The technical field of this invention is methods for screening for transdominant effector peptides and RNA molecules selected inside living cells from randomized pools.
Signaling pathways in cells often begin with an effector stimulus that leads to a phenotypically describable change in cellular physiology. Despite the key role intracellular signaling pathways play in pathogenesis, in most cases, little is understood about a signaling pathway other than the initial stimulus and the ultimate cellular response.
Historically, signal transduction has been analyzed by biochemistry or genetics. The biochemical approach dissects a pathway in a xe2x80x9cstepping-stonexe2x80x9d fashion: find a molecule that acts at, or is involved in, one end of the pathway, isolate assayable quantities and then try to determine the next molecule in the pathway, either upstream or downstream of the isolated one. The genetic approach is classically a xe2x80x9cshot in the darkxe2x80x9d: induce or derive mutants in a signaling pathway and map the locus by genetic crosses or complement the mutation with a cDNA library. Limitations of biochemical approaches include a reliance on a significant amount of pre-existing knowledge about the constituents under study and the need to carry such studies out in vitro, post-mortem. Limitations of purely genetic approaches include the need to first derive and then characterize before proceeding with identifying and cloning the gene.
Screening molecular libraries of chemical compounds for drugs that regulate signaling systems has led to important discoveries of great clinical significance. Cyclosporin A (CsA) and FK506, for examples, were selected in standard pharmaceutical screens for inhibition of T-cell activation. It is noteworthy that while these two drugs bind completely different cellular proteinsxe2x80x94cyclophilin and FK506 binding protein (FKBP), respectively, the effect of either drug is virtually the samexe2x80x94profound and specific suppression of T-cell activation, phenotypically observable in T cells as inhibition of mRNA production dependent on transcription factors such as NF-AT and NF-xcexaB. Libraries of small peptides have also been successfully screened in assays for bioactivity. The literature is replete with examples of small peptides capable of modulating a wide variety of signaling pathways. For example, a peptide derived from the HIV- 1 envelope protein has been shown to block the action of cellular calmodulin.
A major limitation of conventional in vitro screens is delivery. While only minute amounts of an agent may be necessary to modulate a particular cellular response, delivering such an amount to the requisite subcellular location necessitates exposing the target cell or system to relatively massive concentrations of the agent. The effect of such concentrations may well mask or preclude the targeted response.
The present invention provides methods and compositions to create, effectively introduce into cells and screen compounds that affect a signaling pathway. Little or no knowledge of the pathway is required, other than a presumed signaling event and an observable physiologic change in the target cell. The disclosed methods are conceptually distinct from prior library search methods in that it is an in vivo stratagem for accessing intracellular signaling mechanisms. The invention also provides for the isolation of the constituents of the pathway, the tools to characterize the pathway, and lead compounds for pharmaceutical development.
Relevant Literature
Mann et al. (1983) Cell 33, 153-159, Pear et al. (1993) Proc. Natl. Acad. Sci. USA 90(18):8392-6 and copending U.S. patent application Ser. No. 08/023,909 describe the BOSC and BING retroviral systems useful as delivery vectors for the disclosed methods.
Scott and Craig (1994) Current Opinion in Biotechnology 5:40-48 review random peptide libraries. Hupp et al. (1995) describe small peptides which activate the latent sequence-specific DNA binding function of p53. Palzkill et al. (1994) report the selection of functional signal cleavage sites from a library of random sequences introduced into TEM-1-lactamase.
The invention provides methods and compositions for screening for transdominant intracellularly bioactive agents such as pharmaceuticals. The invention accesses molecules or targets within living cells and provides for the direct selection of those bioactive agents with desired phenotypic effects. The general methods involve steps: expressing a molecular library of randomized nucleic acids as a plurality of isolated corresponding randomized expression products in a plurality of cells, each of the nucleic acids comprising a different nucleotide sequence, screening for a cell of the plurality of cells exhibiting a changed physiology in response to the presence in the cell of a transdominant expression product of the corresponding expression products; and detecting and isolating the cell and/or transdominant expression product,
In a particular embodiment, the expressing step comprises translating the nucleic acids and/or corresponding transcripts, and each of the nucleic acids encodes a peptide comprising a different amino acid sequence. The nucleic acids may be joined to sequences encoding polypeptide backbones of artificial design capable of intracellularly presenting randomized peptides as structured domains. The methods may also involve introducing the library into the cells, such as through the use of retroviral vectors, and particularly suitable vectors are also disclosed.