their pre-miRNAs and inhibiting the processing by Dicer. Moreover, we proved that some of these compounds bear anti-proliferative activity toward gastric cancer cells and that this activity is likely linked to a decrease in the production of targeted miRNAs. To date, only few examples of small molecules targeting oncogenic miRNAs have been reported and such inhibitors could be extremely useful for the development of new anticancer therapeutic strategies as well as useful biochemical tools for the study of miRNAs' pathways and mechanisms. Furthermore, this is the first time that a design based on current knowledge about RNA targeting is proposed in order to target miRNAs' production with small molecules.
Previously, we have reported tissue- and stage-specific expression of miR-372 in human embryonic stem cells and so far, not many reports speculate the function of this microRNA (miRNA). In this study, we screened various human cancer cell lines including gastric cancer cell lines and found first time that miR-372 is expressed only in AGS human gastric adenocarcinoma cell line. Inhibition of miR-372 using antisense miR-372 oligonucleotide (AS-miR-372) suppressed proliferation, arrested the cell cycle at G2/M phase, and increased apoptosis of AGS cells. Furthermore, AS-miR-372 treatment increased expression of LATS2, while over-expression of miR-372 decreased luciferase reporter activity driven by the 3' untranslated region (3' UTR) of LATS2 mRNA. Over-expression of LATS2 induced changes in AGS cells similar to those in AGS cells treated with AS-miR-372. Taken together, these findings demonstrate an oncogenic role for miR-372 in controlling cell growth, cell cycle, and apoptosis through down-regulation of a tumor suppressor gene, LATS2.
A graphical method is presented for displaying the patterns in a set of aligned sequences. The characters representing the
sequence are stacked on top of each other for each position in the aligned sequences. The height of each letter is made proportional
to Its frequency, and the letters are sorted so the most common one is on top. The height of the entire stack is then adjusted
to signify the information content of the sequences at that position. From these ‘sequence logos’, one can determine not only
the consensus sequence but also the relative frequency of bases and the information content (measured In bits) at every position
in a site or sequence. The logo displays both significant residues and subtle sequence patterns.
Bacterial resistance development has become a very serious clinical problem for many classes of antibiotics. The 3-aryl-2-oxazolidinones are a relatively new class of synthetic antibacterial agents, having a new mechanism of action which involves very early inhibition of bacterial protein synthesis. We have prepared two potent, synthetic oxazolidinones, U-100592 and U-100766, which are currently in clinical development for the treatment of serious multidrug-resistant Gram-positive bacterial infections caused by strains of staphylococci, streptococci, and enterococci. The in vitro and in vivo (po and iv) activities of U-100592 and U-100766 against representative strains are similar to those of vancomycin. U-100592 and U-100766 demonstrate potent in vitro activity against Mycobacterium tuberculosis. A novel and practical asymmetric synthesis of (5S)-(acetamidomethyl)-2-oxazolidinones has been developed and is employed for the synthesis of U-100592 and U-100766. This involves the reaction of N-lithioarylcarbamates with (R)-glycidyl butyrate, resulting in excellent yields and high enantiomeric purity of the intermediate (R)-5-(hydroxymethyl)-2-oxazolidinones.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
Oxazolidinones represent a novel class of antibiotics that inhibit protein synthesis in sensitive bacteria. The mechanism of action and location of the binding site of these drugs is not clear. A new representative of oxazolidinone antibiotics, linezolid, was found to be active against bacteria and against the halophilic archaeon Halobacterium halobium. The use of H. halobium, which possess only one chromosomal copy of rRNA operon, allowed isolation of a number of linezolid-resistance mutations in rRNA. Four types of linezolid-resistant mutants were isolated by direct plating of H. halobium cells on agar medium containing antibiotic. In addition, three more linezolid-resistant mutants were identified among the previously isolated mutants of H. halobium containing mutations in either 16 S or 23 S rRNA genes. All the isolated mutants were found to contain single-point mutations in 23 S rRNA. Seven mutations affecting six different positions in the central loop of domain V of 23 S rRNA were found to confer resistance to linezolid. Domain V of 23 S rRNA is known to be a component of the ribosomal peptidyl transferase center. Clustering of linezolid-resistance mutations within this region strongly suggests that the binding site of the drug is located in the immediate vicinity of the peptidyl transferase center. However, the antibiotic failed to inhibit peptidyl transferase activity of the H. halobium ribosome, supporting the previous conclusion that linezolid inhibits translation at a step different from the catalysis of the peptide bond formation.
A technique for lead discovery vs RNA targets utilizing mass spectrometry (MS) screening methods is described. The structure-activity relationships (SAR) derived from assaying weak binding motifs allows the pharmacophores discovered to be elaborated via "SAR by MS" to higher affinity ligands. Application of this strategy to a subdomain of the 23S rRNA afforded a new class of compounds with functional activity.
Linezolid is the first of a new class of antimicrobial agents, the oxazolidinones, to be approved for clinical use in the United States and elsewhere. The drug is a totally synthetic compound, which lessens the likelihood of naturally occurring resistance mechanisms. It has excellent activity against virtually all important gram-positive pathogens, including methicillin-resistant staphylococci, penicillin-resistant pneumococci, macrolide-resistant streptococci, and vancomycin-resistant enterococci. Development of resistance to the compound has been infrequent thus far. Linezolid is 100% bioavailable, so it can be given in equal doses orally or parenterally. Its elimination half-life allows dosing twice per day, and alteration of drug dosage is not required in patients with impaired renal or hepatic function. Linezolid has approved indications for skin and soft tissue infections; lower respiratory tract infections; and vancomycin-resistant Enterococcus faecium infections, including cases with concurrent bacteremia. The drug has an acceptable profile of adverse events, but reversible myelosuppression has occurred in patients receiving high doses for more than 2 weeks.