Important Compound Classes
Title
Fragment-Based Screening to Identify Small Molecules that Selectively Bind RNA
Patent Publication Number
WO 2020/264572 A1
Publication Date
December 30, 2020
Priority Application
US 62/867,607
Priority Date
June 27, 2019
Inventors
Disney, M. D.
Assignee Company
The Scripps Research Institute, USA
Disease Area
RNA-based diseases
Biological Target
RNA
Summary
The aberrant expression and mutation of RNAs cause or contribute to nearly every human disease and thus are a critical targets for therapeutic intervention. The identification of small molecules that target RNA is challenging.
The library of small molecule fragments is screened for binding to RNA. A “fragment” is an organic chemical structure or substructure, for which RNA binding affinity is tested. The fragment or substructure comprises a potential RNA-binding moiety (fragment) having a linker group, such as a carboxylic acid group, that can be coupled with an alkyne-containing moiety for Click Chemistry reaction with an azide, and a diazirine-containing moiety. The assembled structure comprising the RNA-binding moiety, the alkyne-containing moiety, and a diazirine-containing moiety is referred to as a substructure reagent.
A method of identifying an RNA-binding substructure comprising a plurality of substructures involves preparing a library comprising members, wherein each member comprises one of the plurality of substructures in the form of a respective substructure reagent, wherein each substructure reagent comprises the respective substructure covalently conjugated to a moiety comprising an alkyne group and a diazirine group; then contacting the library of substructure reagents to the RNA targets, wherein the RNA comprises a radioactive label or a fluorescent label to provide RNA-contacted library; then illuminating the RNA-contacted library with UV light under conditions to cause conversion of a diazirine group to a reactive carbene which reacts covalently with the RNA; then contacting the library substructures associated with radiolabeled RNA and a pull-down reagent comprising a biotin moiety linked to the azide by a linker group comprising a disulfide bond, under conditions comprising the copper catalyst such that azide group of pull-down reagent and alkyne group of substructure reagent react to form a triazole ring, providing a biotinylated substructure reagent covalently bound to RNA only when the RNA-binding substructure is present in that member of the library, then contacting each member of the library with respective streptavidin magnetic bead, pulling down each of the biotinylated substructure reagents, then separating the beads from supernatant liquid magnetically; then measuring radioactivity or fluorescence associated with each member of the library, wherein each substructure reagent covalently associated with RNA that is bound to a magnetic bead is identified as an RNA-binding substructure.
An example is found in the substructure reagent TGP-21-diaz, which was evaluated according to the method of the invention for binding to pre-miR-21. The test substructure TGP-21-acid was coupled with the alkyne-containing moiety and then with the diazirine-containing moiety, diazirine acid, to obtain the substructure reagent TGP-21-diaz.
Key Structures
Biological Assay
The bioactivity of the TGP-21-diaz in binding the pre-miR-21 RNA at various concentrations was evaluated. Further, reaction of TGP-21-diaz with pre-miR-21 RNA was confirmed by in vitro radiolabel assay and fluorescence assay. The radioactive signal associated with the beads and in the supernatant was measured by liquid scintillation counting. The gel was stained with SYBR green and imaged to visualize all RNA (Ex: 497 nm; Em: 520 nm)
Biological Data
The bioactivity of the TGP-21-diaz in binding the RNA pre-miR-21 at various concentrations, with the compound “methyl-diaz” being a nonbinding but alkyne- and diazirine-containing control molecule. A concentration of about 10 μM TGP-21-diaz is sufficient to bring about covalent binding with pre-miR-21 RNA as shown by incorporation of the RNA radiolabel onto the magnetic streptavidin beads. Accordingly, TGP-21-diaz can be identified in the presence of a plurality of other substructure reagents as having affinity for the pre-miR-21 RNA.
Claims
Total claims: 14
Method of identifying claims: 14
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The author declares no competing financial interest.