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The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys[sup 48]-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system. [ABSTRACT FROM AUTHOR]