Adenoviral library assay for adipogenesis genes and methods and compositions for screening compounds

A method for determining whether the expression product of a nucleic acid, capable of inducing lipid droplet formation in a cell transfected with said nucleic acid, is secreted by said cell, comprising:

• (a) infecting producer cells in a medium with an adenoviral vector comprising a unique nucleic acid capable of inducing lipid droplet formation;
• (b) combining said medium with test cells that have not been infected with said vector;
• and
• (c) determining if lipid droplets are formed in said test cells.

A method for identifying a unique nucleic acid capable of inducing lipid droplet formation in a cell, wherein said unique nucleic acid is present in a library, said method comprising:

• (a) growing a plurality of cell cultures containing at least one cell, said one cell expressing adenoviral sequence consisting essentially of E1-region sequences and expressing one or more functional gene products encoded by at least one adenoviral region selected from an E2A region and an E4 region;
• and
• (b) transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one cell in each of said plurality of cell cultures with
• i) an adapter plasmid comprising adenoviral sequence coding, in operable configuration, for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sequences sufficient to allow for homologous recombination with a first recombinant nucleic acid, and not coding for E1 region sequences which overlap with E1 region sequences in said at least one cell, for E1 region sequences which overlap with E1 region sequences in a first recombinant nucleic acid, for E2B region sequences other than essential E2B sequences, for E2A region sequences, for E3 region sequences and for E4 region sequences, and further comprises a unique nucleic acid sequence and promoter operatively linked to said unique nucleic acid sequence;
• and
• ii) a first recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for a functional adenoviral Inverted Terminal Repeat and for sequences sufficient for replication in said at least one cell, but not comprising adenoviral E1 region sequences which overlap with E1 sequences in said at least one cell, and not comprising E2A region sequences or E4 region sequences expressed in said plurality of cells which would otherwise lead to production of replication competent adenovirus wherein said first recombinant nucleic acid has sufficient overlap with said adapter plasmid to provide for homologous recombination resulting in production of recombinant adenoviral vectors in said at least one cell;
• (c) incubating said plurality of cells under conditions which result in the lysis of said plurality of cells facilitating the release of said recombinant adenoviral vectors containing said unique nucleic acid;
• and
• (d) transferring an aliquot of said adenoviral vectors into a corresponding plurality of host cell cultures consisting of cells in which said vectors do not replicate, but in which said nucleic acids are expressible;
• (e) incubating said host cells to allow expression of the product of said nucleic acid;
• and
• (f) observing said host cell for the presence of a lipid droplet

A method for identifying a drug candidate compound useful in the treatment of obesity, said method comprising:

• (a) contacting one or more test compound with a polynucleotide comprising a sequence of SEQ ID NOS:
• 14, 16, 17 or 18, or the corresponding antisense sequence thereof,
• (b) determining the binding affinity of said one or more test compound to said polynucleotide,
• (c) contacting a first subpopulation of host cells transfected with said polynucleotide with one or more of said test compound that exhibits binding affinity for said polynucleotide, and
• (d) identifying, from said one or more test compounds, a candidate compound that inhibits the formation of lipid droplets in said first subpopulation of host cells relative to a second subpopulation of said transfected host cells that have not been contacted with said candidate compound.

A method for identifying a drug candidate compound useful in the treatment of obesity, said method comprising:

• (a) contacting one or more test compound with a polypeptide expression product encoded by the polynucleotide comprising a sequence of SEQ ID NOS:
• 14 or 16,
• (b) determining the binding affinity of said test compound to said polypeptide,
• (c) contacting a first subpopulation of host cells transfected with a polynucleotide expression vector coding for said polypeptide with one or more of said test compound that exhibits binding affinity for said polypeptide, and
• (d) identifying, from said one or more test compounds, a candidate compound that inhibits the formation of lipid droplets in said first subpopulation of host cells relative to a second subpopulation of said transfected host cells that have not been contacted with said candidate compound.