Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

Connelly, Colleen M.; Abulwerdi, Fardokht A.; Schneekloth, John S.

doi:10.1007/978-1-4939-6584-7_11

Cited by 41 publications

(47 citation statements)

References 20 publications

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“…SMM slides were prepared according to established procedures 36,38 . In brief, γ-aminopropyl silane microscope slides (Corning) were functionalized with an Fmoc-protected amino polyethylene glycol spacer (Fmoc-8-amino-3,6-dioxaoctanoic acid) in N,N -dimethylformamide (DMF).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2019

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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 PreQ 1 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2019

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“…To probe nucleic acid binding to known, pharmacologically active compounds, we used an SMM screening platform ( 40 , 45 ). We generated SMMs consisting of the MIPE library ( 41 , 46 , 47 ) (a collection of 1902 FDA approved drugs, compounds currently undergoing clinical trials, and other well-annotated and validated inhibitors) and the NCI Diversity set ( 42 ) (a freely available collection of 1705 chemically diverse compounds).…”

Section: Resultsmentioning

confidence: 99%

Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Calabrese

Zlotkowski

Alden

et al. 2018

Nucleic Acids Research

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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.

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“…primary and secondary alcohols and amines, are spatially arrayed with a robotic microarrayer and covalently linked to a glass surface at high density. 112 The microarray is then incubated with fluorescently-labeled target RNA, followed with washing to remove any unspecific binding. The slides are imaged using a fluorescence scanner, and fluorescence intensity is quantified for each spot on the array.…”

Section: Small Molecule Microarray (Smm)mentioning

confidence: 99%

“…specific to the TAR hairpin. Also, recently, Connelly et al 112 used the SMM approach to identify novel small molecules that selectively bind in proximity to the apical loop of pre-miR-21 hairpin, preventing biogenesis of miR-21 through Dicer inhibition ( Fig. 2A).…”

Section: Small Molecule Microarray (Smm)mentioning

confidence: 99%

Unveiling the druggable RNA targets and small molecule therapeutics

Sztuba-Solińska

Chávez-Calvillo

Cline

2019

Bioorganic & Medicinal Chemistry

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Keywords:RNA structure and function RNA interactions Epitranscriptomics Small molecules RNA-based therapeutics RNA in disease Drug target A B S T R A C T The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets. RNA as a drug targetThe vast diversity of RNAs expanding beyond coding transcripts to several classes of ncRNAs that vary in length, biogenesis, polarity, and putative functions, increases the repertoire of druggable targets. 19,20 Here, specific RNA properties contribute to its attractive, yet challenging makeup as a target molecule. RNA can form complex three-dimensional structures through canonical Watson-Crick base pairing and complex tertiary interactions that are mediated by non-canonical bonds. Such structures can be as intricate and stable as those formed by proteins and can recognize small-molecule ligands, other nucleic acids, and/or proteins with high affinity and specificity. [21][22][23][24] At the same time, the highly dynamic conformation and repetitive character of its surface presents difficulties for drug design. 25,26 In addition, many putative small molecule-binding pockets in RNA are much more polar and solvent exposed than binding sites on proteins, complicating ligand design efforts. Compounding these issues, most target RNAs are expressed at low levels, with the exception of ribosomal (rRNA) and transfer (tRNA) RNAs, which constitute 80-90% and 10-15% of total cellular RNA, respectively. 27 Other abundant RNAs, such as messenger (mRNA), small nuclear (snRNA), and small nucleolar (snoRNA) are present at levels that are about 1-2 orders of magnitude lower than rRNA and tRNA. Certain small RNAs, such as micro (miRNA) and piwi (piRNAs) can be present at very high levels; however, this appears to be cell type dependent. One should keep in mind that if the RNA has catalytic activity or if it acts as a scaffold to regulate https://doi.

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Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

Cited by 41 publications

References 20 publications

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

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

Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Unveiling the druggable RNA targets and small molecule therapeutics

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