TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

Madiraju, Charitha; Welsh, Kate; Cuddy, Michael; Godoi, Paulo H.; Pass, Ian; Ngo, Tram A.; Vasile, Stefan; Sergienko, Eduard; Diaz, Paul; Matsuzawa, Shu‐ichi; Reed, John C.

doi:10.1177/1087057111423417

Cited by 35 publications

(40 citation statements)

References 48 publications

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“…1A). In contrast, previous assays have used the wild-type fluorescent Ub that yields polyubiquitination (31,32). The restriction to a single Ub-Ub linkage eliminates the complexity associated with polyubiquitin chains and allows straightforward quantification of each assay.…”

Section: Discussionmentioning

confidence: 99%

“…S1A). Note that optimal positioning of the fluorophore in Ub is critical, because N-terminally labeled Ub, commonly used in previous publications (31,33), was inactive in our system. Commercial fluorescent Ub proteins do not specify the location of the fluorescent dye in Ub, and it is unclear if such reagents can be generally optimal for HTS FRET assays.…”

Section: Discussionmentioning

confidence: 99%

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Suramin inhibits cullin-RING E3 ubiquitin ligases

Chong

et al. 2016

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

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Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a smallmolecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.protein degradation | K48-polyubiquitination | E3 ligase | E2 enzyme | suramin

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Suramin inhibits cullin-RING E3 ubiquitin ligases

Chong

et al. 2016

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

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“…27,28 The utility of these compounds for the development of therapeutics, however, has been questioned because of their chemical properties. 29 Chronic active BCR signaling, 11 together with MYD88-medated signaling, 13 is largely responsible for the constitutive NF-B activity in ABC-DLBCL cells and controls the proliferation and survival of these cells. Therefore, inhibition of this pathway may provide new therapeutic strategies.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A

Pulvino¹,

Ye²,

Oleksyn

et al. 2012

Blood

127

119

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IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease comprising at least 3 major subtypes with distinct molecular, biologic, and clinical properties: activated B cell-like DLBCL (ABC-DLBCL), germinal center B cell-like DLBCL (GCB-DLBCL), and primary mediastinal B-cell lymphoma. 1,2 Although the overall cure rate for DLBCL reaches more than 50% with the current therapies such as R-CHOP (rituximab plus cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone), less than 40% of ABC-DLBCL patients are cured. [2][3][4] Therefore, new therapy approaches efficient for this and other DLBCL subtypes are highly desirable.The transcription factor NF-B controls the expression of a wide range of genes involved in cell proliferation, survival, stress response, angiogenesis, and inflammation. 5,6 NF-B activity is tightly regulated by multiple signaling pathways, and abnormal NF-B activation has been linked to cancer development and progression. [7][8][9] Constitutive NF-B activation has been observed in high frequency in all of the main DLBCL subtypes, especially in ABC-DLBCL, with more than 90% of the tumors showing nuclear NF-B, the hallmark of its activation. 9-14 A recent genomic study revealed that more than 60% of ABC-DLBCLs and approximately 30% of GCB-DLBCLs harbor somatic mutations in multiple components of NF-B signaling pathways, such as the BCR, CD40, and TLR pathways. 15 Significantly, it has been demonstrated that constitutive NF-B signaling is required for the proliferation and survival of ABC-DLBCL cell lines. 11,13,16,17 All of these observations suggest a primary role for constitutive NF-B signaling in the pathogenesis of DLBCL and, therefore, the NF-B signaling pathway may represent a rational therapeutic target in DLBCL. 9,11,18 Ubiquitination, the covalent attachment of the ubiquitin (Ub) molecule to target proteins, regulates diverse cellular processes. 19 Ubiquitination proceeds through a stepwise enzymatic cascade involving 3 classes of enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The E1 enzyme activates Ub in an ATP-dependent manner and transfers the activated Ub to an E2 enzyme through the formation of a thioester bond between the carboxy terminus of Ub and the active site cysteine of the E2, generating an E2 and Ub thioester conjugate (referred to as E2ϳUb). The E2 then cooperates with an E3 to attach the Ub to a lysine residue of a substrate. Ub itself can serve as a substrate and the process can undergo multiple rounds, resulting in the formation of polyubiquitin chains. 19,20 Because Ub has 7 lysine residues and any one of them can be conjugated to another Ub, polyubiquitin chains of different linkages with distinct functional properties are formed in cells. For example, lysine 48 (K48)-linked polyubiquitin chains typically target substrates for proteasomal degradation, whereas K63-linked polyubiquitin chains function as scaffolds to assemble protein complexes in NF-B signaling and DNA repair. [...

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“…The common formats include the protease-coupled Fluor de Lys (FdL) assay, 5 TR-FRET, 15 and RapidFire chromatography coupled with MS. 7 The FdL assay technology uses enzyme-linked detection of activity on a coumarin-labeled substrate and suffers from a high level of false positive or negatives if compounds interfere with detection. TR-FRET reduces the number of assay artifacts, but relies on antibodies for detection of the reaction product.…”

Section: Resultsmentioning

confidence: 99%

Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry

Patel¹,

Sherrill²,

Mrksich

et al. 2015

SLAS Discovery

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Lysine acetylation plays a critical role in cellular regulation and is implicated in human disease. Sirtuin deacetylases remove acetyl groups from modified lysine residues, and sirtuin 3 (SIRT3) has been identified as a target for cancer therapeutics. Robust and high-throughput screening methods for these targets will be important to the development of therapeutics. This article describes the use of self-assembled monolayer desorption/ionization mass spectrometry, or SAMDI-MS-a label-free drug discovery tool-to characterize SIRT3 activity and discover inhibitors. SAMDI-MS was used to analyze a peptide array having 361 distinct acetylated peptides to identify an active SIRT3 substrate (GYK Ac RGC). This peptide was used in a screen of 100,000 small molecules to identify inhibitors of SIRT3. A total of 306 SIRT3 inhibitors were identified, with one compound, SDX-437, having an IC 50 of 700 nM with >100-fold selectivity for SIRT3 over SIRT1.

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TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

Cited by 35 publications

References 48 publications

Suramin inhibits cullin-RING E3 ubiquitin ligases

Suramin inhibits cullin-RING E3 ubiquitin ligases

Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A

Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry

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