Development of Protacs to Target Cancer-promoting Proteins for Ubiquitination and Degradation

Sakamoto, Kathleen M.; Kim, Kyung B.; Verma, Rati; Ransick, Andy; Stein, Bernd; Crews, Craig M.; Deshaies, Raymond J.

doi:10.1074/mcp.t300009-mcp200

Cited by 320 publications

(255 citation statements)

References 23 publications

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“…However, the fraction of small molecules that both specifically bind to and inhibit the function of a protein target is extremely small. The continuing development of PROTACS (36,37) seeks to potentially exploit the superset of "binders but not inhibitors" by using phosphopeptide-small molecule chimeras to artificially target proteins for ubiquitination. We propose to bypass the ubiquitination step and generate small molecules that will promote the degradation of disease-causing proteins through heterodimerization with the proteasome.…”

Section: Discussionmentioning

confidence: 99%

Localization to the Proteasome Is Sufficient for Degradation

Janse

Crosas

Finley

et al. 2004

Journal of Biological Chemistry

107

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The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.ATP-dependent protease complexes degrade the majority of unstable cellular proteins, a process that is conserved across all three kingdoms of life. These molecular machines function both generally in protein turnover and specifically in the regulation of processes such as transcription, apoptosis, antigen presentation, and cell cycle progression (1). A high degree of conservation is evident among them; the archaebacterial and eukaryotic 20S proteolytic core particles share both sequence and structural homology (2), whereas eubacteria have functionally related complexes: ClpYQ, ClpXP, and ClpAP (3-5). The 20S core particle is composed of four stacked heptameric rings structured in an ␣-␤-␤-␣ configuration. Access to the proteolytic central chamber is obstructed at both ends of the cylindrical assembly by N-terminal projections of the ␣-subunits, thus preventing uncontrolled proteolytic degradation (5, 6). In eukaryotes, docking with the 19S regulatory particle (RP) 1 to form the complete 26S proteasome is sufficient to relieve this block, opening a channel into the core (6, 7).Eukaryotes have evolved an elaborate system that operates in conjunction with the proteasome to facilitate the temporal and specific regulation of intracellular proteolysis. Most proteins are targeted for degradation through ubiquitination, mediated by the E1, E2, E3 ubiquitin (Ub) conjugation machinery. These three consecutively acting enzymes are necessary for target recognition, transfer of a ubiquitin moiety to the substrate, and subsequent elongation of the ubiquitin branched chain (8). Modularity and the large number of E2 Ub-conjugating enzymes and E3 Ub ligases allow for greater specificity and flexibility in recognizing a diverse range of substrates. Once a protein is polyubiquitinated, it is targeted to and degraded by the 26S proteasome.The polyubiqui...

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Section: Discussionmentioning

confidence: 99%

Localization to the Proteasome Is Sufficient for Degradation

Janse

Crosas

Finley

et al. 2004

Journal of Biological Chemistry

107

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“…As originally conceived, PROTACs work constitutively to induce targeted protein ubiquitination/degradation irrespective of any intracellular signaling context (4,6,7). Here, we advance this paradigm by coupling PROTAC-mediated protein degradation to the activation state of a particular signaling pathway.…”

mentioning

confidence: 99%

Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

Hines

Gough

Corson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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131

120

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Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinasesignaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short-and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.A ntagonizing the function of a protein is an effective strategy for determining its role within a cellular context. For some enzymes and/or receptor proteins, this can be accomplished by incubation with a small molecule inhibitor or antagonist. However, specific small molecule inhibitors have not been discovered or developed for many proteins. As alternative strategies, gene deletion and RNAi approaches function at the DNA and mRNA levels to reduce protein expression, thus offering the ability to control function even for those proteins lacking a specific inhibitor (1, 2). However, such nucleic acid-based approaches do not afford the same rapid, direct assessment of protein function as posttranslational intervention; furthermore, in vivo application of these strategies is even today still complicated and relatively cumbersome. Combining the attractive qualities of both RNAi and small molecule inhibitor approaches, we developed a strategy, proteolysis targeting chimeras (PROTACs) for targeted posttranslational knockdown of proteins. Each PROTAC is heterodimeric, consisting of an E3 ubiquitin ligase-binding moiety linked to a ligand that binds to the target protein (3). As such, each PROTAC recruits i...

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“…As these were both wellunderstood systems with implications in prostate [47] and breast [56] cancers, respectively, we anticipated that they would be excellent models to study the action of a PROTAC molecule. DHT-IκBα and estradiol-IκBα PROTACS were synthesized, [49] and subsequent testing clearly indicated that upon microinjection into HEK 293 cells, the PROTAC molecules initiated the degradation of green fluorescent protein (GFP) fusions of the estrogen receptor and androgen receptor, respectively.…”

Section: Proteolysis-targeting Chimeric Molecules (Protacs)mentioning

confidence: 99%

“…[48][49][50] Proteolysis-targeting chimeric molecules (PROTACS) function by forming a complex between the target protein and an E3 ubiquitin ligase. The heterobifunctional molecule is comprised of a recognition element for the target, a linker, and a recognition element for an E3 ligase.…”

Section: Proteolysis-targeting Chimeric Molecules (Protacs)mentioning

confidence: 99%