Mol. interactions, it should be easy to generalize to multiple biological contexts. Technology for rapidly shutting off the production of specific proteins in eukaryotes would be widely useful in research and in gene and cell therapies, but a simple and effective method has yet to be developed. Controlling protein production through repression of transcription is usually slow in onset, as previously transcribed mRNA molecules continue to produce proteins. RNA interference (RNAi) induces mRNA destruction, but RNAi is usually often only partially effective and can exhibit both sequence-independent and sequence-dependent off-target effects1. Furthermore, mRNA and protein abundance are not usually correlated due to translational regulation of specific mRNAs2-4. Lastly, both transcriptional repression and RNAi take days to reverse5,6. To address these limitations, we wished to devise a method for chemical regulation of protein expression at the post-translational level. An ideal method would feature 1) genetic specification of the target protein, 2) a single genetic modification for simplicity, 3) minimal modification of the expressed protein, 4) generalizability to many proteins and cell types, and 5) control by a drug with proven safety and bioavailability in mammals. While methods have been devised with some of these characteristics (Supplementary Results, Supplementary Table 1), none have encompassed all of them. We envisioned that a degron that removes itself in a drug-controllable manner could serve as the basis for a new method with all the desired 3-Formyl rifamycin features. In particular, we reasoned that if a site-specific drug-inhibitable protease and a degron were fused to a protein via an intervening protease site, then by default the protease and degron would be removed and the protein expressed. However, in the presence of protease inhibitor, the degron would remain attached on new protein copies, causing their rapid degradation (Fig. 1a). Open in 3-Formyl rifamycin a separate window Physique 3-Formyl rifamycin 1 Small Molecule-Assisted Shutoff (SMASh) concept and development. (a) SMASh concept. 3-Formyl rifamycin Top, a target protein is fused to the SMASh tag via a HCV NS3 protease recognition site. After protein folding, the SMASh tag is removed by its internal protease activity, and is degraded due to internal degron activity. Bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. (b) Amino acid sequence of the SMASh tag. Sequence derived from NS3 protease (orange), NS3 helicase (gray), and NS4A (red) are shown. Secondary structures in the context of the original HCV polyprotein are underlined. The NS4A/4B protease substrate (green), has an arrow indicating site of cleavage. Dotted line indicates putative degron region. (c) Top, business of fusions of PSD95 with NS3 protease (NS3pro) or NS3pro-NS4A, with predicted protein fragment sizes indicated. Bottom, in the absence of protease inhibitor asunaprevir (ASV), PSD95 was detectable in HEK293 lysates 24 h post-transfection, for both constructs. With asunaprevir, the PSD95-NS3pro fusion was expressed at full-length size, but the PSD95-NS3pro-NS4A failed to exhibit expression. GAPDH served as a loading control. (d) A specific element within NS3pro-NS4A is necessary for degron activity. Transfected HeLa cells expressed either YFP-NS3pro-NS4A, or a variant in which the putative degron (dotted line in b) was mutated to a GGS-repeat linker of the same length (GGS), for 24 h with or without ASV. The GGS mutation restores expression in the ASV condition. -actin served as a loading control. Here, we show that a system of this design using hepatitis C computer virus (HCV) nonstructural protein 3 (NS3) protease enables clinically tested drugs to effectively shut off expression. We termed this method small-molecule assisted shutoff, or SMASh. SMASh enabled drug-induced suppression of various proteins in multiple eukaryotic cell types. In contrast to other single-component methods of post-translational regulation of Ly6a protein expression, SMASh functions robustly in yeast as well. Finally, we used SMASh to confer HCV protease inhibitor sensitivity onto an RNA computer virus currently in clinical trials for cancer but for which no licensed drug inhibitor exists. SMASh thus enables.