In vivo screen using chemical inducers of dimerization

Abstract

A method for identifying a molecule that binds a known target in a cell from a pool of candidate molecules, comprising: (a) covalently bonding each molecule in the pool of candidate molecules to a methotrexate moiety to form a screening molecule; (b) introducing the screening molecule into a cell which expresses a first fusion-protein comprising a binding domain capable of binding methotrexate, a second fusion protein comprising the known target, and a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein; (c) permitting the screening molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; (d) selecting which cell expresses the reporter gene; and (e) identifying the small molecule that binds the known target.

Description

[0001]This application is a divisional of U.S. Ser. No. 09 / 768,479, filed Jan. 24, 2001, which is a continuation-in-part of U.S. Ser. No. 09 / 490,320, filed Jan. 24, 2000, now abandoned, the contents of all of which are hereby incorporated by reference.[0002]This invention has been made with government support under National Science Foundation grants CHE-9626981, CHE-9977402, and CHE-9984928. Accordingly, the U.S. Government has certain rights in the invention.[0003]Throughout this application, various publications are referenced by author or author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.FIELD OF INVENTION[0004]The ...

AI Technical Summary

Benefits of technology

The invention provides a compound that can be used to screen proteins for their ability to catalyze bond cleavage or formation. This compound has a specific formula and can be used to activate or inhibit a cellular readout when the proteins it is designed to bind to dimerize. The method involves expressing a pair of fusion proteins in a cell and then adding the compound to see if it changes the cellular readout. This invention can help identify new proteins with catalytic activity and aid in the development of new treatments for diseases that require the formation or cleavage of bonds.

Problems solved by technology

(Kaiser 1984; Knowles 1987) Despite some successes, (Wharton 1985; Wilks 1988; Hilvert 1985, 1989, 1994; Imperiali 1994; Johnson 1993) protein engineering has proven to be difficult and has suggested that notions of how enzymes work may still be naive.

In vivo complementation of essential enzymes, such as chorismate mutase and triosephosphate isomerase, offers a direct selection for catalysis but is limited to existing reactions.

However, as with proteins, it is difficult to design screens for non-protein catalysts.

FK506, however, is not an ideal chemical handle.

However, it has not heretofore been suggested to use small molecule induced protein dimerization to screen for catalysis in vivo., and specifically, it has not been suggested to use an enzyme cleavable moiety to link two molecules to dimerize proteins.

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