Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-24
2002-09-24
Ponnaluri, Padmashri (Department: 1618)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007900, C435S091500, C435S091500, C435S091500, C436S518000, C436S527000, C436S531000, C436S080000, C436S172000
Reexamination Certificate
active
06455263
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of screening libraries of small molecules such as combinatorial chemical libraries of organic molecules, including peptides and other chemical libraries, for binding to target molecules, using fluoroscence-activated cell sorting (FACS) machines.
BACKGROUND OF THE INVENTION
Traditional approaches to identify and characterize new and useful drug candidates include the isolation of natural products or synthetic preparation, followed by testing against either known or unknown targets. See for example WO 94/24314, Gallop et al., J. Med. Chem. 37(9):1233 (1994); Gallop et al., J. Med. Chem. 37(10):1385 (1994); Ellman, Acc. Chem. Res. 29:132 (1996); Gordon et al., E. J. Med. Chem. 30:388s (1994); Gordon et al., Acc. Chem. Res. 29:144 (1996); WO 95/12608, all of which are incorporated by reference.
The screening of these libraries is done in a variety of ways. One approach involves attachment to beads and visualization with dyes; see Neslter et al., Bioorg. Med. Chem. Lett. 6(12):1327 (1996). Another approach has utilized beads and fluorescence activated cell sorting (FACS); see Needles et al., PNAS USA 90:10700 (1993), and Vetter et al., Bioconjugate Chem. 6:319 (1995).
Fluorescence activated cell sorting (FACS), also called flow cytometry, is used to sort individual cells on the basis of optical properties, including fluorescence. It is generally fast, and can result in screening large populations of cells in a relatively short period of time.
Accordingly, it is an object of the invention to provide methods for the rapid, accurate screening of candidate agents, particularly libraries of agents, using FACS methods.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides methods for screening candidate bioactive agents for binding to a target molecule. The methods comprise contacting a library of candidate bioactive agents covalently attached to a plurality of beads with at least a first population of first target molecules labeled with a first labeling moiety. A fluorescent second labeling moiety capable of binding to said first labeling moiety is then added, and the beads are sorted using a fluorescent activated cell sorter (FACS) machine to obtain a population of fluorescent beads and a population of non-fluorescent beads. The presence of at least one fluorescent bead is indicative that at least one candidate bioactive agent that binds to at least one target molecule.
In an additional aspect, the methods comprise contacting a library of candidate bioactive agents covalently attached to a plurality of beads with at least a first population of first target molecules comprising at least a first subpopulation and a second subpopulation. The first subpopulation is labeled with a first labeling moiety (and optionally with a third labeling moiety) and the second subpopulation is labeled with a second labeling moiety (and optionally with a fourth labelling moiety). The beads are then sorted using a fluorescent activated cell sorter (FACS) machine to obtain a population of fluorescent beads and a population of non-fluorescent beads, wherein the presence of at least one fluorescent bead is indicative that at least one candidate bioactive agent binds to at least one first target molecule.
In a further aspect, the methods comprise contacting a library of candidate bioactive agents covalently attached to a plurality of beads with at least a first population of first target molecules labeled with a first labeling moiety (and optionally a second labeling moiety), and sorting the beads using a fluorescent activated cell sorter (FACS) machine to obtain a population of fluorescent beads and a population of non-fluorescent beads. A competitor moiety known to bind to the first target is then added, and the beads are resorted by a FACS machine to produce a population of beads which are no longer fluorescent, wherein the presence of at least one bead which was fluorescent in step (c) but is no longer fluorescent in step (e) indicates that at least one candidate bioactive agent is a bioactive agent that binds to is said first target molecule.
In an additional aspect, the methods of the invention further comprise chemically modifying the candidate bioactive agents to form new candidate bioactive agents, which may then be rescreened using the methods of the invention.
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Dorsey & Whitney LLP
Ponnaluri Padmashri
Rigel Pharmaceuticals Inc.
Silva, Esq. Robin M.
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