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Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

ABSTRACT: We describe a scalable nuclear magnetic resonance (NMR) screening approach to identify and prioritize small molecule fragments that bind to structured RNAs. This approach is target agnostic and, therefore, amenable to many RNA structures and libraries, and it provides initial hits for further synthetic elaboration and structure-based drug discovery efforts. We demonstrate the approach on the pre-miR-21 stem-loop, which is of significant interest in oncology and metabolic diseases. We screened the pre-miR-21 hairpin using a small (420 compounds) commercially available fragment library and identified 18 hits in the first round of triage screening. This was further refined to four fragments which passed all screening cascade filters. Among these four hits, a thiadiazole fragment was demonstrated to bind the Dicer cleavage site of pre-miR-21 by target-detected NMR experiments and through the observation of clear intermolecular NOEs.

SUBMITTER: Shortridge MD

PROVIDER: S-EPMC8366005 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

Shortridge Matthew D MD Varani Gabriele G

ACS medicinal chemistry letters 20210713 8

We describe a scalable nuclear magnetic resonance (NMR) screening approach to identify and prioritize small molecule fragments that bind to structured RNAs. This approach is target agnostic and, therefore, amenable to many RNA structures and libraries, and it provides initial hits for further synthetic elaboration and structure-based drug discovery efforts. We demonstrate the approach on the pre-miR-21 stem-loop, which is of significant interest in oncology and metabolic diseases. We screened th ...[more]

PMID: 34413954

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Project description:In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

Dataset Information

Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

Publications

Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

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