Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7–Lin28 RNA–Protein Interaction

Lorenz, Daniel A.; Kaur, Tanpreet; Kerk, Samuel A.; Gallagher, Erin; Sandoval, Jorge; Garner, Amanda L.

doi:10.1021/acsmedchemlett.8b00126

Cited by 38 publications

(35 citation statements)

References 40 publications

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“…As the discovery of direct-binding, functional inhibitors of RNA biology is becoming a high priority in RNAtargeted drug discovery campaigns, assays like cat-ELCCA will play an important role in these early stage efforts. Further, as we have also demonstrated the applicability of cat-ELCCA to RNA-protein interactions (Lorenz, Kaur, et al, 2018), another important area of nucleic acid biology with therapeutic potential, we foresee that many important tools for interrogating this new frontier in drug discovery will arise from this platform assay technology. miRNA biogenesis and activity, and the impact of targeting miRNAs with a natural product inhibitor of Dicer-mediated maturation.…”

Section: Discussionmentioning

confidence: 96%

“…To facilitate the discovery of new chemical entities for targeting RNAs, we have developed high-throughput screening (HTS) technology termed catalytic enzyme-linked click chemistry assay, or cat-ELCCA, which is a robust screening platform that does not suffer from compound interference (Garner, 2018a;Lorenz & Garner, 2016Lorenz, Song, & Garner, 2015;Lorenz, Vander Roest, Larsen, & Garner, 2018). Key advantages of cat-ELCCA include its increased sensitivity due to catalytic signal amplification, negligible compound interference in comparison to traditional fluorescence-based assays due to added washing steps, and HTS applicability with Z' factors >0.6 using automated liquid handling (Garner, 2018a;Garner & Janda, 2010, 2011Lorenz & Garner, 2016Lorenz, Kaur, et al, 2018;Lorenz et al, 2015;Lorenz, Vander Roest, et al, 2018;Song, Menon, Mitchell, Johnson, & Garner, 2017;Zhang, Chung, & Oldenburg, 1999). As proof of concept for RNA-targeted probe discovery, we applied cat-ELCCA toward the goal of identifying selective and RNA-binding inhibitors of Dicer-mediated microRNA (miRNA or miR) maturation ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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A click chemistry assay to identify natural product ligands for pre-microRNAs

Garner

Lorenz

Gallagher

2019

Methods in Enzymology

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Despite the great diversity of structure and function and relevance to human health, RNA remains an underexploited area of drug discovery. A major bottleneck toward this goal has been the identification of probes and drug leads that are specific for select RNAs and methods that will facilitate such discovery efforts. Our laboratory has recently developed an innovative approach for assaying RNA-small molecule interactions, catalytic enzyme-linked click chemistry assay or cat-ELCCA, which is a functional assay that takes advantage of the power of catalytic signal amplification combined with the selectivity and bioorthogonality of click chemistry. Importantly, through application of this platform assay technology to the challenging problem of identifying selective inhibitors of pre-microRNA maturation, we identified natural products as a potential source of such compounds. Herein we describe this methodology in addition to the downstream pipeline toward the discovery of natural product ligands for premicroRNAs. Through cat-ELCCA, our goal is to discover novel ligands to facilitate our investigation of RNA recognition by small molecules.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

A click chemistry assay to identify natural product ligands for pre-microRNAs

Garner

Lorenz

Gallagher

2019

Methods in Enzymology

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“…Only two N,N -(1,2-phenylene)-dibenzenesulfonamide derivatives, namely CCG-233094 and CCG-234459, showed dose-dependent inhibition in both cat-ELCCA and EMSA with micromolar IC50s. The binding between Lin28A and these two compounds was confirmed by SPR [59]. Nevertheless, whether the molecules can upregulate let-7 level and lead to any biological consequences in the cellular context remains to be tested.…”

Section: Other Hts Targeting Lin28/let-7 and Conclusionmentioning

confidence: 91%

“…This was followed by click chemistry with methyltetrazine-conjugated horseradish peroxidase (mTet-HRP). Lin28/let-7 interaction was detected by measuring chemiluminescence signals in the presence of an HRP substrate [59]. The screening exhibited a Z' prime of 0.5 and generated 1,468 initial hits with more than 25% inhibition.…”

Section: Other Hts Targeting Lin28/let-7 and Conclusionmentioning

confidence: 99%

RNA-Targeted Therapies and High-Throughput Screening Methods

Zhu

Rooney

Michlewski

2020

IJMS

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RNA-binding proteins (RBPs) are involved in regulating all aspects of RNA metabolism, including processing, transport, translation, and degradation. Dysregulation of RNA metabolism is linked to a plethora of diseases, such as cancer, neurodegenerative diseases, and neuromuscular disorders. Recent years have seen a dramatic shift in the knowledge base, with RNA increasingly being recognised as an attractive target for precision medicine therapies. In this article, we are going to review current RNA-targeted therapies. Furthermore, we will scrutinise a range of drug discoveries targeting protein-RNA interactions. In particular, we will focus on the interplay between Lin28 and let-7, splicing regulatory proteins and survival motor neuron (SMN) pre-mRNA, as well as HuR, Musashi, proteins and their RNA targets. We will highlight the mechanisms RBPs utilise to modulate RNA metabolism and discuss current high-throughput screening strategies. This review provides evidence that we are entering a new era of RNA-targeted medicine.

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“…Estimates suggest that the number of potential human RNA drug targets are an order of magnitude greater than the number of protein targets [6,7]. Compounds that bind RNA have been identified by using screening [8][9][10], structure-based design [11,12], or sequence-based design [13]. However, only a very limited number of these small molecules are bioactive and far fewer are known to target human RNAs and affect their function [4].…”

Section: Introductionmentioning

confidence: 99%

A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways

Benhamou

Angelbello

Wang

et al. 2019

Preprint

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2 HIGHLIGHTS• Intron retention in RNA repeat expansions can be due to repeats binding to proteins • Small molecules that bind RNA repeats and inhibit protein binding can trigger decay • A toxic RNA repeat can catalyze the synthesis of its own inhibitor on-site• On-site drug synthesis most potently affects disease biology eTOC BLURBThe most common way to target RNA is to use antisense oligonucleotides to target unstructured RNAs for destruction. Here, we show for the first time that small molecules targeting structured, disease-causing RNAs can shunt them towards native decay pathways by affecting their processing. SUMMARYMyotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy caused by a toxic expanded repeat of r(CCUG) [heretofore (CCUG) exp ], harbored in intron 1 of CHC-Type Zinc Finger Nucleic Acid Binding Protein (CNBP) pre-mRNA. This r(CCUG) exp causes DM2 via a gain-offunction mechanism that results in three hallmarks of its pathology: (i) binding to RNA-binding proteins (RBPs) that aggregate into nuclear foci; (ii) sequestration of muscleblind-like-1 (MBNL1) protein, a regulator of alternative pre-mRNA splicing, leading to splicing defects; and (iii) retention of intron 1 in the CNBP mRNA. Here, we find that CNBP intron retention is caused by the r(CCUG) exp -MBNL1 complex and can be rescued by small molecules. We studied two types of small molecules with different modes of action, ones that simply bind and ones that can be synthesized by a r(CCUG) exp -templated reaction in cells, that is the RNA synthesizes its own drug.Indeed, our studies completed in DM2 patient-derived fibroblasts show that the compounds disrupt the r(CCUG) exp -MBNL1 complex, reduce intron retention, subjecting the liberated intronic r(CCUG) exp to native decay pathways, and rescue other DM2-associated cellular defects.Collectively, this study shows that small molecules can affect RNA biology by shunting toxic transcripts towards native decay pathways. processing will be required to understand and capture the full potential of compounds to target RNA and rescue disease biology. The development of chemical probes and lead medicines targeting structured RNA, particularly human RNAs, are in their infancy. It will be interesting to see the types of chemical structures, their features, and the functional responses that emerge.

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Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7–Lin28 RNA–Protein Interaction

Cited by 38 publications

References 40 publications

A click chemistry assay to identify natural product ligands for pre-microRNAs

A click chemistry assay to identify natural product ligands for pre-microRNAs

RNA-Targeted Therapies and High-Throughput Screening Methods

A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways

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