Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins

Neklesa, Taavi K.; Tae, Hyun Seop; Schneekloth, Ashley R.; Stulberg, Michael J.; Corson, Timothy W.; Sundberg, Thomas B.; Raina, Kanak; Holley, Scott A.; Crews, Craig M.

doi:10.1038/nchembio.597

Cited by 350 publications

(334 citation statements)

References 42 publications

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“…Futhermore, consistent with our initial report on protein hydrophobic-tagging [10] , degradation of the AR is dependent on the UPS -pretreatment/cotreatment of LNCaP cells with the proteasome-specific inhibitor, epoxomicin, prevents SARD-mediated degradation of the AR. To further explore the mechanism of SARDmediated AR degradation, we investigated the possible involvement of Heat Shock Proteins (HSPs), given their known role in stabilizing misfolded proteins or targeting them for degradation by the UPS.…”

supporting

confidence: 87%

“…This 'hydrophobic tag' may mimic a partially denatured protein folding state, leading to degradation by the UPS. We demonstrated the feasibility of this approach by covalently coupling hydrophobic tags to engineered dehalogenase HaloTag-2 [10][11][12] fusion proteins. Recently, a similar approach was applied to degradation of E. coli DHFR by non-covalent appendage of a hydrophobic tag [13] .…”

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confidence: 99%

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Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

et al. 2015

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Androgen Receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of Selective Androgen Receptor Degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small molecule Androgen Receptor (AR) ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to third generation AR antagonists. KeywordsAntiproliferation; cancer; drug design; hormones; protein degradation Targeted degradation represents an intriguing strategy to regulate the function of therapeutically relevant proteins (e.g., transcription factors and scaffolding proteins) not amenable to traditional small molecule approaches [1,2] . Moreover, targeted protein degradation could overcome resistance mechanisms that modulate the activity of small molecule drugs following target engagement. For instance, point mutants conferring agonist activity to antagonists limit efficacy of androgen-receptor (AR) antagonists for treatment of prostate cancer [3] . While deletion of the disease-causing protein offers a direct solution to this problem, strategies for doing so via genome editing or RNAi remain clinically challenging [4,5] . As an alternative, we have developed several approaches for posttranslational targeting of specific proteins to the ubiquitin-proteasome system (UPS) [6][7][8][9] . For instance, we recently reported a strategy for post-translational protein degradation whereby a hydrophobic moiety appended to the surface of a target protein engages the cellular quality control machinery. This 'hydrophobic tag' may mimic a partially denatured protein folding state, leading to degradation by the UPS. We demonstrated the feasibility of this approach by covalently coupling hydrophobic tags to engineered dehalogenase HaloTag-2 [10][11][12] fusion proteins. Recently, a similar approach was applied to degradation of E. coli DHFR by non-covalent appendage of a hydrophobic tag [13] . A key next step in the development of this nascent technology is to degrade clinically relevant target proteins with a small drug-like molecule. To this end, here we show that coupling a hydrophobic tag to an androgen receptor agonist converts it to a potent Selective Androgen Receptor Degrader (SARD) capable of inducing >50% of AR degradation (DC 50 ) at 1 μM. Remarkably, this SARD retained anti-proliferative activity in cell lines resistant to current standard-of-care drugs for castration-resistant prostate cancer (CRPC).The androgen receptor (AR) [14] is a ...

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confidence: 99%

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

et al. 2015

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“…To overcome these limitations, a variety of systems have been developed that allow the posttranslational degradation of proteins using cell-permeable small molecules (4,(6)(7)(8)(9)(10). Recently, a system for inducible-protein depletion was developed that relies on the transplantation of a ligand-induced degradation system found in plants (4).…”

mentioning

confidence: 99%

Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells

Holland

Fachinetti

Han

et al. 2012

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

302

291

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Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with firstorder kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.proteasome | SCF | ubiquitin

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“…[80][81][82] Another promising application is the regulation of protein functions. Although there have been very few related reports, 83,84) the regulation of intracellular signaling or protein degradation would be useful. Such artificial regulation of cellular functions might become a new trend in next-generation chemical biology.…”

Section: Other Applications Of Bl-tag Technology and Perspectives Of mentioning

confidence: 99%

Development of Molecular Imaging Tools to Investigate Protein Functions by Chemical Probe Design

Mizukami

2011

CHEMICAL & PHARMACEUTICAL BULLETIN

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Molecular imaging technologies, which enable the visualization of the behaviors or functions of biomolecules in living systems, have received considerable attention from life scientists. Novel imaging technologies that overcome the limitations of current imaging techniques are desired. In this review, two independent technologies that were recently developed by the authors are described. The first technology is for smart 19 F magnetic resonance imaging (MRI) probes that were developed for in vivo applications. These probes were developed by exploiting paramagnetic relaxation enhancement in order to detect hydrolase activity. With respect to cellular applications, gene expression in cells was visualized using one of the 19 F MRI probes. It was confirmed that this probe design principle is effective for various hydrolases, and broad applications are expected. The second technology is for practical protein labeling. This labeling method is based on a mutant b b -lactamase and its specific labeling probes. Since the probe is fluorescence resonance energy transfer (FRET)-based, this labeling method achieves both specific and fluorogenic labeling of target proteins. In addition, derivatization of the probe enabled the labeling of intracellular proteins and the modification of various functional molecules.

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Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins

Cited by 350 publications

References 42 publications

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells

Development of Molecular Imaging Tools to Investigate Protein Functions by Chemical Probe Design

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