Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system

Akinjiyan, Favour A.; Carbonneau, Seth; Ross, Nathan T.

doi:10.1016/j.bmcl.2017.08.058

Cited by 8 publications

(5 citation statements)

References 72 publications

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“…To circumvent this, alternative strategies have been developed that involve attaching various tags to target proteins, to enable these to be targeted by a compound capable of inducing interaction with an E3 ligase machinery. 1,6,7 One of the first approaches made use of the plant E3 ligase TIR1, exogenously expressed in non-plant cells, to trigger ubiquitylation and degradation of target proteins fused to an Auxin-inducible degron (AID) tag on addition of the plant hormone Auxin. 8−10 A more recent approach exploits the ability of a phthalimide-based chimeric compound called dTAG to bind proteins fused with a mutant FKBP12 tag and induce degradation via the cereblon (CRBN) E3 ligase.…”

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confidence: 99%

“…PROTACs, however, require a ligand capable of interacting with the desired target, and high-affinity ligands are lacking for the vast majority of human proteins. To circumvent this, alternative strategies have been developed that involve attaching various tags to target proteins, to enable these to be targeted by a compound capable of inducing interaction with an E3 ligase machinery. ,, One of the first approaches made use of the plant E3 ligase TIR1, exogenously expressed in non-plant cells, to trigger ubiquitylation and degradation of target proteins fused to an Auxin-inducible degron (AID) tag on addition of the plant hormone Auxin. − A more recent approach exploits the ability of a phthalimide-based chimeric compound called dTAG to bind proteins fused with a mutant FKBP12 tag and induce degradation via the cereblon (CRBN) E3 ligase. − CRBN is endogenously expressed in most mammalian cells and, therefore, does not need to be overexpressed. Other methods include deGradFP and AdPROM, which rely on the overexpression of the von Hippel–Lindau (VHL) E3 ligase fused to a GFP binding nanobody capable of inducing degradation of GFP-tagged proteins, − or the Trim–AWAY method that enables the TRIM21 E3 ligase to be recruited to targets via a specific antibody .…”

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Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader

et al. 2019

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Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC50 of 3–10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (Dmax of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.

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Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader

et al. 2019

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“…The importance of the UPS in normal and disease states is well characterized, 32,33 and many approaches have been developed recently to further elucidate and target this system for drug discovery. 34 The transient and reversible nature of ubiquitination makes it challenging to accurately measure this posttranslational modification. The most commonly used method for measuring ubiquitination is immunoprecipitation followed by Western blotting, but this approach is not amenable for HTS.…”

Section: Discussionmentioning

confidence: 99%

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs)

Akinjiyan¹,

Fazal²,

Hild³

et al. 2020

SLAS Discovery

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Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

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“…The ubiquitin-proteasome system (UPS) is an important signaling for regulating the degradation or function of intracellular proteins in many diseases, including tumors [18,19]. required for mediating protein ubiquitination, including ubiquitin-activating enzyme E1, ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 [20][21][22]. Among these, E3 ubiquitin ligases represent highly attractive protein targets for drug discovery, because the significance of E3 ligases as prospective targets for small molecule modulation is reinforced by ever growing evidences of their roles in tumors and other diseases [23,24].…”

Section: Discussionmentioning

confidence: 99%

RING finger protein 38 induces gastric cancer cell growth by decreasing the stability of the protein tyrosine phosphatase SHP‐1

Zhang

et al. 2018

FEBS Letters

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The function of the E3 ligase RNF38 is still unknown in gastric cancer. Here, we found that RNF38 is upregulated in gastric cancer, and it is associated with the overall survival of gastric cancer patients. Further studies showed that RNF38 interacts with the nonreceptor tyrosine phosphatase SH2-containing protein tyrosine phosphatase 1 (SHP-1) and induces the polyubiquitination of SHP-1, which leads to destabilization of SHP-1 and promotion of STAT3 signaling in gastric cancer cells. In addition, overexpression or knockdown of RNF38 induces or suppresses gastric cancer cell growth in vitro, respectively, and silencing RNF38 delays tumor growth in vivo. These findings demonstrate that RNF38 is functional in gastric cancer and promotes STAT3 signaling by destabilizing SHP-1; thus, RNF38 could be a novel target for gastric cancer therapy.

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Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system

Cited by 8 publications

References 72 publications

Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader

Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs)

RING finger protein 38 induces gastric cancer cell growth by decreasing the stability of the protein tyrosine phosphatase SHP‐1

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