Development of Protacs to Target Cancer-promoting Proteins for Ubiquitination and DegradationKathleen M. Sakamoto, Kyung B. Kim, Rati Verma et al.|Molecular & Cellular Proteomics|2003 The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCFβ-TRCP-binding phosphopeptide derived from IκBα linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the α isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer. The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCFβ-TRCP-binding phosphopeptide derived from IκBα linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the α isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer. One of the major pathways to regulate protein turnover is ubiquitin-dependent proteolysis. Post-translational modification of proteins with ubiquitin occurs through the activities of ubiquitin activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3), which act sequentially to catalyze the attachment of ubiquitin to lysine residues in an energy-dependent manner (1Deshaies R.J. SCF and Cullin/Ring H2-based ubiquitin ligases.Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar, 2Ciechanover A. Orian A. Schwartz A.L. Ubiquitin-mediated proteolysis: Biological regulation via destruction.Bioessays. 2000; 22: 442-451Google Scholar). Among the hundreds of E3s encoded within the human genome, the Skp1-Cullin-F-box (SCF) 1The abbreviations used are: SCFβ-TRCP, Skp1-Cullin-F-box; Protac, proteolysis targeting chimeric molecule; E2, estradiol; DHT, dihydroxytestosterone; MetAP-2, methionine aminopeptidase-2; ER, estrogen receptor; AR, androgen receptor; DMF, dimethylformamide; DMSO, dimethylsulfoxide; ES, electrospray; GFP, green fluorescence protein; β-TRCP, β-transducin repeat-containing protein. ubiquitin ligases comprise a heterotetrameric group of proteins consisting of Skp-1, Cul1, a RING-H2 protein Hrt1 (also known as Roc1 or Rbx1), and an F-box protein (1Deshaies R.J. SCF and Cullin/Ring H2-based ubiquitin ligases.Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar, 3Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. A family of mammalian F-box proteins.Curr. Biol. 1999; 9: 1180-1182Google Scholar). The mammalian F-box protein β-transducin repeat-containing protein (β-TRCP) of SCFβ-TRCP binds IκBα, the negative regulator of NF-κB, and promotes its ubiquitination and degradation (4Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen J.S. Mann M. Mercurio F. Ben-Neriah Y. Identification of the receptor component of the IκBα-ubiquitin ligase.Nature. 1998; 396: 590-594Google Scholar). A 10-aa phosphopeptide segment of IκBα is both necessary and sufficient to mediate its binding to SCFβ-TRCP and subsequent ubiquitination and degradation (4Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen J.S. Mann M. Mercurio F. Ben-Neriah Y. Identification of the receptor component of the IκBα-ubiquitin ligase.Nature. 1998; 396: 590-594Google Scholar). There is a pressing unmet need to develop effective drugs to treat cancer and other diseases that afflict humans. The recent completion of the human genome sequence coupled with basic studies in molecular and cellular biology have revealed hundreds to thousands of proteins that could conceivably serve as targets for rational drug therapy. Unfortunately, many of these protein targets are not considered to be readily “drugable,” in that they are not enzymes and it is not obvious how to inhibit their function with small molecule drugs. Thus, it would be valuable to have a generic method that would enable specific and efficacious inhibition of any desired protein target, regardless of its biochemical function. Short interfering RNA (siRNA) represents one such method (5Timmons L. The long and short of siRNAs.Mol. Cell. 2002; 10: 435-437Google Scholar, 6Tuschl T. Expanding small RNA interference.Nat. Biotechnol. 2002; 20: 446-448Google Scholar), but it remains unclear whether siRNA will work as therapeutic agents in humans. We sought to develop a different approach, taking advantage of the 10-aa phosphopeptide sequence of IκBα described above to target proteins for ubiquitination and degradation (4Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen J.S. Mann M. Mercurio F. Ben-Neriah Y. Identification of the receptor component of the IκBα-ubiquitin ligase.Nature. 1998; 396: 590-594Google Scholar). As proof of concept, we previously synthesized a chimeric molecule or Protac (proteolysis targeting chimeric molecule) consisting of the IκBα phosphopeptide linked to ovalicin, which covalently binds methionine aminopeptidase-2 (MetAP-2). We showed that this Protac (Protac-1) recruits MetAP-2 to the SCFβ-TRCP ubiquitin ligase resulting in both ubiquitination and degradation of MetAP-2 (7Sakamoto K.M. Kim K.B. Kumagai A. Mercurio F. Crews C.M. Deshaies R.J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8554-8559Google Scholar). MetAP-2 is not known to be an endogenous substrate of SCFβ-TRCP (8Sin N. Meng L. Wang M.Q. Wen J.J. Bornmann W.G. Crews C.M. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6099-6103Google Scholar), and was not ubiquitinated by SCFβ-TRCP in the absence of Protac-1. Although this experiment demonstrated that Protacs could work as envisioned, it left open a number of critical questions. For example, can Protacs be used more generically to target other substrates, including proteins of potential therapeutic interest? Can a Protac recruit a target to SCFβ-TRCP through a noncovalent interaction? Can a Protac work within the context of a cell? Both estrogen receptor α (ER) and androgen receptor (AR) have been demonstrated to promote the growth of breast and prostate cancer cells (9Howell A. Howell S.J. Evans D.G. New approaches to the endocrine prevention and treatment of breast cancer.Cancer Chemother. Pharmacol. 2003; 52 (Suppl 1): 39-44Google Scholar, 10Debes J.D. Schmidt L.J. Huang H. Tindall D.J. P300 mediates androgen-independent transactivation of the androgen receptor by interleukin 6.Cancer Res. 2002; 62: 5632-5636Google Scholar). In fact, there are several treatment modalities such as Tamoxifen and Faslodex, which control breast tumor cell growth through inhibition of ER activity. In early prostate cancer, tumor cells are often androgen responsive. Patients with prostate cancer receive hormonal therapy to control tumor growth. Recent evidence suggests that even in androgen-independent prostate cancer, the AR may promote tumor growth (10Debes J.D. Schmidt L.J. Huang H. Tindall D.J. P300 mediates androgen-independent transactivation of the androgen receptor by interleukin 6.Cancer Res. 2002; 62: 5632-5636Google Scholar). Similarly, many tamoxifen-resistant tumors still express ER (11Levenson A.S. Svoboda K.M. Pease K.M. Kaiser S.A. Chen B. Simons L.A. Jovanovic B.D. Dyck P.A. Jordan V.C. Gene expression profiles with activation of the estrogen receptor alpha-selective estrogen receptor modulator complex in breast cancer cells expressing wild-type estrogen receptor.Cancer Res. 2002; 62: 4419-4426Google Scholar). Thus, new drugs that down-regulate AR and ER by novel mechanisms may be of potential benefit in treating breast and prostate cancers. To address the key questions about Protacs raised by our first study, we set out to develop Protacs comprising the IκBα phosphopeptide linked to either estradiol (E2) or dihydroxytestosterone (DHT) to recruit ER or AR to SCFβ-TRCP to accelerate their ubiquitination and degradation. Recently, both the ER and AR have been shown to be regulated by proteasome-dependent proteolysis (12Cardozo C.P. Michaud C. Ost M.C. Fliss A.E. Yang E. Patterson C. Hall S.J. Caplan A.J. C-terminal Hsp-interacting protein slows androgen receptor synthesis and reduces its rate of degradation.Arch. Biochem. Biophys. 2003; 410: 134-140Google Scholar, 13Lonard D.M. Nawaz Z. Smith C.L. O'Malley B.W. The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation.Mol. Cell. 2000; 5: 939-948Google Scholar, 14Nawaz Z. Lonard D.M. Dennis A.P. Smith C.L. O'Malley B.W. Proteasome-dependent degradation of the human estrogen receptor.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1858-1862Google Scholar). We reasoned that Protacs might mimic the action of the human papillomavirus E6 protein, which accelerates the turnover of the already unstable p53 to the point where p53 can no longer accumulate, resulting in loss of its function (15Zhou P. Bogacki R. McReynolds L. Howley P.M. Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins.Mol. Cell. 2000; 6: 751-756Google Scholar). In this paper, we report the feasibility of using Protacs to target degradation of proteins known to promote tumor growth. We show that Protacs can recruit the ER for ubiquitination and degradation in a cell-free system. Furthermore, our results demonstrate that in cells, Protacs can promote the degradation of AR in a proteasome-dependent manner. Thus, Protacs may be a useful therapeutic approach to destroy proteins that promote tumor growth in patients with cancer. To generate GA-1-monosuccinimidyl suberate, the estradiol derivative, GA-1 (7 mg, 11.5 μmol), was dissolved in 1 ml of anhydrous dimethylformamide (DMF), and disuccinimidyl suberate (21 mg, 57.0 μmol) was added at room temperature. After overnight stirring, DMF was removed under high vacuum, and the resulting white solid was flash-chromatographed to give GA-1-monosuccinimidyl suberate (6.3 mg, 7.3 μmol, 63.5%). For synthesis of GA-1-IκBα phosphopeptide, GA-1-monosuccinimidyl suberate (6 mg, 6.9μmol) in DMSO (1 ml) was added to dimethylsulfoxide (DMSO) solution (0.4 ml) containing IκBα phosphopeptide (1.5 mg, 0.92μmol) and dimethylaminopyridine (0.5 mg). After 30 min stirring at room temperature, the coupling reaction was completed, which was confirmed by a Kaiser test. DMSO was removed under high vacuum, and the resulting crude product was repeatedly washed with dichloromethane and methanol to remove excess GA-1-monosuccinimidyl suberate to give the final product, GA-1-IκBα phosphopeptide (1.5 mg, 0.63 μmol, 68.5%). The final product was characterized by electrospray (ES) mass spectrometry. ES-MS (M+H)+ for GA-1-IκBα phosphopeptide was 2384.0 Da. All other intermediates were characterized by 500-MHz 1H nuclear magnetic resonance spectroscopy. For DHT-Gly-monosuccinimidyl suberate, DMF (28 μl, 0.33 mmol) was added to dichloromethane solution (20 ml) containing Fmoc-Gly-OH (1.06 g, 3.57 mmol) and oxalyl chloride (0.62 ml, 7.10 mmol) at 0 °C. After 3 of stirring at room temperature, dichloromethane was removed under The resulting solid was in dichloromethane ml) and was with g, mmol) and dimethylaminopyridine g, mmol) in dichloromethane (20 ml) at 0 °C. The reaction was overnight at room temperature. After dichloromethane was removed under the resulting was flash-chromatographed to g, g, mmol) was with ml, 1 in at room for and the DMF was removed under high The resulting was flash-chromatographed to mg, disuccinimidyl suberate mmol) was added to DMF solution (1 ml) containing mg, mmol) at room temperature. After overnight stirring, DMF was removed under high vacuum, and the resulting crude product was flash-chromatographed to give DHT-Gly-monosuccinimidyl suberate mg, DHT-Gly-monosuccinimidyl suberate mg, μmol) in DMSO ml) was added to DMSO solution (1 ml) containing IκBα phosphopeptide mg, μmol) and dimethylaminopyridine mg, After min of stirring at room temperature, the coupling reaction was completed, which was confirmed by a Kaiser test. DMF was removed under high vacuum, and the resulting crude product was repeatedly washed with dichloromethane and methanol to remove excess DHT-Gly-monosuccinimidyl suberate to give the final product, phosphopeptide mg, μmol, The final product was characterized by mass spectrometry. ES-MS (M+H)+ for was Da. All other intermediates were characterized by 500-MHz 1H nuclear magnetic resonance spectroscopy. cells were in with and were the to and with of were in on the of were with of and of using method as described (7Sakamoto K.M. Kim K.B. Kumagai A. Mercurio F. Crews C.M. Deshaies R.J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8554-8559Google Scholar). were 30 of a containing the linked to the green protein was into cells at the to were at the of cell were with of 1 from cells with or were for on for After at in an for min at the was added to of which were washed with were with the on a for at by one with A and one with the was by the E2, from ubiquitin or ubiquitin (1.5 Protac final ER from and (1 final in reaction of μl, which was added to of washed (7Sakamoto K.M. Kim K.B. Kumagai A. Mercurio F. Crews C.M. Deshaies R.J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8554-8559Google Scholar). were for 1 at 30 in a with was added to the was by methods using were as described 26S of were added to the ubiquitinated ER on and the reaction was with of 1 of and ubiquitin final as previously described R. Chen S. R. Deshaies R.J. Identification of proteins by mass of Biol. Cell. 2000; Scholar, R. L. R. Deshaies R.J. of in and degradation by the 26S 2002; Scholar). The reaction was for min at 30 with in a For proteasome inhibition 26S were min at 30 with the or at 1 final to to ubiquitinated cells were with a that by of as described were with and in with and to cells were in Protac to in with mass was into cells through a using a The was of the cell For proteasome inhibition cells were with for or with and Protac Kim K.B. Crews C.M. of proteasome as revealed by Cell. 2001; Scholar, R. M. green proteins for proteolysis in Biotechnol. 2000; Scholar). were using a and fluorescence were with a Protacs consisting of the 10-aa on the covalently linked to either estradiol or were synthesized We first in ubiquitination with both but our on to with expression of To whether promotes the ubiquitination of ER by SCFβ-TRCP in a we ubiquitination with of Protac ER was ubiquitinated at a of with at we no longer ubiquitination of ER by SCFβ-TRCP, which may be to a the of excess inhibits the of efficient ubiquitination of ER, we to this for the of our studies as be that we ubiquitination of ER in the absence of A and This may be to the of an SCF ubiquitin ligase in the these were of molecular mass and from the high molecular by and of ubiquitination of ER in of ER ubiquitination by ER was with E2, and SCFβ-TRCP from cells by of on and were with the of for min at 30 and by by with an of high molecular on as that ubiquitin was added in the of ubiquitin estradiol and IκBα phosphopeptide be covalently linked to promote ER The reaction was as described in that IκBα phosphopeptide and estradiol were added to the ubiquitination reaction of and IκBα phosphopeptide and estradiol out Protac activity. as that was used at 1 of IκBα phosphopeptide or of IκBα that is was added to ubiquitination Protacs are target as that and IκBα Protacs were used in of as To address the of action of we whether the IκBα phosphopeptide and estradiol can out and whether these added as can mimic the action of A excess of either IκBα phosphopeptide or estradiol in cells the activity of 1 added as and IκBα phosphopeptide to the of results are with our that as a bridging molecule in that the estradiol with the ER the other the IκBα phosphopeptide, recruits the ER to the We the of with ER were in the of either or a Protac that consisted of the phosphopeptide, which is by the ubiquitin ligase N. Wang P. of a function in 2000; and ovalicin, which binds MetAP-2 (8Sin N. Meng L. Wang M.Q. Wen J.J. Bornmann W.G. Crews C.M. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6099-6103Google Scholar). As shown in ER was not ubiquitinated by SCFβ-TRCP in the of either or are to targeting to the proteasome C.M. in Biochem. Sci. 2000; Scholar), and as a ubiquitinated under the in can be for the proteasome Deshaies R.J. of attachment the rate of Cell. 2003; Scholar). Thus, we sought to whether by were by the 26S To this 26S proteasome R. Chen S. R. Deshaies R.J. Identification of proteins by mass of Biol. Cell. 2000; was added to ubiquitinated ER in the of SCFβ-TRCP and disappearance of high molecular mass ubiquitin was within min and was by the inhibits the activity of the but not by the R. L. R. Deshaies R.J. of in and degradation by the 26S 2002; results with the IκBα Protac demonstrated that a target protein can be to a ubiquitin ligase through noncovalent and be ubiquitinated and in We to whether a Protac could promote the degradation of proteins in For these we used we in with cells that an protein and a cell that was readily to We employed the on the IκBα phosphopeptide its efficient into cells were with 1 and for or absence of by fluorescence A was and degradation was 1 of Protac not We that the of cells with Protac of This was not to cells with were not 1 by the cells shown in To the of we cells and the in 1 of cells demonstrated or disappearance of In cells that to be 1 were experiment was on at with cells of or not in disappearance of from cells not We that the of phosphopeptide and was required for degradation. of IκBα phosphopeptide and into cells not in that Protac is necessary to promote degradation of To whether degradation was on IκBα phosphopeptide and binding to their we with a excess of phosphopeptide or into In both degradation of was All were on with cells The results shown are of the in of cells these the that degradation by targeting to To whether the disappearance of was proteasome cells were with the proteasome for to with In cells with was not that the Protac mediates degradation through a proteasome-dependent were with Protac and in the absence of resulting in inhibition of degradation not The shown is of on 3 different with at 30 cells As demonstrated previously the IκBα but not ubiquitination of ER in The is on the of to be by the ubiquitin ligase as as its to to The of Protac action to in cells, not degradation of The and and targeted critical to the biology of and cells are regulated by ubiquitin-dependent including from of the cell and of which are by degradation of and the regulator IκBα M. Ben-Neriah Y. The control of Rev. 2000; Scholar, is Cell and the Dev. 1999; Scholar). To the of the for therapeutic we are Protacs to recruit proteins to ubiquitin ligases to promote their ubiquitination and degradation. of the Protacs approach is that it in theory can be to any protein in the or of a and may enable the development of a of proteins in the The of our approach is a small molecule that as a to a target protein to a ubiquitin we demonstrated that a Protac comprising a phosphopeptide that binds SCFβ-TRCP and a small molecule that binds MetAP-2 the ubiquitination of MetAP-2 by SCFβ-TRCP ubiquitin ligase in vitro, and targets MetAP-2 for degradation by the proteasome in (7Sakamoto K.M. Kim K.B. Kumagai A. Mercurio F. Crews C.M. Deshaies R.J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8554-8559Google Scholar). in the work were to show that Protacs can the turnover of a target protein in cells, and to the Protacs approach to proteins that a in human diseases. We the estrogen and androgen for our studies to their with estrogen and respectively. Furthermore, both have been with the development and progression of cancer. The results here that Protacs by a bridging to enable efficient and specific of ER in and AR in our in it is that Protacs can be different targets ER, and and that Protacs promote ubiquitination of these targets in a manner that is both target and it is that Protacs can AR turnover in the context of the cellular degradation This degradation was to be specific and on both of the Protac the proteasome the of Protacs to promote AR that the degradation is proteasome specific and not to such as or to other such as To Protacs to cells in the described we employed to the of the SCFβ-TRCP-binding IκBα phosphopeptide A key for Protac technology is to develop cell molecules that can be used to for in cell and of cancer. work in our suggests that Protacs on the of may be used to target ubiquitination and degradation of proteins in cells through the ubiquitin ligase M. R. and C. M. in We that many Protac can be to treat a of diseases. of hundreds of ubiquitin ligases that can be as agents of Protac action have been by the Human Genome it is to that Protacs not be to with such as AR and In any protein that binds a small molecule through high can be a studies that Protacs technology is not but as an to inhibition of proteins that promote human treatment of cancer drugs that inhibit the cell and Protacs a of targeting a protein that is known to regulate growth and of cancer cells, in the that the of patients by inhibiting the agent C.L. H. D.J. R. M. of a specific of the in the of and with the 2001; Scholar). The is that by a generic method that to target the proteins for the regardless of their of action or functional it will be to cancer to We for the expression and of for of the and We are to Mercurio for GA-1-monosuccinimidyl