Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design.

Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP) and 5'-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ∼3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed.

Ligand-observed NMR techniques to probe RNA-small molecule interactions.

A growing understanding of the structure and function of RNA has revealed it as a key regulator of gene expression and disease. A multitude of noncoding functions apart from the central roles of RNA in coding for and facilitating protein biogenesis has stimulated research into RNA as a pharmacological target. Despite many exciting advances, RNA remains an understudied target for small molecules, and techniques to investigate RNA-binding molecules are still emerging. A key stumbling block in this area has been validation of RNA-small molecule interactions. Our laboratory has recently used multiple ligand-observed NMR techniques in this regard, including CPMG and WaterLOGSY. This work describes methods to use these techniques in the context of studying RNA-ligand interactions.

Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure.

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.

Small Molecule Inhibition of MicroRNA miR-21 Rescues Chemosensitivity of Renal-Cell Carcinoma to Topotecan.

Chemical probes of microRNA (miRNA) function are potential tools for understanding miRNA biology that also provide new approaches for discovering therapeutics for miRNA-associated diseases. MicroRNA-21 (miR-21) is an oncogenic miRNA that is overexpressed in most cancers and has been strongly associated with driving chemoresistance in cancers such as renal cell carcinoma (RCC). Using a cell-based luciferase reporter assay to screen small molecules, we identified a novel inhibitor of miR-21 function. Following structure-activity relationship studies, an optimized lead compound demonstrated cytotoxicity in several cancer cell lines. In a chemoresistant-RCC cell line, inhibition of miR-21 via small molecule treatment rescued the expression of tumor-suppressor proteins and sensitized cells to topotecan-induced apoptosis. This resulted in a >10-fold improvement in topotecan activity in cell viability and clonogenic assays. Overall, this work reports a novel small molecule inhibitor for perturbing miR-21 function and demonstrates an approach to enhancing the potency of chemotherapeutics specifically for cancers derived from oncomir addiction.

Characterization of clinically used oral antiseptics as quadruplex-binding ligands.

Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.

Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays.

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRNAs. Here, we report the use of a small molecule microarray (SMM) screening approach to identify low molecular weight compounds that directly bind to a pre-miR-21 hairpin. Compounds identified using this approach exhibit good affinity for the RNA (ranging from 0.8-2.0 µM) and are not composed of a polycationic scaffold. Several of the highest affinity compounds inhibit Dicer-mediated processing, while in-line probing experiments indicate that the compounds bind to the apical loop of the hairpin, proximal to the Dicer site. This work provides evidence that small molecules can be developed to bind directly to and inhibit miR-21.

Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays.

New methods to identify RNA-binding small molecules open yet unexplored opportunities for the pharmacological modulation of RNA-driven biology and disease states. One such approach is the use of small molecule microarrays (SMMs). Typically, SMMs are generated by spatially arraying and covalently linking a library of small molecules to a glass surface. Next, incubation of the arrays with a fluorescently labeled RNA reveals binding interactions that are detected upon slide imaging. The relative ease with which SMMs are manufactured enables the screening of multiple oligonucleotides in parallel against tens of thousands of small molecules, providing information about both binding and selectivity of identified RNA-small molecule interactions. This approach is useful for screening a broad variety of structurally and functionally diverse RNAs. Here, we present a general method for the preparation and use of SMMs to rapidly identify small molecules that selectively bind to an RNA of interest.

The Emerging Role of RNA as a Therapeutic Target for Small Molecules.

Recent advances in understanding different RNAs and unique features of their biology have revealed a wealth of information. However, approaches to identify small molecules that target these newly discovered regulatory elements have been lacking. The application of new biochemical screening and design-based technologies, coupled with a resurgence of interest in phenotypic screening, has resulted in several compelling successes in targeting RNA. A number of recent advances suggest that achieving the long-standing goal of developing drug-like, biologically active small molecules that target RNA is possible. This review highlights advances and successes in approaches to targeting RNA with diverse small molecules, and the potential for these technologies to pave the way to new types of RNA-targeted therapeutics.

Aryl amide small-molecule inhibitors of microRNA miR-21 function.

MicroRNAs (miRNAs) are single stranded RNA molecules of ∼22 nucleotides that negatively regulate gene expression. MiRNAs are involved in fundamental cellular processes, such as development, differentiation, proliferation, and survival. MiRNA misregulation has been linked to various human diseases, most notably cancer. MicroRNA-21 (miR-21), a well-established oncomiR, is significantly overexpressed in many types of human cancers, thus rendering miR-21 a potential therapeutic target. Using a luciferase-based reporter assay under the control of miR-21 expression, a high-throughput screen of >300,000 compounds led to the discovery of a new aryl amide class of small-molecule miR-21 inhibitors. Structure-activity relationship (SAR) studies resulted in the development of four aryl amide derivatives as potent and selective miR-21 inhibitors. The intracellular levels of various miRNAs in HeLa cells were analyzed by qRT-PCR revealing specificity for miR-21 inhibition over other miRNAs. Additionally, preliminary mechanism of action studies propose a different mode of action compared to previously reported miR-21 inhibitors, thus affording a new chemical probe for future studies.

Thiourea-based fluorescent chemosensors for aqueous metal ion detection and cellular imaging.

We describe three significant advances in the use of thioureas as reporting elements for metal-responsive fluorescent chemosensors. First, on the basis of the crystal structure of a chemosensor analogue, we provide a deeper understanding of the details of the thiourea coordination environment. Second, we describe a new generation of chemosensors with higher affinities for Zn(2+) and Cd(2+) than were observed for earlier probes, expanding the scope of this type of probe beyond Hg(2+) detection. Third, we show that a thiourea-based chemosensor can be employed for fluorescence microscopy imaging of Hg(2+) ion concentrations in living mammalian cells.

Control of oncogenic miRNA function by light-activated miRNA antagomirs.

MicroRNAs (miRNAs) are single stranded noncoding RNAs of approximately 22 nucleotides that act as posttranscriptional gene regulators by binding partially complementary sequences in the 3' untranslated region (3'-UTR) of target messenger RNAs (mRNAs). MicroRNAs regulate many biological processes including embryonal development, differentiation, apoptosis, and proliferation and the targets of miRNAs range from signalling proteins and transcription factors to RNA binding proteins. Recently, variations in the expression of certain miRNAs have been linked to a variety of human diseases including cancer and viral infections, validating miRNAs as potential targets for drug discovery. Several tools have been developed to control the function of individual miRNAs and have been applied to study their biological role and therapeutic potential; however, common methods lack a precise level of control that allows for the study of miRNA function with high spatial and temporal resolution. Toward this goal, a light-activated miRNA antagomir for mature miR-21 was developed through the site-specific installation of caging groups on the bases of selected nucleotides. Installation of caged nucleotides led to complete inhibition of the antagomir-miRNA hybridization and inactivation of antagomir function. The miRNA-inhibitory activity of the caged antagomirs was fully restored upon decaging through a brief UV irradiation. The synthesized antagomir was applied to the photochemical regulation of miR-21 function in mammalian cells. Moreover, spatial and temporal control over antagomir activity and thus miR-21 function was obtained in mammalian cells. The presented approach enables the precise regulation of miRNA function with unprecedented spatial and temporal resolution using UV irradiation and can be readily extended to any miRNA of interest.

Cellular microRNA sensors based on luciferase reporters.

Recently, microRNAs (miRNAs) have been linked to a variety of human diseases including cancer and viral infections. Small molecule modifiers of miRNAs could represent new therapeutic agents and be used as tools for elucidating the biological roles of miRNAs. In order to identify small molecule modifiers of miRNAs, functional assays for specific miRNAs must be developed and optimized. Here, we report the construction of a luciferase reporter assay for miRNA miR-122 function and the development of a stable Huh7 cell line that can be used for high-throughput screening of small molecule miR-122 inhibitors. The steps described here can be applied not only to Huh7 cells and miR-122 but also to virtually any cell line and miRNA combination.

Identification of inhibitors of microRNA function from small molecule screens.

Aberrant expression of microRNAs (miRNAs) has been linked to many human diseases including cancer, immune disorders, heart disease, and viral infections. Thus, small molecule inhibitors of miRNAs have potential as new therapeutic agents, as probes for the elucidation of detailed mechanisms of miRNA function, and as tools for the discovery of new targets for the treatment of human diseases. In order to identify small molecule inhibitors of specific miRNAs, functional assays have been developed and applied to the screening of small molecule libraries. Here, we report the application of a luciferase-based reporter assay of miRNA miR-122 function to the discovery of small molecule miR-122 inhibitors.

Spatiotemporal control of microRNA function using light-activated antagomirs.

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators and have been shown to regulate many biological processes including embryonal development, cell differentiation, apoptosis, and proliferation. Variations in the expression of certain miRNAs have been linked to a wide range of human diseases - especially cancer - and the diversity of miRNA targets suggests that they are involved in various cellular networks. Several tools have been developed to control the function of individual miRNAs and have been applied to study their biogenesis, biological role, and therapeutic potential; however, common methods lack a precise level of control that allows for the study of miRNA function with high spatial and temporal resolution. Light-activated miRNA antagomirs for mature miR-122 and miR-21 were developed through the site-specific installation of caging groups on the bases of selected nucleotides. Installation of caged nucleotides led to complete inhibition of the antagomir-miRNA hybridization and thus inactivation of antagomir function. The miRNA-inhibitory activity of the caged antagomirs was fully restored upon decaging through a brief UV irradiation. The synthesized antagomirs were applied to the photochemical regulation of miRNA function in mammalian cells. Moreover, spatial control over antagomir activity was obtained in mammalian cells through localized UV exposure. The presented approach enables the precise regulation of miRNA function and miRNA networks with unprecedented spatial and temporal resolution using UV irradiation and can be extended to any miRNA of interest.

High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function.

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3' untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.

Small molecule modifiers of microRNA miR-122 function for the treatment of hepatitis C virus infection and hepatocellular carcinoma.

MicroRNAs are a recently discovered new class of important endogenous regulators of gene function. Aberrant regulation of microRNAs has been linked to various human diseases, most importantly cancer. Small molecule intervention of microRNA misregulation has the potential to provide new therapeutic approaches to such diseases. Here, we report the first small molecule inhibitors and activators of the liver-specific microRNA miR-122. This microRNA is the most abundant microRNA in the liver and is involved in hepatocellular carcinoma development and hepatitis C virus (HCV) infection. Our small molecule inhibitors reduce viral replication in liver cells and represent a new approach to the treatment of HCV infections. Moreover, small molecule activation of miR-122 in liver cancer cells selectively induced apoptosis through caspase activation, thus having implications in cancer chemotherapy. In addition to providing a new approach for the development of therapeutics, small molecule modifiers of miR-122 function are unique tools for exploring miR-122 biogenesis.