Introduction Session 13PROTACS: CHIMERIC MOLECULES TO TARGET PROTEINS FOR UBIQUITINIntroduction Session 13 PROTACS: CHIMERIC MOLECULES TO TARGET PROTEINS FOR UBIQUITINATION AND DEGRADATION (taken from KM Sakamoto, Mol. Genetics and Metabolism 77 (2002) 44-56) To circumvent the problem of transducing cells at high efficiency, we sought to target deliberately a protein to the SCF complex by developing a chimeric compound, known as proteolysis targeting chimeric molecule (PTCM). We first tested whether the PTCM could recruit methionine aminopeptidase-2 (MetAP-2) to the SCFβ-TRCP for ubiquitination and degradation in vitro. A PTCM was synthesized that contained at one end the minimal 10 aa phosphopeptide sequence of IκB that is recognized by the F-box protein β-TRCP and at the other end, the MetAP-2 binding compound, ovalicin. MetAP-2 binds to ovalicin. MetAP-2 binds to ovalicin covalently. We performed ubiquitination experiments with lysates from 293T cells transfected with Flag tagged β-TRCP and Flag tagged CUL1. The SCF complex was immunoprecipitated using Flag affinity beads followed by addition of purified E1, E2, ATP, and ubiquitin. Our results demonstrate that the PTCM could recruit the MetAP-2 to the SCFβ-TRCP complex resulting in ubiquitination. Addition of PTCM also resulted in degradation of MetAP-2 in Xenopus extracts. To determine whether PTCM could be generalized to other ubiquitin ligases, we performed ubiquitination assays with Cbl. Cbl is a monomeric ubiquitin ligase that attaches ubiquitin to signaling molecules and receptor tyrosine kinases resulting in proteolysis. We generated a PTCM that consisted of ovalicin and the Zap70 phosphopeptide, which binds Cbl. Ubiquitination reactions were performed with purified Cbl, various E1s, Ubch4 (E2), ubiquitin, ATP, MetAP-2, and the Zap70-ovalicin PTCM. We demonstrated that PTCM promotes ubiquitination of MetAP-2 by Cbl in vitro. These results suggest that PTCM can be generalized to other ubiquitin ligases. Future work will focus on testing other targets that promote tumorigenesis, e.g., androgen receptor in prostate cancer cells. If cell permeable PTCMs prove to increase turnover and degrade proteins in cells, this would lead to potential therapeutic applications in patients with cancer and other diseases. Figure by MIT OCW. After Sakamoto, KM. Ubiquitin-dependent proteolysis: its role in the human diseases and the design of therapeutic strategies." Mol Genet Metab 77 1-2 (2002) 44-56. General application of PTCMs. A schematic representation of how different disease-promoting proteins might be recruited to E3 ligases for ubiquitination and degradation by specific PTCMs. UbIIgase-1General application of PTCMs.UbIIgase-2target1target2target3target4target1target2target3target4BORTEZOMIB (also PS-341 or Velcade): A novel, first-in-class proteasome inhibitor for the treatment of multiple myeloma and other cancers (reviewed by P.G. Richardson et al. Cancer con rol (2003) 10: 361-369) t (A full text PDF of this article is available at http://www.moffitt.usf.edu/pubs/ccj/v10n5/pdf/361.pdf) Cancer cells seem to be more sensitive to the proapoptotic effects of proteasome inhibition than are normal cells. It has also been shown that proteasome inhibition enhances the sensitivity of cancer cells to traditional anticancer agents in both in vitro and in vivo preclinical studies. The 20S core is a cylindrical complex made up of four stacked rings. The two outer rings bind to the 19S regulatory particles, and the two inner rings each contain three active sites. These active sites account for the three major proteolytic activities of the proteasome, which have been described as chymotrypsin-like, trypsin-like, and post-glutamyl peptide hydrolytic (PGPH). Synthetic inhibitors of the proteasome include peptide aldehydes such as Z-Leu-Leu-Leu-al (MG132), Z-Ile-Glu(Obut)-Ala-Leu-al (PSI), Ac-Leu-Leu-Nle-al (ALLN), and peptide vinyl sulfones. Natural proteasome inhibitors include lactacystin, epoxyketones and the TMC-95 cyclic peptides. All of these compounds bind to and directly inhibit active sites within the 20S core particle. However, most primarily interfere with the chymotrypsin-like activity of the core particle (the rate-limiting step in proteolysis) and appear to have little effect on the other proteolytic activities. Many of these inhibitors also lack specificity or exhibit unfavorable kinetics for clinical use. For instance, peptide aldehyde inhibitors dissociate rapidly from the proteasome and are inactivated by oxidization, being removed from the cell by the multidrug transporter system. Furthermore, they are also inhibitors of serine and cysteine proteases, including calpains and cathepsins, which can be undesirable for their use in patients. Others, such as the peptide vinyl sulfones and natural inhibitors bind the 20S irreversibly, which can also be detrimental in the long run. These problems were overcome, however, by replacing the aldehyde group of the synthetic peptide inhibitors with boronic acid. The peptide boronates differ from their aldehyde analogs in that they dissociate more slowly from the proteasome, conferring stable inhibition. Further, the weak interaction between boron and sulphur means that the peptide boronates do not inhibit thiol proteases. The peptide boronic acids are also up to 100-fold more potent than their peptide aldehyde analogs. Dipeptide boronic acid bortezomib is of particular interest from a clinical perspective. This small, water-soluble compound is a potent and selective proteasome inhibitor, which offers the additional advantages of low molecular weight and ease of synthesis. Bortezomib is the first molecule in this class to reach clinical trials in cancer patients. (Image removed for copyright reasons. See Figure 1b in Richardson, 2003.) Multiple myeloma is a hematologic malignancy typically characterized by the accumulation of clonal plasma cells at multiple sites in the bone marrow. Although the majority of patients respond to initial treatment with chemotherapy and radiation, most eventually relapse due to the proliferation of resistant tumor cells; despite the advent of high-dose chemotherapy with stem-cell transplantation, MM remains incurable. This cytotoxic resistance reflects both the inherent characteristics of the MM cell and the protective interactions between the tumor and the bone marrow microenvironment. There