Biochemical selectivity profiling against rna helicases
AssigneeBaylor College of Medicine
InventorsThomas F. Westbrook, Kristen Karlin, Calla Olson
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Abstract
Embodiments of the disclosure concern identification of suitable inhibitors of components of RNA metabolism, including those involved with splicing, such as RNA helicases. The disclosure includes in vitro screens for small molecule inhibitors that are selective for a group of RNA helicases but that are counter-selective for another group of RNA helicases. In specific embodiments, the disclosure includes a system for inhibitor analysis that includes assay development and optimization that leads to feasible screening of the intended target(s) and continued counter-selection against undesirable targets throughout the process.
Claims62
A method of screening for inhibitors in vitro for a plurality of RNA helicases, comprising the steps of:
- optionally optimizing conditions for an ATPase assay for each RNA helicase in the plurality;
- subjecting one or more candidate inhibitors to the ATPase assay for each RNA helicase in the plurality to identify candidate inhibitors that inhibit ATPase activity for a first subset of RNA helicases in the plurality but that do not inhibit ATPase activity for a second subset of RNA helicases in the plurality.
The method of any one of the preceding claims, wherein the candidate inhibitors are small molecules, proteins, peptides, nucleic acid, carbohydrate, or a combination thereof.
The method of any one of the preceding claims, wherein the subjecting step comprises high throughput screening.
The method of any one of the preceding claims, wherein the first subset of RNA helicases are of the same sub-family of helicases.
The method of any one of the preceding claims, wherein the RNA helicases in the first subset of RNA helicases share the same function in RNA metabolism.
The method of claim 10, wherein the function is RNA splicing.
The method of any one of the preceding claims, wherein the plurality of RNA helicases comprises two or more of the following RNA helicases:
- DHX8, DHX15, DHX16, DHX35, DHX33, DHX38, DHX40, DHX32, DHX34, DHX37, DHX36, DHX57, DHX29, DHX9, DHX30, UPF1, SMBP2, SETX, MOV10, MOV10L1, DHX58, IFIH1, DDX58, AQR, DDX12, DDX11, HELZ2, ZNFX1, DICER, SUV3, ASCC3, Brr2, SKIV2, MTREX, DDX60, DDX28, DDX18, DDX10, DDX55, DDX31, DDX51, DDX24, DDX56, DDX19A, DDX19B, DDX25, CIF4A1, cIF4A2, cIF4A3, DDX39B, DDX39A, DDX20, DDX6, DDX50, DDX21, DDX1, DDX54, DDX5, DDX17, DDX53, DDX43, DDX23, DDX46, DDX42, DDX41, DDX3Y, DDX3X, DDX4, DDX52, DDX59, DDX47, DDX49, and DDX27.
The method of any one of the preceding claims, wherein the plurality of RNA helicases comprises DHX15.
The method of any one of the preceding claims, wherein the first subset of RNA helicases comprises DHX15.
The method of any one of the preceding claims, wherein the method further comprises analyzing the function of one or more RNA helicases in the plurality.
The method of any one of the preceding claims, wherein the method further comprises analyzing activity of one or more candidate inhibitors in a cell, tissue, and/or organism.
The method of any one of the preceding claims, wherein the method further comprises analyzing activity of one or more candidate inhibitors by a computer.
A method of treating a subject having a medical condition associated with defective RNA metabolism comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a selective inhibitor of one or more RNA helicases, wherein the inhibitor is identified by an in vitro screening method comprising the steps of:
- optionally optimizing conditions for an ATPase assay for an RNA helicase of a plurality of RNA helicases and
- subjecting a test candidate to the ATPase assay for each RNA helicase in the plurality to identify the selective inhibitor that inhibits ATPase activity for a first subset of RNA helicases in the plurality but that do not inhibit ATPase activity for a second subset of RNA helicases in the plurality.
The method of claim 21 or 222, wherein the defective RNA metabolism is accumulation of misprocessed RNA.
The method of any one of claims 21-223, wherein the defective RNA metabolism is defective RNA splicing.
The method of any one of claims 21-227, wherein the test candidates are small molecules, proteins, peptides, nucleic acid, carbohydrate, or a combination thereof.
The method of claim 228, wherein the test candidates are small molecules.
The method of any one of claims 21-229, wherein the subjecting step comprises high throughput screening.
Dependencies are inferred from the claim text.