Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-24
2001-10-30
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S006120, C435S007200, C435S007210, C435S007500, C435S091500, C435S091500, C435S091500, C536S023100, C436S501000, C436S518000
Reexamination Certificate
active
06309842
ABSTRACT:
TECHNICAL FIELD
The invention applies the technical fields of combinatorial chemistry and molecular genetics to the identification of compounds with desired properties, such as capacity to bind to, agonize or antagonize a cellular receptor.
BACKGROUND OF THE INVENTION
Several methods have been reported for producing and screening large libraries to identify compounds having specific affinity for a target. These methods include the phage-display method in which randomized peptides are If displayed from phage and screened by affinity chromatography to an immobilized receptor. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; Ladner, U.S. Pat. No. 5,223,409 (incorporated by reference in their entirety for all purposes). In another approach, combinatorial libraries of polymers immobilized on a chip are synthesized using photolithography. See, e.g., U.S. Pat. No. 5,143,854; WO 90/15070 and WO 92/10092. The immobilized polymers are contacted with a labelled receptor and scanned for label to identify polymers binding to the receptor.
A general, and particularly useful, method for synthesizing and screening large libraries of compounds is the encoded synthetic library method (ESL) of Dower et al. In this method, the different compounds in the library are usually synthesized attached to separate supports (e.g., beads) by stepwise addition of the various components of the compounds in several rounds of coupling. A round of coupling can be performed by apportioning the supports between different reaction vessels and adding a different component to the supports in the different reaction vessels. The particular component added in a reaction vessel can be recorded by the addition of a tag component to the support at a second site. After each round of synthesis, supports from the same reaction vessel can be apportioned between different reaction vessels and/or pooled with supports from another reaction vessel in the next round of synthesis. In any, and usually in all rounds of synthesis, the component added to the support can be recorded by addition of a further tag component at a second site of the support. After several rounds of synthesis, a large library of different compounds is produced in which the identities of compounds are encoded in tags attached to the respective supports bearing the compounds. The library can be screened for binding to a target. Supports bearing compounds having a specific affinity for the target are isolated, and the identity of such compounds can be determined by decoding the tags.
All of the above methods have proved successful in isolating compounds having specific affinity for a target of interest. For example, the methods have been used to isolate compounds that bind to a cellular receptor for use as antagonists of receptor-ligand interactions. However, the repertoire of compounds that can be identified by some of the above methods is somewhat limited by the fact that compounds are screened in a tethered format. Furthermore, existing screening methods generally have not been used to distinguish between compounds that merely bind to a receptor, and compounds that are capable of transducing a biological signal through the receptor. The latter compounds are expected to have particularly useful properties, such as the capacity to agonize normal ligand-receptor interactions. These properties can be exploited in many applications such as stimulation of cell or tissue growth, and enzyme, growth factor or hormone replacement therapy.
The present invention provides methods for screening compounds for capacity to transduce a signal through a cellular receptor, thereby allowing the isolation of novel pharmaceuticals.
SUMMARY OF THE INVENTION
The invention provides methods for screening libraries of compounds for a desired activity. In many of the methods, a library of complexes is produced in which each complex comprises a compound under test, a tag recording at least one step in the synthesis of the compound, and a tether susceptible to modification by a reporter molecule. One or more, but not all, of the complexes have a tether that has been modified by the reporter enzyme, the modification indicating that the one or more complexes bear compound(s) having the desired property. At least one complex having a modified tether is separated from the library by virtue of the modified tether. The tag of the complex having the modified tether is then decoded to identify at least one step in the synthesis of a compound having the desired property.
In one aspect, the invention provides methods of screening compounds for capacity to transduce a signal through a cellular receptor. The methods entail providing a plurality of complexes, each complex comprising a compound under test, a tag recording at least one step in synthesis of the compound, and a tether susceptible to modification by a reporter molecule. The complexes are contacted with cells having a receptor and a DNA segment encoding the reporter molecule. At least one compound transduces a signal through the receptor of a cell causing release of reporter molecule expressed from the DNA segment from the cell. The complex having the modified tether is then isolated. The tag of this complex identifies at least one step in the synthesis of the compound transducing the signal.
Optionally, complexes comprise compounds linked to supports by a photocleavable linker and the compounds are freed from the supports by exposure to radiation to allow free compounds to diffuse into contact with cellular receptors. Examples of compounds that can be screened include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. One class of receptor of interest for screening compounds for transducing activity are G-protein coupled receptors. Often, compounds are contacted with supports in a medium that limits diffusion of released reporter, such as a gel matrix. The selection of tether and reporter molecule are interdependent; for example, in one format, the reporter molecule is a protease and the tether bears a site for the protease. In a variation, a second reporter molecule is induced by signal transduction, and the second reporter molecule modifies or induces the reporter molecule which modifies the tether.
In a further variation, the cells further comprise a second DNA segment encoding a site-specific recombinase, and the DNA segment contains a gene encoding the reporter molecule, which gene is disrupted by an inactivating sequence flanked by sites recognized by the site-specific recombinase. The transducing compound causes expression of the site-specific recombinase, which excises the inactivating sequence from the gene resulting in expression of the reporter molecule, which is released from the cell and modifies the tether of the complex from which the compound was released.
In a second aspect, the invention provides additional methods for screening compounds for capacity to transduce a signal through a cellular receptor. In these methods, a plurality of supports are provided, each support bearing a compound under test, and a tether susceptible to modification by a reporter molecule, however, the supports need not have tags. The supports are contacted with cells having a receptor and a DNA segment encoding the reporter molecule. The compounds are partially freed from the supports, whereby at least one compound transduces a signal through the receptor of a cell causing expression of the reporter molecule, which reporter molecule is released from the cell and modifies the tether of the support from which the compound transducing the signal was partially freed. The support having the modified tether is isolated, which support bears the compound transducing the signal.
In another aspect, the invention provides a further method for screening compounds for capacity to transduce a signal through a cellular receptor. In these methods, compounds are provided as an arr
Dower William J.
Gates Christian M.
Heinkel Gregory L.
Lalonde Guy
Mattheakis Larry C.
Glaxo Wellcome Inc.
Ponnaluri Padmashri
Townsend & Townsend & Crew
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