Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in XXXX cells.

@entity1 @entity20127 @entity137 @entity263 @entity5 @entity993 @entity8985 @entity15308 @entity31137 @entity23886 @entity994
BACKGROUND: The cyclin-dependent kinase (CDK) inhibitor @entity993 (Kip)   is downregulated in a majority of @entity1 @entity5 due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of @entity993 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF(SKP)  , KPC, @entity31137 ), which regulate @entity993 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that @entity993 protein expression is restored in @entity5 cells. RESULTS: We developed a cell-based assay for measuring the levels of endogenous nuclear @entity993 in a high throughput screening format employing @entity263 cells engineered to overexpress @entity20127 . The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of @entity993 at low micromolar concentrations. SMIPs (small molecule inhibitors of @entity993 depletion) also upregulate @entity994 ( @entity23886 )  , inhibit cellular @entity15308 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential @entity137 in LNCaP cells relative to normal @entity1 fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate @entity20127 and to stabilize @entity993 , although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear @entity993 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on @entity993 and @entity994 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and @entity8985 . CONCLUSIONS: Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear @entity993 as an endpoint, presents an effective means of identifying bioactive molecules with @entity5 selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on @entity993 and novel leads for chemotherapeutics targeting a prominent molecular defect of @entity1 @entity5 .
