Identification of Hydrophobic Tags for the Degradation of Stabilized Proteins

Tae, Hyun Seop; Sundberg, Thomas B.; Neklesa, Taavi K.; Noblin, Devin J.; Gustafson, Jeffrey L.; Roth, Anke G.; Raina, Kanak; Crews, Craig M.

doi:10.1002/cbic.201100793

Cited by 82 publications

(102 citation statements)

References 25 publications

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“…We then tested their ability to degrade GFP-HaloTag7, (stably expressed in HEK 293 cells) by flow cytometry, measuring changes in mean fluorescence intensity. This assay (previously developed to test hydrophobic tags) 26 was chosen as our primary assay rather than immunoblotting as it offered more sensitive and reliable quantitation. Furthermore, the relatively high throughput nature of the assay allowed us to rapidly test multiple treatment conditions and obtain more complete dose response curves.…”

Section: Resultsmentioning

confidence: 99%

“…25, 26 An ideal control compound was therefore the enantiomers of the specific HaloPROTACs as they would have identical physical properties (such as hydrophobicity, solubility and passive membrane permeability) but would lack the ability to bind to VHL. We synthesized five key ent -HaloPROTACs with both amide and phenol linkers of various lengths and found that none resulted in any significant degradation of GFP-HaloTag7, providing strong evidence that the HaloPROTACs work through a VHL dependent mechanism (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

“…HEK 293 Flp-In cells stably expressing GFP-HaloTag7 were obtained as described in the literature. 26 To generate HEK293T cells stably expressing HT7-ERK1 or HT7-MEK1, each of the HT7 fusion constructs was cloned into the retroviral pBabe-puro vector. The pHTN vector was used as HT7 template, and the Proquest human spleen cDNA library was used as a source of ERK-1 and MEK-1 cDNA.…”

Section: Methodsmentioning

confidence: 99%

“…24 We have previously developed an alternative degradation technology that uses hydrophobic tags to mimic protein unfolding, leading to the degradation of HaloTag2 fusion proteins. 25 However, these molecules were far less capable of degrading proteins fused to HaloTag7, 26 a variant developed by Promega to increase the protein’s stability. 27 Due to HaloTag7’s resistance towards alternative methods of induced degradation and the existence of a potent small molecule ligand capable of accommodating long linkers, we concluded that HaloTag7 fusion proteins would make an ideal model system to develop novel small molecule HaloPROTACs.…”

Section: Introductionmentioning

confidence: 99%

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HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

Buckley¹,

Raina²,

et al. 2015

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Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knockdown widely used HaloTag7 fusion proteins in a general fashion.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

Buckley¹,

Raina²,

et al. 2015

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“…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] .…”

mentioning

confidence: 99%

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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Cellular Principles of Targeted Protein Degradation

Diamantino

Hellerschmied

2022

Inducing Targeted Protein Degradation

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Identification of Hydrophobic Tags for the Degradation of Stabilized Proteins

Cited by 82 publications

References 25 publications

HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

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

Cellular Principles of Targeted Protein Degradation

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